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UBC Theses and Dissertations

Humatopoietic effects of photofrin Hunt, David William Carey 1996

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HEMATOPOIETIC EFFECTS OF PHOTOFRIN® by DAVID W I L L I A M CAREY HUNT B . S c , Univers i ty o f W inn ipeg , 1 9 7 4 M . S c , Univers i ty o f Man i toba , 1 9 7 8 A THESIS SUBMITTED' IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY W e accept th is thesis as con fo rm ing to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apr i l 1 9 9 6 ® DAVID W I L L I A M CAREY HUNT In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2788) ABSTRACT In the presence of visible l ight , cer ta in natura l ly -occurr ing and syn the t ic non-metal l ic porphyr ins and their der ivat ives can cause tox ic e f fects w i t h i n cel ls. This aspect of these c o m p o u n d s has been harnessed for the e l iminat ion of mal ignant t issue, a therapeut ic approach te rmed pho todynamic therapy (PDT). The l ikel ihood tha t photosensi t iz ing porphyr ins m igh t s igni f icant ly in f luence the s ta tus of t issues other than those targeted by PDT, has not been r igorously inves t iga ted . W h e n given to normal male mice , the photosensi t izer Photofr in® (d ihematoporphyr in ether) p roduced s ign i f icant increases in spleen w e i g h t , nuc leated cell numbers , indices of cell pro l i fera t ion, and levels of g ranu locy te -macrophageand ery thro id progeni tors . Concordan t w i t h these changes, w a s an increase in the number of spleen cells w h i c h expressed the receptor (CD71) for the i ron t ranspor t prote in t ransfer r in but no change in express ion of the receptor (CD25) for the T cell g r o w t h fac tor in ter leuk in-2, suggest ing tha t the compound had no t e f fec ted splenic l y m p h o c y t e ac t i va t ion . Further f l o w cy tomet r i c studies demons t ra ted tha t Photofrin® elevated the number of nuc leated spleen cells w h i c h expressed ant igens recognized by the monoc lona l an t ibody L R - 1 , the erythro id l ineage-speci f ic an t ibody TER-119, and those tha t did no t express the leukocyte c o m m o n ant igen C D 4 5 or major h is tocompat ib i l i t y Class I molecu les. The ant igen recognized by the LR-1 ant ibody w a s subsequent ly ident i f ied as the g lycosy lphosphat idy l inos i to l -anchoredheat stable ant igen (HSA) by enzyme digest , f l o w c y t o m e t r i c and Wes te rn immunob lo t s tud ies. The increased expression of HSA in the spleens of Photofr in®-treated mice w a s associated w i t h heightened ii ery thropoies is in the o rgan . Granulopoiesis w a s the dominan t ac t iv i ty in the m a r r o w of mice g iven Photofr in®. This w a s represented by a large increase in the propor t ion of cells w h i c h expressed the myelo id-associated Gr-1 and C D 1 1 b ant igens and a decrease in the propor t ion of cells w h i c h expressed CD71 or the B cel l - restr icted isofo rm of C D 4 5 (B220) . In con t ras t to Photofr in®, o ther photosensi t iz ing porphyr ins inc lud ing benzoporphyr in der ivat ive (BPD, ve r tepor f in ) , hyd roxye thy l v iny l deu te ropo rphy r in , hematoporphyr in IX, and p ro toporphyr in IX had no discernable hematos t imu la to ry e f fec t . Cul ture of mur ine ery th ro leukemic cells w i t h Photofr in®, but not BPD, increased cell sur face CD71 levels and binding of t ransfer r in to these cel ls, bu t did not cause ery thro id d i f fe ren t ia t ion . Photofrin® also increased sur face CD71 expression by non-ery thro id P815 m a s t o c y t o m a cel ls, suggest ing tha t Photofr in® can inf luence cellular iron s ta tus . A l t hough the mechan ism by w h i c h Photofrin® inf luences hematopoies is w a s no t ident i f ied, it never the less wi l l be an in terest ing agent w i t h w h i c h to s tudy mur ine hematopoies is . iii T A B L E O F C O N T E N T S page ABSTRACT ii LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xx i A C K N O W L E D G E M E N T xx iv 1 . INTRODUCTION 1 1.1 Photodynamic therapy 1 1.2 Photofr in® (d ihematoporphyr in ether) 3 1.3 Second generat ion porphyr in photosensi t izers 5 1.4 Hematopoies is 5 1.4.1 Organizat ion of the hematopo ie t ic t issues 6 1.4.1.1 Bone m a r r o w 6 1 .4 .1 .2 Hematopoie t ic s tem cells (HSC) 7 1 .4 .1 .3 Hematopoie t ic d i f ferent ia t ion and 9 prol i ferat ion 1 .4 .1 .4 Cont r ibu t ion of the spleen to hematopoies is 14 1 .4 .1 .5 Hematopoie t ic s t imula t ion by exogenous 16 fac tors 1.5 I roh-heme metabo l ism and erythropoies is 17 1.5.1 Erythropoiet in (Epo), e ry thropo ie t in receptor (EpoR), and 17 regulat ion of ery thropoies is 1.5.2 The t ransfer r in (Trf ) - t ransferr in receptor (TrfR) sys tem 21 iv page 1.5.3 Heme metabo l ism 2 3 1.5.4 Inf luence of porphyr in photosensi t izers on the 27 heme metabol ic p a t h w a y and hematopoie is is 1.5.5 Inf luence of heme on hematopoies is 2 9 1.5.6 Inf luence of i ron-binding prote ins on hematopoies is 3 0 1.6 Thesis ob ject ives 31 2. MATERIALS A N D METHODS 33 2.1 An ima ls 3 3 2 .2 Porphyr ins 3 4 2 .2 .1 Iron p ro toporphyr in IX (hemin) 3 4 2 . 2 . 2 Pro toporphyr in IX (PPIX) 3 4 2 . 2 . 3 Deuterporphyr in IX 2 ,4 (4,2) h y d r o x y e t h y l v iny l (HVD) 3 4 2 . 2 . 4 Benzoporphyr in der ivat ive mono-ac id r ing A 35 (BPD, ver topor f in ) 2 . 2 . 5 Hematoporphyr in IX (Hp) 35 2 . 2 . 6 Iron hematoporphyr in IX (FeHp) 3 6 2 . 2 . 7 Photofrin® (d ihematoporphyr in ether, por f imer sodium) 3 6 2 .3 Exper imental p ro toco ls 38 2 .3 .1 Porphyr in e f fec ts in naive mice 38 2 . 3 . 2 Inf luence of Photofrin®, FeHp, and HVD on 38 hematopo ie t ic recovery af ter sub- lethal i r radiat ion 2 . 3 . 3 Inf luence of Photofrin® on hematopo ie t ic recovery after 3 9 5- f luorouraci l (5-FU) t rea tmen t v page 2 . 4 Tissue sampl ing 4 0 2 .4 .1 Peripheral b lood 4 0 2 . 4 . 2 Spleen 41 2 . 4 . 3 Bone m a r r o w (BM) 4 2 2 . 4 . 4 T h y m u s 4 2 2 . 4 . 5 Peritoneal cells 4 2 2 . 4 . 6 Lymph node cells 4 3 2 .5 Hematopo ie t ic progeni tor assays 4 3 2 .5 .1 Granu locy te-macrophage progeni tor assay 4 3 2 . 5 . 2 Erythro id progeni tor assays 4 4 2 .6 Prol i ferat ion assays 4 5 2 .6 .1 Responses t o m i togens 4 6 2 . 6 . 2 Responses to hematopo ie t ic cy tok ines 4 7 2 . 6 . 3 Evaluat ion of rest ing cellular metabol ic ac t iv i ty 4 8 2 . 6 . 4 Prol i ferat ive responses to a prote in ant igen 4 8 2 .6 .5 M ixed leukocy te react ion (MLR) 50 2 .7 Cell l ines 51 2 .8 F low c y t o m e t r y 52 2 .9 Cell cyc le analysis 53 2 . 1 0 Erythroid d i f ferent ia t ion of mur ine ery thro leukemia (MEL) cells 59 2 .11 Transferr in b inding 6 0 v i page 2 . 1 2 Ana lys is of per i toneal cell hematopo ie t ic accessory ac t iv i ty 6 0 2 . 1 2 . 1 Granu locy te-macrophage progeni tor assay and 6 0 determinat ion of per i toneal cell co lony-s t imula t ing act iv i ty 2 . 1 2 . 2 Peritoneal cell FACS analysis 6 2 2 . 1 2 . 3 Determinat ion of ni tr ic oxide (NO) p roduc t ion 62 2 . 1 3 Evaluat ion of the impac t of Photofrin® and BPD 63 on the immune response 2 . 1 3 . 1 An t igen sensi t izat ion 63 2 . 1 3 . 2 Determinat ion of serum ant ibody levels 6 4 2 . 1 3 . 3 Evaluat ion of an t i -OVA IgG an t ibody spec i f ic i ty 65 2 . 1 4 Character izat ion of the LR-1 ant igen 65 2 . 1 4 . 1 Tissue d is t r ibut ion and co-express ion studies 65 2 . 1 4 . 2 Pur i f icat ion of leukocytes for LR-1 ant igen 67 expression studies 2 . 1 4 . 2 . 1 Peripheral b lood e ry th rocy tes 67 2 . 1 4 . 2 . 2 Splenic dendr i t ic cells (DC) 67 2 . 1 4 . 2 . 3 Splenic B cells 68 2 . 1 4 . 2 . 4 Splenic T cells 6 9 2 . 1 4 . 3 Evaluat ion of g lycosy l -phosphat idy l inos i to l 6 9 (GPI)-l inkage 2 . 1 4 . 4 Wes te rn blot analysis 7 0 2 . 1 5 Data presentat ion and stat ist ical analysis 71 vii page 3. RESPONSE OF THE INTACT MURINE HEMATOPOIETIC 72 SYSTEM TO PHOTOSENSITIZING PORPHYRINS 3.1 In t roduc t ion 7 2 3 .2 Results 7 2 3 .2 .1 Splenic response to photosensi t iz ing porphyr ins 7 2 3 . 2 . 2 Evaluat ion of the in vitro CFU-GM 79 progeni tor tes t sys tem 3 . 2 . 3 Inf luence of Photofrin® and Hp on splenic 8 0 ery thropo ie t ic ac t iv i ty 3 . 2 . 4 FACS analysis of spleen leukocy te ant igens 85 3 .2 .5 Cell cyc le analysis 9 0 3 . 2 . 6 Spleen cell responses to d i f fe rent hematopo ie t ic 9 0 g r o w t h fac to rs 3 . 2 . 7 Spleen cell expression of ac t iva t ion ant igens 95 3 . 2 . 8 F low cy tomet r i c ev idence of a splenic ery thro id 105 progeni tor response to Photofrin® 3 . 2 . 9 Splenic response to Photofrin® by DBA/1 mice 11 5 3 . 2 . 1 0 Regulatory aspects of the splenic hematopo ie t ic 119 response to Photofrin® 3 . 2 . 1 1 Inf luence of BPD on splenic hematopo ie t ic 119 ac t iv i ty 3 . 2 . 1 2 Inf luence of Photofrin® and BPD on m a r r o w 121 hematopo ie t ic ac t iv i ty 3 . 2 . 1 3 Inf luence of Photofrin® on CD71 express ion 121 in the m a r r o w 3 . 2 . 1 4 Inf luence of Photofrin® on m a r r o w cel lular i ty 126 and express ion of d i f ferent leukocy te ant igens viii page 3 . 2 . 1 5 Peripheral b lood parameters af ter Photofrin® 134 admin is t ra t ion 3 .3 Discussion 137 4 . INFLUENCE OF PORPHYRINS ON THE HEMATOPOIETIC 147 RECOVERY OF MICE TREATED W I T H r -RADIATION OR 5-FLUOROURACIL 4 .1 In t roduc t ion 147 4 . 2 Results 150 4 . 2 . 1 Hematopoie t ic recovery in i rradiated mice 150 t reated w i t h Photofrin® (10 mg/kg) 1 and 4 days post- i r radiat ion 4 . 2 . 2 Hematopo ie t ic recovery by i r radiated mice 154 t reated w i t h Photofrin® or HVD (10 mg/kg) 1 , 4 and 7 days post- i r radiat ion 4 . 2 . 3 Hematopoie t ic recovery of i rradiated mice 155 t reated w i t h FeHp 4 . 2 . 4 Inf luence of Photofrin® on the myelopoie t ic 158 recovery of mice t reated w i t h 5-FU 4 . 3 Discussion 163 5. INFLUENCE OF PHOTOFRIN® ON THE HEMATOPOIETIC 169 ACCESSORY ACTIVITY OF PERITONEAL CELLS 5.1 In t roduc t ion 169 5 .2 Results 172 5 .2 .1 Inf luence of Photofrin® on the cel lular i ty and 172 leukocy te prof i le w i t h i n the per i toneal cav i t y 5 . 2 . 2 Hematopo ie t ic accessory act iv i ty of 173 per i toneal cells ix page 5.3 Discussion 185 6. INFLUENCE OF PHOTOFRIN® A N D BPD ON THE 189 IMMUNE RESPONSE 6.1 In t roduc t ion 189 6 .1 .1 The immune response 189 6 . 1 . 2 The role of iron and heme in the 193 immune response 6 . 1 . 3 Impact of porphyr in photosensi t izers on 194 the immune response 6 . 1 . 4 Exper imental approach 195 6.2 Results 195 6 .2 .1 Evaluat ion of Photofrin® and BPD for 196 in vitro mi togenic act iv i ty 6 . 2 . 2 Inf luence of Photofrin® and BPD admins t ra t ion 196 on spleen cell mi togenic responses 6 . 2 . 3 Lympho id prol i ferat ive responses to 199 an adminis tered ant igen 6 . 2 . 4 Ef fect of Photofrin® admin is t ra t ion on the 201 fo rmat ion of ant igen-speci f ic humora l immun i t y 6 .3 Discussion 2 0 6 7. CHARACTERIZATION OF THE LR-1 ANTIGEN 2 0 9 7.1 In t roduc t ion 2 0 9 7 .2 Results 2 1 0 7 .2 .1 Tissue d is t r ibut ion of the LR-1 ant igen 2 1 0 x page 7.2.2 Expression of the LR-1 antigen on normal 217 blood erythrocytes, splenic dendritic cells (DC) and splenic B lymphocytes 7.2.3 Evaluation of glycosyl-phosphatidylinositol 220 (GPI)-linkage 7.2.4 Western blot analysis 223 7.2.5 LR-1 binding specificity for HSA 226 7.2.6 Infl uence of Photofrin® and Hp on spleen cell 226 expression of HSA as recognized by four different anti-HSA reagents 7.2.7 Spleen cell HSA co-expression studies 229 - influence of Photofrin® 7.2.8 Influence of Photofrin® on BM HSA and 237 CD71 expression 7.3 Discussion 240 8. IN VITRO STUDIES 254 8.1 Introduction 254 8.2 Results 255 8.2.1 Influence of Photofrin® on splenic 255 granulocyte-macrophage colony formation 8.2.2 Splenic responses to Photofrin® in combination 256 with different recombinant cytokines 8.2.3 Response of MEL cells cultured with 258 hemin, DMSO, or Photofrin® 8.2.4 Influence of Photofrin® on transferrin binding 269 8.2.5 Influence of BPD on MEL cell expression 273 of CD71 and Trf binding xi page 8 . 2 . 6 Response of P815 cells to Photofrin® 2 7 5 8 .3 Discussion 2 7 9 9. GENERAL DISCUSSION A N D CONCLUSIONS 2 8 9 10. REFERENCES 2 9 6 xii Table 1.1 Table 2.1 Table 2 .2 Table 2 .3 Table 2 . 4 Table 3.1 Table 3 .2 Table 3 .3 Table 3 . 4 Table 4 .1 Table 6.1 Table 7.1 Table 7 .2 Table 7.3 LIST OF TABLES page Secondary hematopo ie t ic regulatory fac to rs 14 Recombinant mur ine and human cy tok ines 4 9 uti l ized in these studies Monoc lona l ant ibodies used for leukocy te 55 sur face analysis by f l o w c y t o m e t r y Pur i f ied, unlabel led and con jugated isotyp ic 57 con t ro l ant ibodies employed in th is s tudy F luorochrome-con jugated secondary monoc lona l 58 and polyc lonal an t ibody reagents Spleen parameters of mice admin is tered d i f fe rent 7 4 photosens i t iz ing porphyr ins Compar ison of 3 d i f fe rent Photofrin® doses on 8 9 spleen parameters Compar ison of spleen cell expression of HSA 106 w i t h t ha t of the LR-1 marker Spleen and BM ery thro id l ineage-associated 113 ant igen exprss ion Spleen cell expression of the LR-1 ant igen in normal 161 mice and mice t reated w i t h 5-FU and g iven 5 % dext rose or Photofrin® Summar ized spleen and l ymph node parameters 2 0 2 of naive and OVA- immun ized mice Summar ized results of spleen cell double label 2 3 2 FACS analyses Compar ison of the inf luence of Photofrin® on 2 3 3 spleen cell co-express ion of CD71 and HSA, as labelled by LR-1 and J 1 1 d monoc lona l ant ibodies Inf luence of Photofrin® on BM HSA express ion 2 4 0 and HSA/CD71 co-express ion levels Table 8.1 Inf luence of Photofrin® on MEL cell express ion of CD71 2 7 3 xiii LIST OF FIGURES page Figure 1.1 Pa thways of hematopo ie t ic d i f ferent ia t ion 10 Figure 1.2 Heme (iron p ro toporphyr in IX) synthes is 26 Figure 2.1 Molecular s t ruc ture of the porphyr ins 37 uti l ized in these studies Figure 2 .2 Ver i f ica t ion of the f l o w c y t o m e t r y co lour 5 4 compensa t ion procedure Figure 3.1 Inf luence of Photofrin® on spleen size and appearance 73 Figure 3 .2 Time course of the splenic response to Photofr in® 7 6 Figure 3.3 Compar ison of t w o d i f fe rent doses of Photofr in® 77 on splenic CFU-GM levels Figure 3 .4 Morpho log ic appearance of splenic CFU-GM 78 Figure 3.5 Expansion of spleen cells w i t h P W M - S C C M 81 in a l iquid cul ture sys tem Figure 3 .6 Inf luence of Photofrin® and Hp on splenic e ry thro id 8 2 progeni tor levels Figure 3.7 Morpho log ic appearance of splenic CFU-E and BFU-E 8 3 Figure 3 .8 Summar ized spleen cell sur face ant igen 87 express ion studies Figure 3.9 Cell cyc le analysis 91 Figure 3 . 1 0 Cell cyc le analyses - f l o w cy tomet r i c prof i les 92 Figure 3 .11 Spleen cell responses to d i f fe rent hematopo ie t ic 9 3 g r o w t h fac to rs Figure 3 . 1 2 Spleen cell responses to rEpo 9 4 Figure 3 . 1 3 Spleen cell expression of CD25 and CD71 ant igens 97 fo l l ow ing Photofrin® admin is t ra t ion x iv page Figure 3 . 1 4 Spleen cell expression of CD25 and CD71 sur face 9 8 ant igens - FACS prof i les Figure 3 . 1 5 Spleen cell expression of CD71 as recognized by 9 9 the monoc lona l ant ibodies C2F2 and R17 2 1 7 Figure 3 . 1 6 Spleen cell CD71 expression - FACS prof i les 100 Figure 3 .17 Spleen cell CD71 co-express ion pat terns 101 Figure 3 . 1 8 Inf luence of Photofrin® and PPIX on spleen w e i g h t , 103 cel lular i ty and expression of the t ransfer r in receptor (CD71) Figure 3 . 1 9 Inf luence of Photofrin® and PPIX on spleen cell CD71 104 expression - FACS prof i les Figure 3 . 2 0 Spleen cell expression of the ery thro id TER-119 ant igen 108 Figure 3 .21 Spleen cell HSA (J11d ant igen) and LR-1 ant igen 109 express ion Figure 3 . 2 2 Inf luence of Photofrin® on levels of 110 C D 7 1 + / L R - 1 + spleen cells Figure 3 . 2 3 Co-expression of CD71 w i t h the LR-1 ant igen 111 - FACS prof i les Figure 3 . 2 4 Spleen and BM cell ery thro id-assoc ia ted ant igen 112 express ion prof i les Figure 3 . 2 5 Numbers of LR-1 + / C D 4 5 " / | 0 cells are increased in the 114 spleens of Photofr in®-treated mice Figure 3 . 2 6 Spleen parmeters of Photofr in®-injected DBA/1 mice 116 Figure 3 . 2 7 LR-1 expression by DBA/1 mouse spleen cells 117 Figure 3 . 2 8 Trf-F b inding by spleen cells f r o m DBA/1 mice 118 Figure 3 . 2 9 Inf luence of indomethac in on the splenic response 120 to Photofrin® Figure 3 . 3 0 Inf luence of BPD on d i f ferent spleen parameters 122 xv page Figure 3 .31 Inf luence of Photofrin® on levels o f BM CFU-GM 123 Figure 3 . 3 2 Inf luence of BPD on BM CFU-GM levels 1 2 4 Figure 3 . 3 3 Inf luence of Photofrin® on BM BFU-E levels 125 Figure 3 . 3 4 Inf luence of Photofrin® on BM cell CD71 expression 127 as recognized by the monoc lona l an t ibody ER-MP-21 Figure 3 . 3 5 Inf luence of Photofrin® on BM CD71 express ion 128 as recognized by monoc lona l an t ibody ER-MP-21 - FACS prof i les Figure 3 . 3 6 Inf luence of Photofrin® on BM cell CD71 express ion 129 as labelled by the monoc lona l an t ibody C2F2 Figure 3 .37 Inf luence of Photofrin® on BM CD71 express ion 130 as recognized by the C2F2 monoc lona l an t ibody - FACS prof i les Figure 3 . 3 8 Inf luence of Photofrin® on m a r r o w cel lu lar i ty 131 Figure 3 . 3 9 Inf luence of Photofrin® on BM leukocy te 132 ant igen expression Figure 3 . 4 0 BM cell expression of C D 4 5 R - B 2 2 0 , C D 1 1 b , G r - 1 , 133 and LR-1 ant igens after Photofrin® admin is t ra t ion - FACS prof i les Figure 3 .41 Peripheral b lood analysis 135 Figure 3 . 4 2 Serum IL-6 levels of Photofr in®-treated mice 136 Figure 4 .1 Chemical s t ruc ture of 5- f luorouraci l (5-FU) 149 Figure 4 . 2 Inf luence of Photofrin® on the hematopo ie t ic recovery 152 of sub- lethal ly (4 Gy) irradiated mice Figure 4 . 3 Prol i ferat ive response to Con A by spleen cells 153 f r o m irradiated mice Figure 4 . 4 Inf luence of Photofrin® and HVD on the hematopo ie t ic 156 recovery of sub- lethal ly i rradiated (4 .8 Gy) mice xv i page Figure 4 . 5 Inf luence of FeHp on the hematopo ie t ic recovery 157 of sub- lethal ly i rradiated (7 Gy) mice Figure 4 . 6 Hematopo ie t ic responses to Photofrin® by 160 mice t reated w i t h 5-FU Figure 4 . 7 F low cy tomet r i c prof i les of LR-1 express ion on spleen 162 cells f r o m mice t reated w i t h 5-FU Figure 5.1 Peritoneal cell numbers af ter Photofrin® admin is t ra t ion 174 Figure 5 .2 Expression of d i f fe rent leukocy te ant igens by per i toneal 175 cells af ter int raper i toneal Photofrin® admin is t ra t ion Figure 5.3 F low cy tomet r i c prof i les of per i toneal cells obta ined 176 6 hours af ter the i.p. in ject ion of Photofrin® Figure 5 .4 Hematopo ie t ic accessory act iv i ty of per i toneal cells 178 Figure 5.5 GM-CSF levels in per i toneal ce l l -condi t ioned med ium 179 Figure 5 .6 Capaci ty of per i toneal ce l l -condi t ioned med ium to 180 suppor t the in vitro g r o w t h of normal BM CFU-GM Figure 5.7 Inf luence of per i toneal cells on BM CFU-GM 181 deve lopment in the presence or absence of P W M - S C M Figure 5.8 Peritoneal cell ni tr ic oxide (NO) fo rmat ion 183 Figure 5 .9 Compar ison of the route of Photofrin® admin is t ra t ion 184 on per i toneal cell numbers Figure 6.1 Evaluat ion of Photofrin® for mi togenic ac t iv i ty 197 Figure 6 .2 Evaluat ion of Photofrin® and BPD for 198 co-mi togen ic ac t iv i ty Figure 6.3 Spleen cell mi togenic responses fo l l ow ing 2 0 0 Photofrin® admin is t ra t ion Figure 6 .4 Spleen and l ymph node cell prol i ferat ive 2 0 3 responses to O V A Figure 6.5 Inf luence of Photofrin® on serum an t i -OVA IgG levels 2 0 4 xvi i page Figure 6 .6 Speci f ic i ty of serum ant i -OVA IgG ant ibodies 2 0 5 Figure 7.1 Expression of the LR-1 ant igen on normal DBA/2 2 1 2 mouse bone m a r r o w , t h y m o c y t e s , sp lenocy tes and l ymph node cells Figure 7 .2 Co-expression of var ious sur face ant igens w i t h the 2 1 3 LR-1 marker on normal DBA/2 BM cel ls, t h y m o c y t e s and sp lenocytes Figure 7 .3 LR-1 and J 1 1 d labell ing of normal mouse spleen cells 2 1 4 Figure 7 .4 Co-expression of var ious leukocy te ant igens w i t h 2 1 5 the ant igen recognized by LR-1 or the HSA-speci f ic monoc lona l an t ibody J 1 1 d on normal D B A / 2 spleen cells Figure 7.5 Co-expression of T and B cell ant igens w i t h the 2 1 6 LR-1 marker and HSA, as recognized by J 1 1 d , on sp lenocy tes prepared f r o m d i f fe rent mouse strains Figure 7 .6 Surface labell ing of HSA on normal mouse peripheral 2 1 7 b lood e ry th rocy tes by M 1 / 6 9 , J 1 1 d and LR-1 monoc lona l ant ibodies Figure 7.7 Immunos t imu la to ry ac t iv i ty of pur i f ied splenic DC 2 1 8 Figure 7 .8 An t igen expression pat tern of splenic DC 2 1 9 Figure 7 .9 Ef fect of LPS act iva t ion on the express ion of the 221 LR-1 ant igen by splenic B cells Figure 7 . 1 0 Phosphat idy l inosi to l -speci f ic phosphol ipase C (PI-PLC) 2 2 2 enzyme d igest ion studies Figure 7 .11 LR-1 immunob lo t analysis of t issue ex t rac ts 2 2 4 Figure 7 . 1 2 HSA binding speci f ic i t ies of L R - 1 , J 1 1 d , and 2 2 7 M 1 / 6 9 monoc lona l ant ibodies Figure 7 .13 Numbers of spleen cells w h i c h express HSA as 2 2 8 determined by 4 d i f fe rent ant i -HSA monoc lona l ant ibodies xvi i i page Figure 7 . 1 4 Spleen cell expression of HSA as labelled w i t h 2 3 0 4 d i f fe rent ant i -HSA ant ibodies - FACS prof i les Figure 7 .15 Inf luence of Photofrin® on spleen cell co-express ion 2 3 4 of HSA w i t h var ious leukocyte ant igens Figure 7 . 1 6 Spleen cell co-express ion of CD71 w i t h HSA 2 3 6 Figure 7 . 1 7 Inf luence of Photofrin® on BM cell HSA express ion 2 3 8 land numbers of BM cells w h i c h co-express HSA and CD71 Figure 7 .18 Molecular organizat ion and amino acid sequence 2 4 5 of mur ine heat stable ant igen (HSA) Figure 8.1 Inf luence of Photofrin® on spleen cell 2 5 7 g ranu locy te -macrophage co lony (CFU-GM) fo rmat ion Figure 8 .2 M T T dye reduct ion ac t iv i ty in spleen cell cu l tures 2 5 9 conta in ing d i f fe rent recombinant g r o w t h fac to rs Figure 8 .3 M T T dye reduct ion ac t iv i ty in spleen cell cu l tures 2 6 0 conta in ing d i f fe rent recombinant g r o w t h fac to rs and Photofrin® Figure 8 . 4 Prol i ferat ive response of MEL cells to Photofr in® 261 Figure 8 .5 Inf luence of D M S O , hemin , and Photofrin® on the 2 6 3 g r o w t h and ery thro id d i f ferent ia t ion of MEL cells Figure 8 .6 Cell sur face ant igen expression by the 2 6 5 MEL cell line D1B Figure 8 .7 Expression of C D 4 5 , HSA, and CD71 by MEL cells 2 6 6 cu l tured w i t h D M S O , hemin or Photofr in® Figure 8 .8 F low cy tomet r i c prof i les of CD71 express ion by MEL 2 6 7 cells cu l tured w i t h D M S O , hemin or Photofr in® Figure 8 .9 Relative expression levels of HSA and CD71 by MEL 2 6 8 cells cu l tured w i t h D M S O , hemin or Photofrin® Figure 8 . 1 0 Inf luence of Photofrin® on MEL cell g r o w t h 2 7 0 x ix page Figure 8 .11 Inf luence of t w o d i f fe rent concent ra t ions of Photofrin® 271 on MEL cell express ion of H-2D d and C D 7 1 ant igens Figure 8 . 1 2 Inf luence of Photofrin® on MEL cell 2 7 2 b inding of t ransferr in Figure 8 . 1 3 Inf luence of BPD on MEL cell CD71 express ion and 2 7 4 Trf-F b inding Figure 8 . 1 4 Di f ferent cell sur face ant igens of mur ine P815 2 7 6 m a s t o c y t o m a cells Figure 8 . 1 5 Inf luence of Photofr in® on the g r o w t h o f P815 cells 2 7 7 Figure 8 . 1 6 Inf luence of Photofrin® on P815 cell express ion 2 7 8 of C D 4 5 and CD71 markers xx LIST OF ABBREVIATIONS A L A ^-aminolevul in ic acid A L A S Amino levu l in ic acid syn thase BFU-E Burst fo rm ing un i t -ery thro id BM Bone m a r r o w BPD Benzoporphyr in der ivat ive monoac id Ring A BSA Bovine serum a lbumin CD Cluster of d i f ferent ia t ion (cluster designat ion) CFA Complete Freund's ad juvant CFU-E Colony fo rming un i t -ery thro id CFU-GM Colony fo rming un i t -g ranu locy te -macrophage CFU-S Spleen co lony- fo rming uni t Con A Concanaval in A DC Dendri t ic cell DMSO Dimethy l su l fox ide DNA Deoxyr ibonucle ic acid ELISA Enzyme-l inked immunosorben t assay Epo Erythropoiet in EpoR Erythropoiet in receptor FCS Fetal calf serum FeHp Iron hematoporphyr in IX FITC Fluorescein iso th iocyanate 5-FU 5-f luorouraci l 9 Gravit ies G-CSF Granulocyte co lony-s t imu la t ing fac to r GM-CSF Granu locy te -macrophageco lony-s t imu la t ing f actor Gy Grays (1 Gray = 1 0 0 rads) HO Heme oxygenase Hp Hematoporphyr in IX HpD Hematoporphyr in der ivat ive xx i HSA - Heat stable ant igen (CD24) HS-alb - Human serum albumin HSC - Hematopoie t ic s tem cell HVD - Hyd roxye thy l v iny l deu te roporphyr in IX ICAM-1 - Intercellular adhesion molecule-1 (CD54) Ig - Immunoglobu l in IL - Inter leukin IL-2R - ln ter leukin-2 receptor (CD25) i.p. - Intraper i toneal i.v. - In t ravenous IRE - Iron responsive e lement IRE-BP - Iron responsive e lement b inding prote in LPS - L ipopolysacchar ide A M-CSF - Macrophage co lony-s t imu la t ing fac to r 2-ME - 2-/3 mercaptoe thano l MEL - Mur ine ery thro leukemia M H C - Major h is tocompat ib i l i t y comp lex MLR - M ixed leukocy te react ion mRNA - Messenger r ibonucleic acid M T T - 3-[4,5-dimethyl thiazol-2-yl ] -2,5-diphenyl tetrazol ium bromide NK - Natural killer NO - Nitr ic ox ide O V A - Ova lbumin PBS - Phosphate-buf fered saline PDT - Photodynamic therapy PE - Phycoery thr in PI - Propid ium iodide PPIX - Pro toporphyr in IX P W M - S C C M - Pokeweed mi togen spleen cell-condit ioned medium r - Recombinant xxi i Stem cell fac tor (c-kit l igand) Standard dev iat ion Transferr in Transferr in receptor (CD71) xxi i i ACKNOWLEDGEMENT No research s tudy can be accompl ished in iso lat ion. M a n y individuals have s u p p o r t e d , ass is ted, and encouraged me over the past six and three-quarter years . First, I t hank my commi t tee member Dr. Ross Pet ty w h o conv inced me tha t a doc to ra l thes is w a s a w o r t h w h i l e pursui t and permi t ted me to cont inue part - t ime research w o r k in his laboratory . In the Rheumato logy lab, I obta ined exper ience w i t h the techn iques of cellular immuno logy , w h i c h great ly fac i l i ta ted the execut ion of the present s tud ies . The ins ight fu l suggest ions prov ided by other commi t tee members , Dr. Pauline Johnson and Dr. Wi l f red A . Jef fer ies , are gratefu l ly a c k n o w l e d g e d . In all s incer i ty , the relevance of some quest ions t o o k mon ths to become clear - obv ious ly a sign tha t educat ion w a s occur r ing . Funding suppor t prov ided by the B.C. Science Counci l ( 1 9 8 9 - 1 9 9 2 ) and the David H e m i n g w a y Foundat ion ( 1 9 8 9 - 1 9 9 1 ) is great ly apprec ia ted. The major i ty of the thesis w o r k w a s carr ied ou t at QLT PhotoTherapeut ics . The presence of excel lent sc ient i f ic equ ipment , laboratory mater ia ls , and a superb ly run animal care faci l i ty great ly fac i l i ta ted my research. I a c k n o w l e d g e the f reedom given to me by the QLT admin is t ra t ion to fo l l ow my research ins t inc ts . In the lab, I received ski l l ful assistance f r o m David Granvi l le, Diane King, S imon Leong, Mar t in Renke, and Claire Smi ts . I w o u l d like to especial ly thank Dr. Agnes Chan for her suppor t and encouragement , and Rob Sorrent i for help w i t h the radiat ion p ro tec t ion exper iments , d iscuss ions, and assistance in the edi t ing of var ious manuscr ip ts . I value useful d iscuss ions held w i t h Dr. Suzanne Chappel (Terry Fox Labs) and for the prov is ion of reagents. I t hank lan Haidl (Jef fer ies Lab) for cons t ruc t i ve dia logues on dendr i t ic cel ls, macrophages , FACS analysis, and graduate schoo l . Finally, I am indebted to Hui- jun J iang w h o assisted good-na tu red ly w i t h many labor ious analyses and came up w i t h ef f ic ient , a l ternat ives to exper imenta l p rob lems, not only in th is s tudy bu t also w i t h our human dendr i t ic cell w o r k . W h e n I f i rs t approached Jul ia Levy about graduate w o r k , she grac ious ly s ta ted t h a t my lab exper ience and seemingly advanced years were an a t t r ibute rather than a d r a w b a c k . The research oppor tun i t ies prov ided by Jul ia have more than exceeded my expec ta t ions . I t hank her for her suppor t and the con f idence she has s h o w e d in me over the years. Her abi l i ty to a lways see a br ight side - of even a d isappoin t ing exper imenta l o u t c o m e has been a source of assurance and the recogni t ion tha t the scient i f ic endeavour does not balance on the o u t c o m e of any one exper iment . M o s t impor tan t l y , I could have not comple ted th is w o r k w i t h o u t the love and suppor t prov ided by my w i fe Georg ina, our chi ldren Ryan and Olivia and my parents Carol and Dave Sr. Over these years, Olivia and Ryan have quick ly g r o w n f r o m y o u n g chi ldren to the d a w n of the teenage years. I acknow ledge tha t these were not the easiest t imes for the fami ly and on many evenings an empty spot sat at the d in ing room table. However , th is thesis is the cu lminat ion of a fami ly e f fo r t . It is my hope tha t Ryan and Olivia wi l l have access to similar educat iona l oppor tun i t ies tha t have been available to me. xx i v C H A P T E R O N E I N T R O D U C T I O N 1.1 P h o t o d y n a m i c therapy Photodynamic therapy (PDT) is a cancer t rea tmen t based upon the pr inciple t ha t cer ta in l ight -absorb ing c o m p o u n d s wi l l accumula te in mal ignant t issues and ini t iate a necrosis of the t u m o u r w h e n ac t iva t ing l ight is appl ied to the area. This techn ique has gained w idespread acceptance for the t rea tment of cancers to w h i c h a l ight source can be readily d i rec ted , such as those of the bladder, esophagus or sk in . Certain natural ly occur r ing and a var ie ty of synthet ic porphyr ins possess phys icochemica l propert ies w h i c h permi t their use as pho tochemothe rapeu t i c agents . The heme precursor p ro toporphyr in IX (PPIX) can behave as a photosensi t izer and the over -accumula t ion of th is c o m p o u n d in t issues, character is t ic of cer ta in human porphyr ias , is associated w i t h an exquisi te sensi t iv i ty to sunl ight in a f f l ic ted indiv iduals. A l t h o u g h photosensi t izers accumula te in normal and neoplast ic t i ssues, the site at w h i c h these c o m p o u n d s are act ivated can be de termined by speci f ic ta rget ing w i t h an ac t iva t ing wave leng th of l ight (Henderson and Dougher ty , 1 9 9 2 ) . However , at pho todynamica l l y e f fec t ive doses, photosens i t iz ing porphyr ins cause no s igni f icant tox ic e f fec ts in the absence of l ight (Richter etal., 1 9 8 7 ; Dougher ty , 1 9 8 9 ) . In the presence of speci f ic wave leng ths of l ight , non-metal l ic porphyr ins undergo a t rans i t ion f r o m the ground state leading to the fo rmat ion of s inglet o x y g e n in the t issue. This event is dependent upon the presence of 1 molecular o x y g e n in the immediate cell env i ronment . The initial absorpt ion of l ight pho tons raises the ch romophore of a photoac t iva tab le d rug f r o m its g round state to an exc i t ed , shor t - l ived ( 1 0 9 to 10" 6 seconds) singlet s tate (Gomer e f al., 1 9 8 8 ) . The photosensi t izer can decay back to the g round state and emi t l ight in the f o r m of f luorescence. H o w e v e r , the exc i ted singlet state may also undergo an inter-sys tem cross ing to a more stable and relat ively more pers is tent ( 1 0 3 seconds) t r ip let s tate of the molecule. This species may react d i rect ly w i t h other molecules by e lect ron or hydrogen a tom t ransfer to produce radical fo rms of the photosensi t izer and subst ra te generat ing superox ide ( 0 2 ) , perox ide ( H 2 0 2 ) , and hydroxy l (-OH) radicals ( termed the Type I pho tochemica l react ion) . However , if the energy of the ac t iva ted t r ip let is d i rect ly t ransfer red to g round state o x y g e n , singlet o x y g e n ( 1 0 2 ) is p roduced (Type II pho tochemica l react ion) . Singlet oxygen is a highly react ive , e lectrophi l ic species w h i c h can in teract w i t h e lect ron-r ich regions of a w i d e range of biological molecules inc luding meth ion ine , cho les tero l , guanosine and many unsatura ted c o m p o u n d s such as lipids (Gomer et al., 1 9 8 8 ) . Type I and II pho to -ox ida t i ve react ions may occur s imul taneously and are in f luenced by oxygen concen t ra t i on , the ident i ty of the sensit izer, and the nature of the subst ra te . H o w e v e r , s inglet o x y g e n is bel ieved to be the dominan t damag ing molecular species generated by PDT. It is the pervading belief t ha t t umour des t ruc t ion by porphyr ins is a result of a p h o t o d y n a m i c e f fec t on the t u m o u r mic rovascu la ture as we l l as a result of d i rect des t ruc t ion of t u m o u r cel ls. It has been s h o w n tha t PDT may reduce t u m o u r b lood f l o w , cause vasocons t r i c t i on , platelet aggregat ion , and endothel ial cell damage . H o w e v e r , it is also l ikely tha t d i rect pho todynamic e f fec ts also occur at the t u m o u r cell level (Henderson and Dougher ty , 1 9 9 2 ) . 1.2 P h o t o f r i n ® ( d i h e m a t o p o r p h y r i n e t h e r ) Photofr in®, regarded as the pro to typ ic porphyr in photosensi t izer , has been used cl inical ly for a number of years at a large number of medical cent res. The deve lopment of th is pho tochemotherapeu t i c agent has had a c i rcu i tous h is tory . Modern medic ine recognizes Meyer-Betz (1913) for sel f -administer ing hematoporphy r in (Hp) and d iscover ing tha t th is f o r m of " t h e r a p y " w a s associated w i t h the deve lopmen t of skin photosens i t i v i t y . Hp can be prepared f r o m who le b lood or hemin by the act ion of hydobromic acid in glacial acid acid causing the release of i ron and the convers ion of the t w o v iny l side chains to hydroxye thy l g roups . Later s tudies s h o w e d tha t Hp prepared in th is fash ion w a s a crude mix ture of many porphyr ins inc luding s igni f icant amounts of o l igomer ic porphyr ins as wel l as m o n o m e r s such as hyd roxye thy l v iny l deu te roporphyr ins (HVD) and PPIX (Schwar tz , 1 9 5 5 ) . The initial b iomedical use of porphyr ins w a s in the area of t u m o u r d iagnosis . Tumours could be visual ized by t issue f luorescence w h e n exposed to v io le t l ight af ter sys temic admin is t ra t ion of Hp (Figge etal., 1948 ) . A der ivat ive of Hp (HpD) w a s prepared by t rea tmen t of crude Hp w i t h a mix ture of acet ic and sulphur ic ac ids, mix ing w i t h NaOH, and neutral izat ion to a physio logical pH (Lipson etal., 1 9 6 1 ) . Upon ster i l izat ion, the fo rmu la t ion became acceptable for exper imenta l use in humans . This s tudy also repor ted tha t HpD had s igni f icant p h o t o d y n a m i c act iv i ty in mice. A n enr ichment of the tumour- loca l iz ing 3 and photosens i t iz ing c o m p o n e n t and a reduct ion in the relat ive p ropor t ion of the monomer ic c o m p o n e n t w a s accompl ished by f rac t ionat ion of HpD by gel f i l t rat ion c h r o m a t o g r a p h y (Dougher ty , 1 9 8 7 ) . On the basis of chemical and spect roscop ic studies it w a s conc luded tha t the principal tumour- loca l iz ing c o m p o n e n t s of HpD were d ihematoporphy r in ethers (Dougher ty , 1 9 8 7 ) . Current ly , Photofrin® is manu fac tu red f r o m pig b lood and enr iched for d imer ic and t r imer ic porphyr ins ( > 8 0 % ) th rough a d iaf i l t rat ion process, a l though o l igomers con ta in ing up to nine porphyr in r ings may be present . In addi t ion, Photofr in® conta ins Hp ( 5 - 1 0 % ) , HVD ( 5 - 1 0 % ) and PPIX ( 3 - 7 % ) , a l though these monomer ic c o m p o u n d s possess weaker photosensi t iz ing ac t iv i ty than the o l igomer ic c o m p o n e n t s (Dougher ty , 1 9 8 4 ; G o m e r e f a / . , 1 9 8 8 ) . Photofrin® and HpD intensely absorb l ight of a w a v e l e n g t h of approx imate ly 4 0 0 nm ( termed the Soret band) . Several o ther smaller absorpt ion peaks are observed at higher wave leng ths . A w a v e l e n g t h of 6 3 0 n m is typ ica l ly employed for PDT w i t h Photofrin®. In the mouse , detec tab le amoun ts of Photofrin® are retained for ex tended periods ( > 75 days) in mos t organs after its admin is t ra t ion thereby accoun t ing for the pro longed skin photosens i t i v i t y associated w i t h th is c o m p o u n d (Bellnier et al., 1 9 8 9 ) . For the normal mouse , Photofrin® levels are h ighest in the liver f o l l owed by the adrenal g lands, ur inary bladder, pancreas, k idney, sp leen, s t o m a c h , bone, muscle and lowes t in brain t issue 2 4 hours af ter admin is t ra t ion (Bellnier et al., 1 9 8 9 ) . Radioisotope studies indicated tha t the monomer ic c o m p o n e n t s of Photofrin® are el iminated rapidly v ia the hepat ic-bi l iary route but the o l igomer ic mater ial exhib i ts a pro longed c learance (Bellnier et al., 1 9 8 8 ) . It w a s fu r ther demons t ra ted tha t 4 re tent ion of Photofr in® in the spleen and the liver is s t r ik ingly p ronounced . 1.3 Second generat ion porphyr in photosensi t izers The pro t rac ted skin photosens i t i v i ty associated w i t h HpD and Photofr in® admin is t ra t ion inst igated the search for n e w photosensi t izers . The deve lopmen t of single c o m p o u n d photosensi t izers seeks to maximize tumour -local izing proper t ies , minimize their propens i ty to sensit ize the skin of t reated sub jec ts , and the capac i ty to be act ivated w i t h longer and therefore more penet ra t ing wave leng ths of visible l ight. Second generat ion porphyr in or porphyr in -like photosensi t izers are represented by c o m p o u n d s inc luding benzoporphyr in der ivat ive m o n o acid Ring A (BPD, R ich te re f al., 1 9 8 7 ) , mono-L-aspar ty l -ch lo r ine6 , (Nelson etal., 1987) and Sn et iopurpur in (Morgan et al., 1 9 8 8 ) . BPD is a po ten t photosensi t izer w h i c h has been extens ive ly studied in th is , and other laborator ies. BPD w a s chemical ly synthes ized f r o m Hp (Pangva et al., 1 9 8 6 ) . In con t ras t to Photofrin®, BPD is a single wel l -def ined c o m p o u n d , and is rapidly cleared f r o m the body . BPD produces on ly short - l ived skin photosens i t i za t ion , absorbs l ight at a more penetra t ing w a v e l e n g t h ( 6 9 0 nm) , is non- tox ic in the absence of l ight , and is 10 -20 t imes more pho to tox ic than Photofrin® against a var ie ty of t u m o u r cell l ines w h e n assayed in vitro (Richter et al., 1 9 8 7 ) . 1.4 Hematopoies is The major goal of th is thesis is to determine w h e t h e r d i f ferent 5 porphyr ins m igh t in f luence hematopo ie t ic ac t iv i ty w i th in the m a r r o w and spleen of the mouse . To bet ter unders tand progeni tor and phenotyp ic changes w h i c h might occur w i t h i n these t issues, it wi l l be useful to rev iew w h a t is k n o w n about the d i f fe rent c o m p o n e n t s of the hematopo ie t ic apparatus and h o w these elements respond to d i f fe rent s t imul i . 1.4.1 Organizat ion of the hematopo ie t ic t issues 1.4.1.1 Bone m a r r o w In the normal adul t , the bone m a r r o w (BM) represents the major locat ion of b lood cell p roduc t ion and the only site in w h i c h lymphopoies is , myelopoies is and ery thropoies is proceed s imul taneous ly . BM is pr imari ly located w i t h i n the cy l indr ica l cavi t ies of the long bones and the spongy bone of the s t e r n u m , r ibs, ver tebra l bodies, c ran ium and pelvis. Developing blood cell precursors are indiv idual ly p rogrammed to in teract w i t h f i xed , s t romal cells in d is t inct ive BM mic roenv i ronments . The non-hematopo ie t i c , sessile s t romal cells of the medul lary cav i t y f o r m a scaf fo ld ing for the hematopo ie t i c cel ls. Mul t ip le cell t ypes compr ise the s t roma and as a m in imum include the endothel ia l cells l ining the s inusoids, the f ibroblasts of the endos teum, advent i t ia l layer and nerve sheaths, and ad ipocy tes . The cells of the s t roma produce a w i d e var ie ty of fac tors w h i c h regulate s teady state myelopoiesis and lymphopo ies is . These cells may also respond in s i tuat ions of "hematopo ie t i c s t ress" w h i c h cou ld occur upon exposure to radiat ion, c y t o t o x i c drugs or p roduc ts of in fec t ious agents . The con t r ibu t ion of s t romal cells to the g r o w t h and d i f ferent ia t ion 6 of hematopo ie t ic progeni tor cells can be evaluated in vitro ut i l iz ing normal bone m a r r o w s t romal e lements or s t romal cell l ines. The extracel lu lar matr ix (EM) of the BM also in f luences the migra t ion , pro l i ferat ion, shape and metabol ic func t ions of deve lop ing cel ls. The EM, composed of mul t ip le col lagen t y p e s , f i b ronec t in , laminin and p ro teog lycans , may a f fec t b lood cell deve lopment by act ing as binding subst ra tes for hematopo ie t ic cells and modula t ing the d is t r ibu t ion of g r o w t h fac tors (Roberts etal., 1 9 8 8 ) . Add i t iona l ly , roles for macrophages (Dorshk ind , 1 9 9 0 ; W a n g et al., 1992) and T l ymphocy tes (Torok-Storb et al., 1982) in the regulat ion of hematopoies is have been ident i f ied. Both of these cell t ypes may release a diverse assor tment of hematopo ie t ic regulatory fac to rs . 1 .4 .1 .2 Hematopo ie t ic s tem cells (HSC) The deve lopment and main tenance of normal levels of c i rculat ing leukocy tes is dependent upon the act iv i ty of p lur ipotent ia l HSC. Dur ing mur ine fetal deve lopmen t , the sites at w h i c h hematopoies is occur sh i f t dramat ica l ly . Erythropoiesis domina tes w i t h i n the yo lk sac b e t w e e n days 7 and 12 of embryon ic l i fe. Hematopoies is c o m m e n c e s in the liver after day 1 1 , presumably after HSC take up residence in the organ and by 1 5 days of ges ta t ion , hematopoies is commences in the spleen and BM (Ikuta et al., 1 9 9 2 ) . Whi le BM is the major reservoir of HSC in the adul t mouse , phenotyp ica l ly and funct iona l ly equiva lent HSC can also be isolated f r o m the spleen and peripheral b lood , a l though in m u c h f e w e r numbers (Bodine et al., 1 9 9 3 ) . HSC may be mobi l ized f r o m the BM to the blood and spleen by bacter ial p roduc ts (eg. endo tox in , A i n s w o r t h , 1 9 8 8 ) , c y t o t o x i c drugs (Craddock 7 et al., 1992) or the admin is t ra t ion of recombinant cy tok ines inc luding granu locyte co lony s t imula t ing fac tor (G-CSF) (Bungart et al., 1990) and s tem cell fac tor (SCF, steel fac to r , c-kit l igand) (Bodine etal., 1 9 9 3 ; Fleming etal., 1 9 9 3 a ) . Intr insical ly, HSC possess the proper ty of se l f - renewal and the capac i ty to reconst i tu te all hematopo ie t ic l ineages of heavi ly i rradiated syngeneic or congen ic mouse st ra ins. Conceptua l ly , HSC sel f - renewal may be envisaged at the single cell level as the ex is tence of an asymmetr ica l cell d iv is ion creat ing one daughter cell ident ical to the mother , w i t h the other c o m m i t t e d to d i f fe rent ia t ion. Under the con t ro l of an intr icate n e t w o r k of cy tok ines and s t romal cells HSC generate all cells of the ery th romye lo id and lympho id l ineages. A systemat ic approach to the naming of leukocy te sur face molecules w a s made w i t h the "c luster of d i f fe ren t ia t ion" (CD) designat ion for these ant igens as def ined w i t h monoc lona l ant ibodies (Barclay etal., 1 9 9 2 ) . Mur ine HSC are def ined as rare (1 per 1 0 4 or 10 5 ) BM cells t ha t do not express s igni f icant levels of l ineage ( l in)-specif ic cell sur face ant igens inc luding C D 4 , CD8 (T l ymphocy tes ) , C D 4 5 R - B 2 2 0 (B l ymphocy tes ) , TER-119 (ery throcy tes) Gr-1 (granulocytes) or C D 1 1 b / M a c - 1 (monocy tes /macrophages , granu locytes) (Spangrude et al., 1 9 8 8 ; Uchida and W e i s s m a n , 1 9 9 2 ) . This v i e w has been chal lenged by others w h o s h o w e d tha t mur ine HSC may express low- to -med ium a m o u n t s of the G r - 1 , TER-119, B 2 2 0 , or CD4 markers (Szi lvassy and Cory, 1 9 9 3 ) . H o w e v e r , the sur face of these cells does express l ow levels of Thy-1 and high levels of Ly6-A/E (s tem cell an t igen, Sca-1) , major h is tocompat ib i l i t y complex (MHC) Class I ant igens (Ogata e r a / . , 1 9 9 5 ) , and leukosial in (CD43) and can be isolated by cell sor t ing f l o w c y t o m e t r y on the basis of th is pheno type (Spangrude 8 etal., 1 9 8 8 ; Moore et al., 1 9 9 4 ) . The T h y - 1 l 0 , Sca-1 + , lin" HSC f rac t ion may be fu r ther par t i t ioned on the basis of the uptake of the mi tochondr ia l membrane dye rhodamine 123 (Spangrude and J o h n s o n , 1990) and cell cyc le analysis (Fleming et al., 1 9 9 3 ) . The accumula t ion of rhodamine 123 by HSC correlates inversely w i t h the expression of the mul t i -d rug resistance gene (MDR1) P-glycoprote in. MDR1 p romotes the ef f lux of a w ide var ie ty of c y t o t o x i c molecules f r o m the cell (Chaudhary and Roninson, 1 9 9 1 ) . HSC in the G 0 / G 1 phases of the cell cyc le and exhib i t ing l o w rhodamine 123 uptake p roduced bo th shor t t e r m radiat ion pro tec t ion and long- te rm mult i - l ineage reconst i tu t ion whereas HSC in the S / G 2 / M phases of the cell cyc le demonst ra ted a d imin ished capac i ty for bo th rad ioprotect ion and long- te rm hematopoiet ic reconst i tu t ion (Fleming etal., 1 9 9 3 ) . In add i t ion , express ion of the ty ros ine kinase t ype receptor c-kit (CD117) for SCF by mur ine HSC corre lated d i rect ly w i t h the abi l i ty to p roduce long t e r m , mult i - l ineage reconst i tu t ion (Orlic et al., 1 9 9 3 ) . The receptor for t ransfer r in (CD71) marks prol i ferat ing cells but is l ikely not present or at very l ow levels on mur ine (Ogata et al., 1995) and rat (Jef fer ies et al., 1985) HSC. Co-express ion of Thy-1 and the s ia log lycoprote in C D 3 4 combined w i t h low expression of C D 4 5 R A and CD71 character ize the human HSC (Lansdorp and D r a g o w s k a , 1 9 9 2 ) . 1.4.1 .3 Hematopo ie t ic d i f ferent ia t ion and prol i ferat ion All e ight major b lood cell l ineages arise f r o m the small centra l pool of HSC (Figure 1.1). The cont ro l of s tem cell d i f ferent ia t ion is achieved th rough the 9 Hematopoietic Stem Cell Hematopoietic Progenitor Thymus Figure 1.1 P a t h w a y s o f hematopo ie t ic d i f fe ren t ia t ion . Format ion of the major b lood leukocy te group ings f r o m the p lur ipotent ia l hematopo ie t ic s tem cell is ou t l ined. Cytok ines w h i c h have been s h o w n to regulate and p romote progeni tor cell d i f ferent ia t ion and/or prol i ferat ion are ind ica ted. Diagram w a s adapted f r o m Ikuta et al. ( 1 9 9 2 ) . 10 act ion of t w o over- lapping sys tems. The f i rst level is prov ided by s t romal cells w h i c h t h r o u g h cel l - to-cel l con tac t inf luence the fo rmat ion of c o m m i t t e d progeni tor cells f r o m HSC (Metca l f , 1 9 9 1 ) . A second level of cont ro l is exer ted th rough the act ion of local ly p roduced or systemical ly released hematopo ie t ic g r o w t h fac tors w h i c h s t imulate the prol i ferat ion of c o m m i t t e d progeni tor cells and their o f fspr ing and d i rect their passage into mature blood cells (Metca l f , 1 9 8 9 ) . In vitro s tudies have indicated tha t a synergis t ic in teract ion among several cy tok ines is required to p romote HSC 1) surv ival (prevent ion of death by apoptos is ) , 2) progression f r o m a quiescent G 0 s tate to G^IS phases of the cell cyc le , 3) d i f fe rent ia t ion into c o m m i t t e d progeni tors and 4) se l f - renewal (Moore and Shapiro, 1 9 9 3 ) . It has been s h o w n tha t inter leukin (IL)-1 (Stanley et al., 1 9 8 6 ) , IL-3 ( S u d a e f a / . , 1 9 8 5 ) , IL-6 ( Ikebuchi etal., 1 9 8 7 ) , IL-11 (Musashi et al., 1 9 9 1 ) , IL-12 (Hirayama etal., 1 9 9 4 ; Jacobsen etal., 1993) and G-CSF ( Ikebuchi etal., 1988) and SCF (de Vr ies et al., 1991) can inf luence mur ine HSC pro l i fera t ion. Recent ly, a l igand for the f l k -2 / f l t -3 ty ros ine kinase receptor ( M a t t h e w s et al, 1990) of HSC w a s ident i f ied. This pro te in , s t ructura l ly- re lated to SCF, enhances the response of mur ine feta l l iver and bone m a r r o w HSC to myelo id g r o w t h fac to rs including IL-3, IL-6 and g ranu locy te -macrophage co lony s t imula t ing fac tor (GM-CSF) but does not p romote the fo rma t ion of ery thro id precursors (Lyman et al., 1 9 9 4 ; Hannum et al., 1 9 9 4 ) . A n assor tment of cy tok ines d i rect the d i f ferent ia t ion and prol i ferat ion of c o m m i t t e d hematopo ie t ic progeni tor cel ls. For example , G-CSF, M-CSF and ery th ropo ie t in (Epo) d i rect the f inal matura t ion of g ranu locy tes , m o n o c y t e s and 11 ery th rocy tes respect ive ly (Miyaj ima et al., 1 9 9 2 ) . H o w e v e r , considerable redundancy in the act ion of many of these fac tors is apparent in tha t individual cy tok ines may el ici t ident ical prol i ferat ive and d i f ferent ia t ive responses in progeni tor cells (Metca l f , 1 9 9 3 ) . This may be part ly explained by the ex is tence of c o m m o n receptor sub-un i t signal t ransduc ing polypept ide chains w h i c h associate w i t h cy tok ine-spec i f i c receptor a cha ins. For example , the heterodimer ic high af f in i ty cell sur face receptors for GM-CSF, IL-3 and IL-5 uti l ize the same /? chain indicat ing tha t the intracel lular s ignal l ing p a t h w a y s t r iggered by each of these g r o w t h fac tors likely are ident ical (Miya j ima et al., 1 9 9 3 ) . In add i t ion , the b inding of IL-3 to its receptor is c o m p e t e d by GM-CSF and the b inding of IL-5 to its receptor can be b locked by either IL-3 or GM-CSF (Lopez et al., 1 9 9 1 ) . A n analogous receptor organizat ion occurs w i t h i n the high af f in i ty receptors for the hematopo ie t ic cy tok ines IL-6, I L - 1 1 , leukemia inh ib i tory fac tor (LIF) and oncos ta t in M w h i c h possess a c o m m o n /? chain (gp130) and exhib i t similar p le iotropic act ions (Gearing et al., 1 9 9 2 ; Metca l f , 1 9 9 3 ) . A c o m m o n s t ruc tura l organizat ion and amino acid sequence homology exists for the extracel lu lar domains , consis t ing of approx imate ly 100 amino acids, for the receptors (R) of many hematopo ie t ic g r o w t h fac to rs inc luding the IL-2R (/? and y cha ins) , IL-3R (a and J3 cha ins) , IL-4R, IL-5R, IL-6R g p 1 3 0 , IL-7R, IL-9R, G M -CSF (a and /? cha in) , G-CSFR EpoR, pro lact in R and LIFR (a cha in) . A t r y p t o p h a n -ser ine-x- t ryptophan-ser ine mot i f (x = one of several d i f fe rent amino acids) sequence mot i f occurs prox imal to the cell membrane and the presence of four cyste ine residues in the N-terminal region of the extracel luar domain character ize 12 members of th is receptor super fami ly (Miyaj ima et al., 1 9 9 2 ) . These features suggest t ha t these receptors may have d iverged f r o m a c o m m o n ancestral receptor prote in (Metca l f , 1 9 9 3 ) . Thus , the hematopoie t ic sys tem displays remarkable over lap in the molecular arch i tecture of many of its cell sur face receptors and cellular responses w h i c h fo l l ow the in teract ion of a var ie ty of cy tok ines w i t h these receptors . A heterogeneous assor tment of fac to rs posi t ive ly and negat ively regulate the fo rma t ion of the blood cel ls. Some of these agents are cy tok ines w h o s e pr imary act ion are not as hematopo ie t ic g r o w t h fac to rs but nevertheless are regulatory c o m p o n e n t s of the hematopo ie t ic apparatus. It may o f ten be unclear w h e t h e r an indiv idual fac tor a f fec ts progeni tor cells d i rect ly and/or exer ts its act ion th rough in teract ion w i t h hematopo ie t ic accessory cel ls. Therefore careful exper imenta t ion is required in order to ident i fy at w h i c h level a fac tor exerts its e f fec ts on hematopo ies is . In add i t ion , some fac to rs may produce negat ive or posi t ive pro l i ferat ive responses, dependent on fac tor concen t ra t ion and/or the presence of o ther cy tok ines . Anci l lary fac tors w i t h demonst rab le hematoregu la tory ac t iv i ty in the mouse are l isted in Table 1 . 1 . In relat ion to the present s tudy , the inf luence of ferr i t in and t ransfer r in prote ins in the regulat ion of hematopoies is war ran ts special cons idera t ion . The role of these i ron-binding prote ins in cellular metabo l ism and regulat ion of hematopoies is are in t imate ly interrelated w i t h those of heme ( A b r a h a m , 1 9 9 1 ) . These features wi l l be fur ther d iscussed be low. 13 Table 1.1 Secondary hematopo ie t ic regulatory fac to rs . Factor (abbreviation) Source Hematopoietic Effects Reference Interferon-/ (IFN-y) Activated T cells, NK cells, macrophages Negative regulator Eng et al., 1995 Transforming growth factor-^ (TGF-£) Platelets, bone Bi-functional effects on myelopoiesis Inhibitor of erythropoiesis Hatzfeld et al., 1991; Dybedal and Jacobsen, 1995 Tumour necrosis factor-a Activated T cells, monocytes, endothelial cells Direct inhibitor of progenitors; indirect stimulator through growth factor induction Jacobsen et al., 1992 Prostaglandin^ (PGE2) Monocytes-macrophages, fibroblasts Inhibitor of myelopoiesis. Pelus, 1989 Macrophage inflammatory proteins (MIP-1 a and MIP-20) Monocytes-macrophages Stimulators of myelopoiesis. Broxmeyer etal., 1989 Ferritin Most cells Inhibitor of myelopoiesis. Broxmeyer et al., 1989 Transferrin Liver Inhibitor of myelopoiesis Gentile and Broxmeyer, 1983 1.4 .1 .4 Cont r ibu t ion of the spleen to hematopoies is In the adul t mouse , the spleen serves t w o major purposes. The f i rst is tha t of a l ympho id organ w h e r e b y speci f ic immune responses to fore ign ant igens may be p ropaga ted . A second role is t ha t of an hematopo ie t ic o rgan . In cont ras t to o ther l ympho id o rgans , the spleen conta ins a diverse col lect ion of l ymphohematopo ie t i c cel ls. The spleen is s t ructura l ly d iv ided into an ery thro id red 14 pulp w h i c h acts in the scaveng ing of senescent red b lood cells and serves as a reserve site for hematopoies is and the w h i t e pulp w h i c h possesses an organizat ion and immuno log ic role comparab le to tha t of a l ymph node cor tex (Hood et al., 1 9 8 4 ) . This l ympho id t issue sur rounds the splenic arter io les. T l ymphocy tes are concen t ra ted in periarter iolar l y m p h o c y t e sheaths (PALS) wh i le B l ymphocy te fol l ic les appear as o u t g r o w t h s of the PALS, usual ly at arterial branch po in ts . Sur round ing the PALS is the marginal zone lympho id c o m p a r t m e n t , conta in ing a p redominance of B cells in associat ion w i t h T cells (Hood et al., 1 9 8 4 ) . The marginal zone l ikely represents the major site at w h i c h T and B cells init ially encounter b lood-borne ant igens. The route of ent ry of l ympho id cells into the wh i te pulp occurs at the border be tween the red and w h i t e pu lp . Kinetic studies have indicated tha t splenic macrophages have a dual or ig in , cons is t ing of cells der ived w i t h i n the spleen and also f r o m the inf lux of m o n o c y t e s into the organ (van Furth and Diesselhof f -den Dulk, 1 9 8 4 ) . Based upon their relat ive express ion of the Ly-6C ant igen, splenic macrophage progeni tors were s h o w n to be phenotyp ica l ly d is t inc t f r o m those of the BM (McCormack et al., 1 9 9 3 ) . This f ind ing suggests tha t macrophages tha t arise in the spleen and BM may be der ived f r o m d is t inc t , t issue-speci f ic p rogen i to rs . Due to vo lume const ra in ts placed by the sur round ing bone, the m a r r o w may have l imi tat ions in its abi l i ty to expand in s i tuat ions of hematopoiet ic s t ress. In these c i rcumstances , the mur ine spleen may a c c o m m o d a t e the demand for increased hematopo ie t ic cell o u t p u t . Many studies have s h o w n tha t certain recombinan t g r o w t h fac to rs great ly increase mur ine splenic hematopo ie t ic ac t iv i ty , 15 whi le p roduc ing minor or even inhib i tory e f fec ts w i th in the B M . Fur thermore, l ineage-speci f ic fac to rs such as G-CSF may elevate levels of splenic granu locy te-macrophage , ery thro id and megakaryocy te progeni tors (Tamura etal., 1 9 8 7 ; Ponja et al., 1 990) ind icat ing tha t the spleen can be t rans fo rmed into a highly favourable site for mul t i - l ineage hematopoies is . Whe ther an increase in splenic hematopoiet ic ac t iv i ty is the consequence of progeni tor cell migrat ion f r o m the m a r r o w and/or as a consequence of the act ivat ion of resident progeni tors is not a lways ev ident . One s tudy suggested tha t a fac to r -dependent hematopo ie t ic m ic roenv i ronment is generated w i t h i n the spleen upon G-CSF admin is t ra t ion (Fukushima et al., 1 9 9 2 ) . The spleen in the adul t mouse has a we l l -documented capac i ty to suppor t HSC and th is f o rms the fundamenta l basis of the classical spleen co lony - fo rm ing uni t (CFU-S) assay used to de tec t early s tem cell ac t iv i ty (Till and M c C u l l o c h , 1 9 6 1 ) . 1.4.1.5 S t imula t ion of hematopoies is by microbial fac to rs It has been k n o w n for some t ime tha t cer ta in microbia l p roduc ts exert s t imu la tory e f fec ts on hematopoies is in the m a r r o w and spleen of normal and myelosuppressed mice. The major i ty of these c o m p o u n d s , such as the yeast po lysacchar ides g lucan (Patchen et al., 1990) and A M 5 (Real et al., 1992 ) , bacter ial l ipopolysacchar ide (LPS, endo tox in ; A i n s w o r t h , 1988) and w h o l e , heat-killed Lactobacillus case/' (Nomoto et al., 1 9 9 1 ) , indirect ly in f luence hematopoies is t h r o u g h in teract ions w i t h macrophages p romot ing the release of hematos t imu la to ry g r o w t h fac to rs by these cel ls. 16 1.5 I ron-heme metabo l ism and erythropoies is 1.5.1 Ery thropoiet in (Epo), e ry thropo ie t in receptor (EpoR) and regulat ion of ery thropoies is Since ery thro id cells synthesize high amoun ts of the porphyr in heme as the centra l mo ie ty of the hemoglob in molecular comp lex , the response of th is leukocy te l ineage to admin is tered porphyr in photosensi t izers may be of part icular impor tance . Epo is the pr incipal g r o w t h fac tor t ha t p romotes the v iabi l i ty , pro l i ferat ion and d i f ferent ia t ion of c o m m i t t e d mammal ian ery thro id progeni tor cells (Youssouf ian etal., 1 9 9 3 ) . A l t h o u g h Epo may be produced by macrophages (Wang et al., 1 9 9 2 ) , it is p redominant ly synthesized by the k idney in response to hypox ia and anemia. Epo exer ts its cellular e f fec ts t h rough fac tor -spec i f ic cell sur face EpoR. The c D N A for the mur ine EpoR encodes a protein of 5 0 7 amino acids conta in ing a single t ransmembrane domain and a membrane prox imal t ryp tophan-ser ine-x-t ryp tophan-ser ine mot i f and four cyste ine residues in the extracel luar region (D 'Andrea et al., 1 9 8 9 ) , character is t ic of members of the cy tok ine receptor super fami ly (Miyaj ima et al., 1 9 9 2 ) . A l t h o u g h it has no t been unequivocal ly d e m o n s t r a t e d , it is l ikely tha t the 65 kDa EpoR exists as part of a complex w i t h a second po lypept ide (D 'Andrea et al., 1 9 8 9 ; Miura and Ihle, 1 9 9 3 ) . The pauc i ty ( < 1 0 0 0 sites per cell) of cell sur face EpoR on ery thro id progeni tors and the lack of purely Epo-responsive cell l ines s lowed the elucidat ion of the signal t ransduc t ion p a t h w a y uti l ized by the EpoR. A l t h o u g h the EpoR lacks an intracel lular kinase d o m a i n , the b inding of Epo to its receptor s t imulates mult ip le intracel lular ty ros ine phosphory la t ion events w h i c h precede gene t ranscr ip t ion and 17 cell pro l i ferat ion (Damen et al., 1 9 9 2 ) . These ty ros ine phosphory la ted proteins include She, Grb, R a f - 1 , a regulatory sub-uni t of phosphat idy l inos i to l 3-kinase and the EpoR itself (Ohashi etal., 1 9 9 5 ) . It w a s suggested tha t the Janus kinase, J a k 2 , physical ly associates w i t h the membrane prox imal region of the EpoR cy top lasmic domain and tha t Epo b inding causes the ty ros ine phosphory la t ion of J a k 2 and act ivates its in vitro k inase ac t iv i ty (Wi t thuhn et al., 1 9 9 3 ) . Recent ly , it has been indicated tha t upon b inding of Epo to immature mur ine splenic ery thro id cel ls, Signal T ransduc ing and Ac t i va to rs of Transcr ip t ion (STAT) prote ins become act iva ted (Penta and Sawyer , 1 9 9 5 ) . STAT1 and S T A T 5 prote ins are rapidly phosphory la ted af ter the in teract ion w i t h Epo w i t h its receptor and t rans located to the nucleus w h e r e they bind speci f ic regions of DNA (Penta and Sawyer , 1 9 9 5 ) . Thus , J A K 2 kinase ac t iv i ty is l inked to genet ic regulatory e lements t h rough STAT prote ins. A prote in ty ros ine phosphatase conta in ing a Src homo logy uni t 2 domain (SH-PTP1) associates w i t h the EpoR and its de-phosphory la t ion of J a k 2 counterac ts the prol i ferat ive signals generated th rough the act ivated EpoR (Klingmuller et al., 1 9 9 5 ) . T h u s , in s i tuat ions of SH-PTP1 def ic iency or impa i rment there is an enhanced sens i t iv i ty to Epo by Epo-responsive ery thro id (CFU-E) precursor cells (Klingmuller et al., 1 9 9 5 ) . Erythro id progeni tors are generated f r o m the HSC and the earliest e ry thro id precursors ident i f iable in cul ture are the s low ly pro l i ferat ing burs t - fo rming uni ts (BFU-E), w h i c h are init ial ly unresponsive to Epo and do not express the EpoR. These cells require SCF, GM-CSF or IL-3 in order to express the EpoR and thereby become responsive to Epo (Swanda et al., 1 9 9 0 ) . Fo l lowing several more days in 18 cu l tu re , these Epo-responsive progeni tor cells f o r m the smaller CFU-E w h i c h generate e ry th rob las t colonies (Koury and Bondurant , 1 9 8 8 ) . Progeni tors f r o m these t w o stages of e ry thro id deve lopment have d is t inc t physical propert ies and can be highly enr iched using a series of cell separat ion techn iques (Landschulz et al., 1 9 8 9 ; Sawada et al., 1 9 9 0 ) . The addi t ion of Epo to BM cell suspension cul tures s t imulates i ron incorporat ion into heme leading to g lobin synthes is (Lut ton et al., 1 9 9 1 ) . Expression of the EpoR decl ines as ery thro id matura t ion proceeds and cells beyond the ery throb las t stage are no longer dependent upon Epo (Koury and Bondurant , 1 9 8 9 ) . Thus , Epo suppl ies a prol i ferat ive signal at the level of the BFU-E and a d i f ferent ia t ive signal at the CFU-E stage (D 'Andrea and Z o n , 1 9 9 0 ) . Transgenic mice w i t h delet ions of the Epo or EpoR genes produce f e w mature e ry th rocy tes and die dur ing fetal deve lopment indicat ing tha t there are no other l igands or receptors w h i c h can compensate for these e lements of the ery thropoiet ic p a t h w a y (Wu et al., 1 9 9 5 ) . Never theless, in the absence of Epo or the EpoR, normal levels of BFU-E and CFU-E progeni tors were present in the livers of these mice ind icat ing tha t the Epo-EpoR sys tem is required for terminal ery thro id d i f fe rent ia t ion rather than for ery thro id c o m m i t m e n t or the g r o w t h of BFU-E or their t rans i t ion to CFU-E (Wu et al., 1 9 9 5 ) . It has been s h o w n for an EpoR-transfected mur ine IL-3 dependent cell line (Ba/F3) t ha t at l ow rEpo concent ra t ions there w a s a pro longat ion of the G 1 phase of the cell cyc le and ev idence of ery thro id d i f fe rent ia t ion (/J-globin mRNA accumula t ion) (Carrol et al., 1 9 9 5 ) . However , in the presence of high concen t ra t ions of recombinant Epo (rEpo), w a s shor tened and an increase in cell 19 g r o w t h rates occu r red . r lL-3 s t imula ted g r o w t h of these cells but did not induce /?-globin and suppressed rEpo- induced yff-globin induc t ion (Carrol et al., 1 9 9 5 ) . These studies indicate tha t Epo and IL-3 exer t d i f ferent ia t ive and prol i ferat ive ef fects in ery thro id deve lopmen t and the equi l ibr ium b e t w e e n these p a t h w a y s is sensit ive to levels of these cy tok ines w i t h i n the hematopoie t ic env i ronment . Other cy tok ines , inc luding TGF-/?1, have been s h o w n to inf luence the matura t ion of normal puri f ied human BFU-E by augment ing their t rans i t ion to the CFU-E stage and hence terminal ery thro id d i f fe rent ia t ion (Krystal et al., 1 9 9 5 ) . SCF and Epo delay th is matura t ion thereby permi t t ing addi t ional rounds of cell d iv is ion and increased yields of e ry th rocy tes (Krystal et al., 1 9 9 5 ; M u t a et al., 1 9 9 5 ) . Developing ery thro id cells uti l ize ery thro id-spec i f ic cont ro l fac tors w h i c h bind to speci f ic sequences of DNA ups t ream of the a- and /?-globin c o m p o n e n t s , t e rmed locus cont ro l regions, regulate the t ranscr ip t ion of genes required for g lobin fo rmat ion and ery thro id matura t ion ( A n d r e w s et al., 1 9 9 3 ) . A number of c/s-act ing sequences, operat ive only in ery thro id cel ls, have been p inpo in ted and include the t ranscr ip t ion fac tors GATA-1 (Evans and Felsenfeld, 1 9 8 9 ; Tsai etal., 1 9 8 9 ) , ery thro id-speci f ic nuclear fac tor (NF-E2, A n d r e w s etal., 1 9 9 3 ) , and the ery thro id Kruppel- l ike fac tor (EKLF, Bieker and S o u t h w o o o d , 1995) w h i c h bind DNA G A T A mot i f s , AP-1- l ike sequences or CACC-l ike sequences, respect ive ly . These fac to rs have been ident i f ied in e ry th ro id , bu t not in myelo id or l ympho id cell l ines and in the adul t mouse expression is con f ined to ery thropoiet ic t issues (spleen and BM) . G A T A - 1 w a s subsequent ly s h o w n to bind to a site in the promoter region of the ery thro id l ineage-restr icted EpoR (Zon et al., 1 9 9 1 ) . EKLF 2 0 appears dispensible for ery thro id c o m m i t m e n t and the prol i ferat ion of ery thro id precursors bu t appears essential at the f inal s tages (hemoglob in format ion) of ery thro id matu ra t ion (Perkins etal., 1 9 9 5 ) . Thus , e ry th rocy te fo rmat ion is in f luenced by the act ion of specif ic DNA-b ind ing fac to rs ( G A T A - 1 , EKLF, NF-E2), a l ineage-speci f ic cy tok ine (Epo), a l ineage-restr ic ted receptor (EpoR), the interplay of ple iotropic cy tok ines (IL-3, SCF) and the avai labi l i ty of a metal l ic e lement ( F e + 2 ) . 1.5.2 The t ransfer r in (Tr f ) - t ransferr in receptor (TrfR) s y s t e m M a n y fundamenta l cellular events are dependent upon i ron. A l though all cells require i ron , ery thro id cells have by far the greatest requ i rement for iron since hemoglob in-assoc ia ted iron accounts for as m u c h as 7 0 % of the to ta l iron con ten t in the adul t (Ponka and Schu lman, 1 9 9 3 ) . This e lement is incorporated into the heme prosthet ic group of prote ins such as hemog lob in , the c y t o c h r o m e s and a number of enzymes inc luding cyc lo -oxygenase , catalase and peroxidase (Ponka and Schu lman , 1 9 9 3 ) . Iron also serves as a c o m p o n e n t of a number of non-heme prote ins, o f ten in associat ion w i t h sulphur. Iron is t ranspor ted among sites of absorp t ion , s torage and ut i l izat ion by means of t ransfer r in (Tr f ) . This g lycopro te in consis ts of a single po lypept ide chain w i t h an approx imate molecular w e i g h t of 8 0 kDa (MacGi l lvary , 1983) w h i c h t igh t ly ( ~ 1 0 2 3 M"1) binds t w o ferr ic ( F e + 3 ) a toms. Trf shares amino acid homology w i t h at least three other i ron-binding g r o w t h fac to rs : lac to t ransfer r in , ovot ransfer r in and melanot ransfer r in (melanoma-associated ant igen p97) (Food etal., 1 9 9 4 ) . The 21 l iver is the major source of c i rcu lat ing Tr f a l though recent ev idence indicates tha t mur ine macrophages may also synthesize and release t ransfer r in (Djeha et al., 1 9 9 5 ) . Trf is del ivered into the cell after b inding to speci f ic sur face receptors. TrfR are expressed at a l o w level on mos t cells but at high levels on rapidly d iv id ing cells and placental brush border cel ls. In add i t ion , TrfR are expressed at especial ly high levels on ery thro id precursors in order to meet the high iron requi rements for heme and subsequent ly hemoglob in synthes is w i th in these cel ls. The TrfR is a homodimer ic s t ruc ture cons is t ing of po lypept ide chains of ~ 9 0 kDa (Trowbr idge et al., 1 9 8 2 ) . The mur ine TrfR gene has been c loned and sequenced (Stearne et al., 1985) and a large bat te ry of ant i -mouse TrfR monoc lona l ant ibodies have been prepared (Lesley et al., 1 9 8 4 ; Kemp et al., 1 9 8 7 ; Leenan et al., 1 9 9 0 ) . The impor tance of the Tr f -Tr fR p a t h w a y in cellular g r o w t h is under l ined by the observat ion tha t many ant i -TrfR monoc lona l ant ibodies b lock cell prol i ferat ion in vitro (T rowbr idge et al., 1 9 8 2 ; Kemp et al., 1 9 8 7 ; Leenan et al., 1 9 9 0 ) . Tr fR are posi t ioned w i th in c la thr in-coated pits and receptor- l igand complexes are endocy tosed by an energy dependent mechan ism. T r f -bound iron is released w i t h i n acidic ( < p H 5.5) non- lysosomal vessic les (Daut ry-Varsat e r a / . , 1 9 8 3 ) . The receptor -apot ransfer r in complex returns to the cell sur face where apot ransfer r in is released, becoming available for addi t ional iron binding and endocy tos is (Klausner et al., 1 9 8 3 ) . Cytop lasmic iron is uti l ized immediate ly , or s tored in comp lex w i t h the cy top lasmic mul t imer ic prote in fer r i t in . This molecule consis ts of 2 4 sub-un i ts conta in ing H (acidic) and L (basic) chains in vary ing 2 2 propor t ions , depend ing upon the t issue examined. The main func t i on of ferr i t in is to serve as a reservoir of intracel lular iron w h i c h can be tapped for the synthesis of i ron-conta in ing prote ins. TrfR are expressed on develop ing ery thro id cells f r o m the p ronormob las t stage to the re t icu locyte phase, w i t h their dens i ty decreasing dur ing ery thro id matu ra t ion in bo th the mouse (Lesley et al., 1984) and human (Loken et al., 1 9 8 7 ) . Tr fR are not present on the mature red blood cel l . For the mouse , it w a s demons t ra ted tha t BM cells w i t h BFU-E act iv i ty are posi t ioned w i t h i n the TrfR" f rac t ion wh i le cells w i t h CFU-E act iv i ty reside in the T r f R + f rac t ion (Lesley e r a / . , 1 9 8 4 ) . In nonery th ro id cells TrfR numbers are posi t ively corre lated w i t h prol i ferat ion but in hemoglob in-synthes iz ing cells TrfR numbers increase dur ing d i f ferent ia t ion and there fore are negat ive ly corre lated w i t h pro l i ferat ion (Ponka and Schu lman, 1 9 9 3 ) . Senescent red cells are phagocy tosed by cells of the ret iculoendothel ia l sys tem and the released iron becomes available to the iron metabol ic p a t h w a y for reut i l izat ion. 1.5.3 Heme metabo l i sm Vi r tua l ly all unicel lular organisms and mos t cells of mult i -cel lular organ isms w i t h the except ion of mature e ry th rocy tes and other terminal ly d i f fe rent ia ted cel ls, synthesize the i ron-conta in ing porphyr in heme (Ponka and Schu lman , 1 9 9 3 ) . In mammals , heme synthes is is greatest in deve lop ing erythro id cells and the liver where large amounts of heme are needed in the fo rmat ion of the prosthet ic g roup for the mi tochondr ia l c y t o c h r o m e s and other heme prote ins. 23 H o w e v e r in the adul t , a lmost 9 0 % of all heme resides w i t h i n ery thro id cells. In add i t ion , the rate of heme synthes is in these cells exceeds tha t of all o thers (Ponka and Schu lman , 1 9 9 3 ) . Heme synthes is is a mul t i -s tep process requir ing the par t ic ipat ion of four mi tochondr ia l and four cy toso l ic enzymes. Dist inct regulatory mechanisms for th is p a t h w a y exist in non-ery thro id and ery thro id t issues. The initial event in heme synthes is occurs w i t h i n the mi tochondr ion where ^-aminolevul in ic acid synthase (ALAS) cata lyzes the fo rmat ion of ©"-aminolevulinic acid (ALA) by l inking glyc ine and succ iny l coenzyme A (Figure 1.2). A L A S , w h o s e synthes is and ac t iv i ty is negat ively regulated by heme appears to represent the rate- l imi t ing enzyme for hepat ic heme synthes is . H o w e v e r , ery thro id cells also conta in a t issue-speci f ic f o r m of A L A S (ALAS-E) (Dandekar et al., 1 9 9 1 ) . Hemin (a halide salt of heme) s t imulates the synthes is of ALAS-E and ALAS-E gene expression and resul tant heme fo rmat ion in f luences the regulat ion of o ther genes involved in the synthes is of heme (Meguro e r a / . , 1 9 9 5 ; Mele fors e r a / . , 1 9 9 3 ) . Since Trf represents the sole source of i ron, its avai labi l i ty l imits heme synthes is and consequent ly hemoglob in fo rmat ion since the presence of heme is ob l igatory for g lobin mRNA t rans la t ion . Fur thermore, since the 5 ' -un t rans la ted region of ery thro id A L A S mRNA conta ins an i ron-responsive e lement (IRE) (Dandekar et al., 1 9 9 1 ) , the a v a i l a b l y of iron may cont ro l ALAS-E express ion in ery thro id cel ls. In non-ery thro id cel ls, the removal of iron f r o m Trf is not cont ro l led by levels of intracel lular heme, the avai labi l i ty of iron does not cont ro l the overal l heme synthes is rate and the A L A S mRNA lacks the IRE (Ponka and Schu lman , 1 9 9 3 ) . 2 4 A n addi t ional cont ro l po int in heme synthes is , also w i t h d is t inct ery thro id and non-ery thro id features, occurs at the level of porphobi l inogen deaminase (PBG-D), one of t w o enzymes act ing in concer t to produce uroporphr inogen III. PBG-D exists in t w o isoforms t rans la ted f r o m t w o separate mRNAs w h i c h arise f r o m the same st ructura l gene (Ponka and Schu lman , 1993 ) . One PBG-D var iant is f ound in all cells and the second only in ery thro id cells. In regards to the fou r th and f i f th enzymes of the heme b iosynthet ic p a t h w a y , u roporphyr inogen synthase and uroporphyr inogen decarboxy lase , there is no ev idence tha t non-ery thro id and ery thro id fo rms are d i f fe rent (Ponka and Schu lman, 1 9 9 3 ) . The s ix th and seventh enzymes of the heme synthe t ic p a t h w a y , cop roporphy r inogen oxidase synthase and coproporphr inogen decarboxylase enzymes are sub jec t to unique pat terns of regulat ion dur ing the d i f ferent ia t ion of MEL cells (Conder et al., 1 9 9 1 ) . Ferrochelatase inserts F e + 2 into PPIX to complete the fo rma t ion of heme. Ferrochelatase mRNA is present as 2 .2 and 2 .8 ki lobase (kb) t ranscr ip ts in mur ine t issues (Chan et al., 1 9 9 3 ) . The smaller t ranscr ip t was associated w i t h greater mRNA stabi l i ty and predominated in re t icu locy tes and the spleen suggest ing tha t fer rochelatase ac t iv i ty is also speci f ical ly regulated in ery thro id t issues (Chan et al., 1 9 9 3 ) . Thus , the regulatory cont ro l of the heme b iosynthet ic p a t h w a y in ery thro id cells has several cont ro l po in ts , rather than at one single dominan t site (ALAS) as demons t ra ted for non-ery thro id t issues. Heme is catabol ized to bi l iverdin IX th rough the act ion of heme oxygenase (HO) leading to the release of i ron. In the mouse , HO act iv i ty is greatest in the spleen f o l l o w e d by the liver and BM (Abraham, 1 9 9 1 ) . In hematopo ie t ic cel ls, 25 H ALA-Synthase COO' CH, CH, C : 0 5-AMINOLEVULINIC ACID (ALA) CH, HOOC - CH, H E M E Ferrochelatasei Fe" CH, HOOC - CH, P R O T O P O R P H Y R I N IX Protoporphyrinogen Oxidase Coproporphyrinogen Oxidase CH, CH, HOOC - CH, C H . - C O 0 H P R O T O P O R P H Y R I N O G E N III ALA-Dehydralase Heme Oxygenase BILIVERDIN IX BILIRUBIN IX Fe(?) jo o O coo-coo CH, CH, CH, K P O R P H O B I L I N O G E N Porphobilinogen Deaminase CH, CH, HOOC - CH, CH," COOH H Y D R O X Y M E T H Y L B I L A N E I Uroporphyrinogen Synthase HOOC - CH, CH,C0O' COOH CH - C H , CH, <:oo - V CH, CH, HOOC - CH, CH,- COOH U R O P O R P H Y R I N O G E N III HOOC - CH, | Uroporphyrinogen Decarboxylase CH, HOOC - CH, C O P R O P O R P H Y R I N O G E N I Figure 1.2 Heme (iron p ro toporphyr in IX) synthes is . Eight ident i f iable enzymat ic steps occur in the fo rmat ion of heme of w h i c h the f i rs t and the f inal three of these stages occur in the m i tochondr ion . The f i rs t s tep is the condensat ion of g lyc ine and succ iny l coenzyme A to f o r m 6-aminolevul in ic acid (ALA) by ^-aminolevul in ic acid syn the tase (ALAS) . A L A then passes into the cy toso l where A L A dehydratase catalyzes the condensat ion of t w o molecules of A L A into the pyrrole porphobi l inogen (PBG). Four molecules of PBG are comb ined to f o r m the te t rapyr ro le u roporphyr inogen III (URO III) th rough the act ion of PBG deaminase and URO III cosyn thase . URO decarboxy lase conver ts the four acet ic acid side chains to methy l g roups , y ie ld ing coproporphyr inogen III. The remaining syn the t i c s teps take place w i th in m i tochondr ia . The f inal stage in th is b iosynthet ic p a t h w a y is the insert ion of fer rous ( F e + 2 ) i ron into p ro toporphyr in IX by fer rochelatase to yield heme. The major degradat ion p a t h w a y for heme is cont ro l led by heme oxygenase w h i c h produces bi l iverdin IX w h i c h is subsequenly metabol ized to bi l i rubin. Iron released in th is process becomes available for re-use. 2 6 the level of HO may play a cr i t ical role in the uncoup l ing of progeni tor cell d i f fe rent ia t ion f r o m prol i ferat ion (Abraham, 1 9 9 1 ) . It w a s demons t ra ted for murine BM CFU-E cu l tu red in the presence of Epo, HO act iv i ty w a s elevated in the early stages of e ry th ro id g r o w t h but progressively decl ined as ery thro id deve lopment progressed indicat ing tha t HO act iv i ty correlates inversely w i t h ery thro id d i f fe rent ia t ion (Lut ton et al., 1 9 9 1 ) . 1.5.4 Inf luence of porphyr in photosensi t izers on the heme metabol ic p a t h w a y and hematopoies is Despite a w idespread interest in the use of non-metal l ic porphyr ins as photosens i t izers , surpr is ingly l itt le is k n o w n about the interplay of these compounds w i t h the i ron-heme metabol ic p a t h w a y or the hematopo ie t ic s y s t e m . This c i rcumstance is largely due to the ove rwhe lm ing emphasis on the s tudy of these c o m p o u n d s as pho tochemotherapeu t i c agents rather than whe the r these c o m p o u n d s migh t in f luence the act iv i ty of cellular metabol ic p a t h w a y s . However , the heme b iosynthe t ic p a t h w a y can be " s u b v e r t e d " for pho todynamic purposes by admin is ter ing large amoun ts of the heme precursor A L A . This subsequent ly photosensi t izes the subject by increasing the fo rmat ion of PPIX (Kennedy et al., 1 9 9 0 ) . Since t u m o u r s and normal skin conta in relat ively l ow levels of fe r roche la tase, PPIX accumula tes in these t issues rather than being conver ted to heme (Kr iemer-Bi rnbaum, 1 9 8 9 ) . Iron chelat ion w i t h desfer r ioxamine enhanced the photosens i t iza t ion process in mur ine P A M 2 1 2 cells cu l tured w i t h A L A by reducing the convers ion of PPIX to heme (Ortel e r a / . , 1 9 9 3 ) . 27 Studies by Cant i et al. (1984) indicated tha t admin is t ra t ion of the photosensi t izer HpD increased spleen and BM cel lular i ty in normal mice and accelerated the hematopo ie t ic recovery (spleen and BM cel lu lar i ty , peripheral b lood w h i t e cells numbers) of mice immunosuppressed w i t h ionizing radiat ion or chemotherapeu t i c agents . This group subsequent ly s h o w e d tha t the hematos t imu la to ry propert ies of HpD were shared by its pur i f ied vers ion Photofrin® (Canti et al., 1 9 8 9 ) . H o w e v e r , no cellular p a t h w a y s were examined t h r o u g h w h i c h these porphyr ins migh t have produced these hematopo ie t ic responses. Gomer et al. (1991) s h o w e d tha t incubat ion of Chinese hamster f ibroblasts w i t h Photofr in®, either in the presence or absence of d i rected l ight, increased express ion of the catabol ic enzyme HO mRNA and pro te in . It w a s unclear w h e t h e r the increased levels of HO in l igh t -pro tected cu l tures w a s related to Photofrin® degradat ion or as a consequence of a general ized ox idat ive stress response w i t h i n these cel ls. Hp has been s h o w n not to undergo chemical degradat ion before e l iminat ion, possibly because the lack of a coord ina ted metal ion prevents its convers ion to bile p igments by the HO sys tem (Bellnier et al., 1989 ) . Pharmokinet ic studies in DBA/2 mice w i t h radio-labelled Photofrin® indicated tha t some degradat ion of the mater ial may occur , a l though b r e a k d o w n products were not character ized (Bellnier et al., 1 9 8 9 ) . Fur thermore, it is u n k n o w n whe the r c o m p o n e n t s of Photofr in® migh t undergo metal lat ion of the te t rapyrro l ic r ing in vivo. These observat ions indicate tha t there is a pauc i ty of in format ion concern ing the ex ten t to w h i c h porphyr in photosensi t izers migh t in f luence the iron-28 heme metabol ic p a t h w a y or the hematopoie t ic s y s t e m . 1.5.5 Inf luence of heme on hematopoies is In the absence of ev idence concern ing the impac t of porphyr in photosensi t izers on the heme metabol ic p a t h w a y and hematopo ies is , it is necessary to d r a w f r o m ex is t ing data on the e f fec ts of natura l ly -occurr ing porphyr ins on these processes. H o w e v e r , it cou ld be misleading to predict biological responses to a porphyr in on the basis of exper imenta l results obta ined w i t h hemin or PPIX, since d i f ferences in side chain moiet ies or the presence or absence of a metal s ign i f icant ly in f luence the phys icochemica l propert ies of a te t rapyr ro l ic c o m p o u n d . Direct regulatory e f fec ts on hematopoies is appear restr ic ted to heme and PPIX. Hemin (ferric chlor ide PPIX) s t imulated heme synthes is and itself w a s incorporated into the hemoglob in of mur ine ery thro leukemia (MEL) cells (Chang and Sassa, 1 9 8 2 ) . Co PPIX, on the other hand increased heme b iosynthes is but was not incorpora ted into heme. H o w e v e r , nei ther M n , M g , Ni , Sn , Cd , Zn or Cu PPIX were act ive in th is sys tem (Chang and Sassa, 1 9 8 2 ) . It w a s subsequent ly s h o w n tha t hemin and to a lesser degree CoPPIX could increase the expression of genes regulated by an enhancer e lement conta in ing b inding sites for NF-E2 in human ery th ro leukemia (HEL) K 5 6 2 cells (Palma et al., 1 9 9 4 ) . PPIX w a s init ially more e f fec t ive than hemin for the induct ion of hemoglob in fo rmat ion in K 5 6 2 cel ls, bu t w a s u l t imate ly tox i c at the concent ra t ions employed (Palma et al., 1 9 9 4 ) . In cu l ture s tud ies, hemin enhanced CFU-E (Porter et al., 1979) and BFU-E (Holden e r a / . , 1983) fo rmat ion as wel l as the g r o w t h of large mixed CFU-29 granu locy te -macrophage-megakaryocy te -e ry th ro id (CFU-GEMM) colonies (Monet te and S igounas, 1988) by mouse BM cells. Admin is t ra t ion of hemin (6 .5 mg/kg) to normal mice increased BFU-E, but not CFU-S levels (Monet te e r a / . , 1 9 8 4 ) . Hemin exer ted a pro tec t ive e f fec t on a number of hemato log ica l parameters (BM cel lu lar i ty , b lood plate lets, b lood hematocr i t ) and hematopo ie t ic progeni tors (CFU-S, BFU-E) in mice chronica l ly suppressed w i t h az idothymid ine (AZT) (Harrison et al., 1 9 9 3 ) . Coadmin is t ra t ion of hemin and rEpo fur ther improved th is pro tec t ive e f fec t against A Z T (Harr ison et al., 1 9 9 3 ) . In add i t ion , hemin appears to also act posi t ive ly at the s t romal cell level in mur ine long t e r m BM cu l ture sys tems by increasing s t romal cel lu lar i ty and elevat ing levels of CFU-S and myelo id progeni tors (Ab raham, 1 9 9 2 ) . H o w e v e r , levels of ery thro id progeni tors were not al tered in th is s y t e m . It w a s postu la ted tha t heme may be a central molecule for the cont ro l of the fo rmat ion and release of hematopo ie t ic g r o w t h fac to rs by BM accessory cells (Ab raham, 1 9 9 2 ) . Fur thermore, the inf luence of hemin on cell g r o w t h and d i f fe rent ia t ion appears to be d is t inc t f r o m its requi rement as a prosthet ic group for hemepro te ins (Sassa, 1 9 8 8 ) . 1.5.6 Inf luence of i ron-b inding prote ins on hematopoies is In the cons iderat ion of the impact of porphyr ins on hematopoies is , it is also impor tan t to address h o w i ron-binding pro te ins , associated w i t h i ron-heme metabo l i sm, m igh t also inf luence the hematopo ie t ic apparatus . W h e n i ron-saturated Trf w a s admin is tered to mice, decreases in splenic and femora l cel lu lar i ty and CFU-G M occured (Genti le and Broxmeyer , 1 9 8 3 ) . Ac id ic , heavy chain (H-) ferr i t in has 3 0 been impl icated as a negat ive regulator of myelopoies is (Broxmeyer , 1992 ) . Admin is t ra t ion o f recombinant human H-ferri t in p roduced s ign i f icant reduct ions in BM and splenic CFU-GM, BFU-E and CFU-GEMM w i th in 2 4 hours (Broxmeyer et al., 1 9 8 9 ) . H-ferri t in receptors have been ident i f ied on human ery throb las ts der ived f r o m BFU-E co lon ies. In add i t ion , H-ferri t in suppressed fo rmat ion of BFU-E by normal human BM cells and the prol i ferat ion of human K 5 6 2 ery thro leukemic cells (Fargion et al., 1 9 9 2 ) . The mechan ism by w h i c h H-ferri t in inhibi ts hematopoiet ic ac t iv i ty has not been establ ished. It w a s suggested tha t H-ferri t in m igh t accompl ish th is by not a l lowing Trf t o donate iron to the cell by keeping iron in the unavailable ferr ic s tate (Broxmeyer , 1 9 9 2 ) . Despite the ex is tence of an extens ive l i terature, w o r k by o thers quest ioned the val id i ty of the H-ferri t in studies per formed by Broxmeyer et al. (Sala et al., 1 9 8 6 ) . A recent demons t ra t ion tha t i ron-loaded lac to fer r in , present in epithel ial secret ions and neutrophi l ic granules, d i rect ly binds speci f ic DNA sequence mot i f s and act ivates t ranscr ip t ion indicates tha t th is i ron-b inding prote in l ikely has a d i rect role in cellular regulat ion (He and Furmanski , 1 9 9 5 ) . Indeed, lactoferr in w a s s h o w n to bind to the GM-CSF promoter of IL-1 s t imula ted cells and down- regu la te its ac t iv i ty (Penco e r a / , 1 9 9 5 ) . 1.6 Thesis ob ject ives Litt le in fo rmat ion is available concern ing the in teract ion of porphyr in photosensi t izers w i t h the hematopo ie t ic apparatus or e lements of the i ron-heme b iosynthet ic p a t h w a y . It w a s therefore a major goal of the thesis to ident i fy photosens i t iz ing porphyr ins w h i c h migh t alter these act iv i t ies in the mouse . Upon 31 the exper imenta l eva luat ion of Photofrin®, BPD, Hp, and PPIX, only Photofrin® produced a detec tab le posi t ive hematopo ie t ic response in the BM or spleen of normal mice. The hematopo ie t ic response to Photofrin® w a s dominated by granulopoieis is in the m a r r o w and by ery thropoies is in the spleen. The ef fect iveness of Photofr in® as a hematos t imu la to ry agent w a s also evaluated in mice mye losuppressed w i t h sub- lethal doses of ^-radiat ion or the chemotherapeut i c drug 5 - f luorouraci l . Photofrin® p romoted the hematopo ie t ic recovery o f animals g iven either one of these t rea tmen ts , a l though th is ac t iv i ty w a s more p ronounced in i rradiated animals. The observat ion tha t Photofrin® marked ly enhanced splenic ery thropoies is in normal mice p rompted invest igat ion into the impact of th is c o m p o u n d on the in vitro d i f ferent ia t ion of mur ine ery thro leukemia (MEL) cel ls. A l t h o u g h Photofr in® did no t cause terminal ery thro id d i f fe rent ia t ion or p romote the prol i ferat ion of these cel ls, it did produce an increase in the express ion of the cell sur face receptor for the i ron- t ranspor t prote in t rans fer r in . This last observat ion indicated tha t cer ta in cons t i tuen ts w i th in Photofrin® may in teract w i t h e lements of the i ron-heme metabol ic p a t h w a y , mod i fy ing the s ta tus of the receptor sys tem responsible for del iver ing iron into the cel l . However , it is uncer ta in whe the r a mod i f i ca t ion of cellular iron s ta tus represents a mechan ism by w h i c h Photofrin® could in f luence the act iv i ty of the mur ine hematopo ie t ic s y s t e m . 32 CHAPTER T W O MATERIALS A N D METHODS M a n y of the procedures descr ibed in th is sect ion have been uti l ized in studies per ta in ing to more than one chapter . There fore , in the quest for brev i ty , all exper imenta l p rocedures employed in the thesis w o r k are detai led w i th in this chapter . 2.1 An ima ls Male D B A / 2 (H-2 d ) mice of 6-8 w e e k s of age were obta ined f r o m Charles River Canada (Mont rea l , Quebec) . In a l imited number of exper iments , male PL (H-2 U ) , DBA/1 (H-2 q ) (Jackson Laborator ies, Bar Harbor, ME) or Balb/c (H-2 d ) mice (Charles River Canada) were emp loyed . An imals were housed in plastic cages (5 - 8 per cage) under pathogen- f ree cond i t ions in the animal ho ld ing faci l i t ies of Quadra Logic Techno log ies , Inc. under 12 hours l ight / 12 hours dark. Mice were g iven s tandard laboratory rodent c h o w (Ralston Purina) and acidi f ied wa te r ad libitum. An imals w e re rout inely screened for ev idence of exposure to a bat tery of mur ine pa thogens using the ImmunoComb® assay s y s t e m . For radiat ion recovery exper iments , mice were mainta ined under a similar regimen w i t h i n the animal unit of the Depar tment of M ic rob io logy and Immuno logy , Univers i ty of Brit ish Columbia. Mice w e re of 8 -12 w e e k s of age at the star t of all exper iments . An imals were we ighed at the s tar t and comple t ion of all exper iments . 3 3 2 .2 Porphyr ins Porphyr in preparat ions were handled under l ow l ight condi t ions in a laminar f l o w h o o d . The barrels of 0 .5 ml plastic syr inges conta in ing photosensi t izer w e r e masked w i t h l ight -opaque adhesive tape. Syr inges w e r e f i t ted w i t h 2 6 gauge needles for in ject ions. 2 . 2 . 1 Iron p ro toporphyr in IX (hemin) Hemin chlor ide (Sigma H-2250) , a halide salt of heme, w a s solubil ized in 0 .2 M N H 4 0 H . The pH of the solut ion w a s lowered to 9.5 w i t h the s low addi t ion of 12N HCI and cons tan t s t i r r ing. The 15 m M hemin s tock so lut ion w a s f i l ter-steri l ized t h r o u g h a 0.22/J Mi l l ipore f i l ter and stored at - 7 0 °C unt i l required for cell cu l ture exper iments . The molecular s t ruc ture of heme is s h o w n in Figure 1A. 2 . 2 . 2 Pro toporphyr in IX (PPIX) PPIX d isod ium salt (Sigma P-5889, Figure 1B) w a s d issolved in 4 m M NaOH (pH 11.7) w i t h s t i r r ing. The solut ion w a s neutral ized to a pH of approx imate ly 7 .4 w i t h the s low addi t ion of 0.1 N HCI. The porphyr in w a s fur ther di luted w i t h a neutral NaOH/HCI solut ion (pH ~ 7 . 4 ) and f i l ter steri l ized th rough a 0 .22 / / Mi l l ipore f i l ter. PPIX w a s adminis tered int raper i toneal ly (i.p.) to normal mice. Cont ro l mice received the neutral NaOH/HCL solut ion (Solvent C). 2 . 2 . 3 Deuterporphyr in IX 2 ,4 (4,2) hyd roxye thy l v iny l (HVD) HVD (Figure 2C) , a monomer ic cons t i tuen t of Photofr in®, w a s obta ined 3 4 f r o m Porphyr in Products (HVD 5 8 0 - 9 ) , Logan UT. The compos i t i on of HVD w a s analyzed by reverse phase ch romatog raphy over an analyt ical C 1 8 Ul t rasphere HPLC co lumn (Beckman Ins t ruments , Inc., San Ramon CA) and indicated tha t HVD was of a pur i ty of 8 6 . 5 % (mean of three analyses). For in ject ions, HVD w a s solubil ized over one hour at 10 mg /m l in DMSO and then di luted s low ly to 1 mg /m l w i t h PBS at 6 0 °C using a vo r tex mixer . The mix ture w a s steri l ized by passage th rough a 0 .22 / / f i l ter (Mil l ipore) and adminis tered i.p. to sub- lethal ly i rradiated mice. Contro l mice received in ject ions of the steri l ized so lvent so lut ion ( 1 0 % DMSO : 9 0 % PBS). 2 . 2 . 4 Benzoporphyr in der ivat ive mono-ac id r ing A (BPD, ver tepor f in) BPD (Figure 3D) (Richter et al., 1987) w a s suppl ied as a b lue-green, lyophi l ized p o w d e r f r o m QLT PhotoTherapeut ics , Inc. , Vancouver , B.C.). Immediate ly prior to use, BPD w a s dissolved at 2 0 mg /m l in 1 0 0 % t issue cul ture grade d imethy l su l fox ide (DMSO, D - 2 6 5 0 , Sigma Chemical C o m p a n y , St. Louis MO) then fu r ther d i lu ted to 1 mg /m l w i t h steri le phosphate-bu f fe red saline (PBS, pH 7 .3 ) . BPD w a s adminis tered in t ravenous ly (i.v.) at 10 m g / k g to normal mice. Exper imental cont ro l mice received the cor respond ing so lvent so lu t ion ( 5 % DMSO : 9 5 % PBS). 2 . 2 . 5 Hematoporphy r in IX (Hp) Immediate ly before use, Hp (Figure 1E) d ihydroch lor ide (H 5 9 8 - 9 , Porphyr in Products Inc.) (pur i ty > 9 0 % ) w a s d issolved in 0.1 N NaOH at 25 mg/ml and then d i lu ted to 2 .5 mg /m l w i t h 0.1 M phosphate (pH 7 .4 ) . The resul tant 3 5 solut ion (pH ~ 7.4) w a s steri l ized t h o u g h a 0 .2 / /Ac rod isc f i l ter. Hp w a s administered i.p. at a dose of 25 m g / k g to normal mice. Contro l mice received the solvent (Solvent B). 2 . 2 . 6 Iron hematoporphy r in IX (FeHp) FeHp (Figure 1 F) of a pur i ty of approx imate ly 8 5 % w a s prepared f r o m Hp in the U.B.C. laboratory of Dr. David Dolphin (Depar tment of Chemist ry) by Dr. Lily Xie (Depar tment of Chemis t ry , UBC) as descr ibed (Hunt et al., 1 9 9 4 ) . Before use, FeHp w a s dissolved in 0.1 N NaOH at 2 .5 m g / m l , d i luted to 1.25 mg/ml w i t h phosphate buf fer (pH 7.0) and son ica ted. HCI (0.1 N) w a s added d ropw ise unti l the so lut ion reached a pH of 7 . 3 - 7 . 5 . FeHp w a s fur ther d i lu ted to 1 mg/ml w i t h phosphate buf fer and then f i l ter-ster i l ized. This porphyr in w a s adminis tered i.p. at 10 m g / k g to sub- lethal ly i rradiated mice. Contro l animals received the so lvent alone. 2 . 2 . 7 Photofr in® (d ihematoporphyr in ether , por f imer sod ium) Clinical-grade Photofrin® w a s obta ined f r o m QLT PhotoTherapeuut ics . Photofrin® is suppl ied as a lyophi l ized, reddish powder and w a s reconst i tu ted at 2 .5 mg /m l in ster i le, pyrogen- f ree 5 % dext rose in wa te r (Baxter Corp . , To ron to , Ontario) immedia te ly before use. Endotox in con tamina t ion w i th in all Photofrin® preparat ions w a s < 2 . 5 uni ts per mg (1 endotox in uni t = 0.1 ng) by the Limulus amoebocy te lysate m e t h o d . The proposed s t ruc ture of the major porphyr in s t ruc ture conta ined in Photofrin® is s h o w n in Figure 2 .1G. W h e n lower doses of the drug were requi red, Photofr in® w a s fur ther di luted w i t h 5 % dex t rose . Photofrin® was 3 6 H O O C - C H 2 H , C - C O O H Figure 2.1 Molecular structures of the porphyrins evaluated in these studies. The depicted compounds possess in vivo photosensitizing activity, with the exception of the metallic porphyrins (A) iron protoporphyrin IX (heme) and (F) Fe hematoporphyrin IX (FeHp). The remainder including (B) protoporphyrin IX, (C) deuteroporphyrin IX 2,4 (4,2) hydroxyethyl vinyl (HVD), (D) benzoporphyrin derivative monoacid Ring A (BPD), (E) hematoporphyrin IX (Hp), and (G) dihematoporphyrin ether (Photofrin®) exhibit various degrees of photosensitizing activity. These porphyrins, with the exception of heme, were evaluated for their ability to influence murine hematopoietic activity in vivo in the absence of activating levels of light. Heme was studied for its capacity to induce the erythroid differentiation of MEL cells. 37 typ ica l ly admin is tered by the i.p. in ject ion of a 0 .2 - 0 . 2 5 ml so lut ion per mouse a l though in select exper iments Photofrin® w a s g iven i.v. t h r o u g h a tai l ve in . 2.3 Experimental Protocols To assess in vivo e f fec ts of porphyr ins upon the murine immunohematopo ie t i c ax is , three basic exper imenta l p ro toco ls we re uti l ized as out l ined b e l o w : 2.3.1 Porphyrin effects in naive mice Mice were g iven a single in ject ion of Photofrin® ( i .p. or i .v.) , PPIX ( i .p . ) , Hp (i.p.) or BPD-MA ( i .v . ) . Contro l animals we re adminis tered the appropr ia te ly -matched so lvent . In a select exper iment per fo rmed to invest igate the regulat ion of the hematopo ie t ic response to Photofr in®, animals we re t reated w i t h indomethac in (Indocid®, Merck Frosst Canada, Inc.) . This an t i - in f lammatory agent w a s prepared by suspending drug capsule con ten ts at 1 m g / m l in a solut ion of PBS, 0 . 5 % T w e e n 2 0 . Mice received 0.1 ml ( 1 0 0 /yg/mouse) of th is mix ture orally t h r o u g h a stainless steel gavage tube f i t ted on to a 1 ml syr inge 1 day before, and 1 and 3 days af ter the admin is t ra t ion of Photofrin® (25 mg/kg) or 5 % dext rose. Contro ls received the appropr iate so lvent in paral lel. 2.3.2 Influence of Photofrin®, FeHp, and HVD on hematopoietic recovery after sub-lethal irradiation Mice received a single bi lateral , sub- lethal dose ( 4 - 7 Gy; 1 Gy = 100 rads) of w h o l e body i r radiat ion f r o m a 6 0 C o source (Gammacel l 2 2 0 , A t o m i c Energy 3 8 Canada L imi ted , O t t a w a , Ontario) located in the UBC Depar tment of Chemist ry at a dose rate of 2 .9 Gy /m inu te . Prel iminary exper iments indicated tha t a radiat ion dose of 4 . 0 - 5 . 0 Gy caused a rapid ( < 2 4 hrs) and susta ined ( > 10 days) reduct ion ( 8 5 - 9 5 % ) in spleen cel lular i ty and levels of spleen and BM c o m m i t t e d granu locyte-macrophage progen i to rs , thereby furn ish ing a p ro found ly suppressed hematopoiet ic sys tem in w h i c h to evaluate the inf luence of the tes t c o m p o u n d on myelopoiet ic recovery . Photofr in® (10 mg/kg) or 5 % dext rose w a s adminis tered i.p. either 1 and 4 days or 1 , 4 and 7 days post - i r rad ia t ion. HVD (10 mg/kg) w a s g iven 1,4 and 7 days post - i r rad ia t ion. W h e n Photofrin® w a s given in parallel w i t h H V D , it w a s d issolved in PBS (2 .5 mg /m l ) , d i lu ted to 1 mg/ml w i t h PBS conta in ing DMSO (final DMSO concen t ra t ion = 1 0 % ) . FeHp (10 mg/kg) w a s g iven 2 4 hours before and either 4 6 hours or 4 8 and 9 6 hours after sub-lethal (7 Gy) i r radiat ion. Mice were analyzed for myelopo ie t ic recovery up to 21 days post - i r rad ia t ion. 2 . 3 . 3 Inf luence of Photofrin® on hematopo ie t ic recovery af ter 5- f luorouraci l (5-FU) t rea tment To produce a myelosuppressed s ta te , mice were g iven 5-FU. 5-FU (Sigma F-6627) w a s d issolved at 10 mg/ml in w a r m PBS, f i l ter-steri l ized and adminis tered i.v. at a dose of 1 5 0 m g / k g . This dosage of 5-FU has been s h o w n to drast ical ly reduce numbers of myelo id and lympho id cells bu t largely spares pr imi t ive s tem cells w h i c h suppor t the hematopoie t ic recovery (Van Zant , 1 9 8 4 ) . One and three days after 5-FU admin is t ra t ion , mice received an i.p. in ject ion of 5 % dext rose or Photofr in® (10 m g / k g ) . Myelopo ie t ic recovery w a s mon i to red for up to 3 9 10 days af ter the 5-FU in ject ion. 2 . 4 Tissue sampl ing Mice were sacr i f iced by an overdose of ei ther Halothane® (M.T .C. Pharmaceut ica ls , Cambr idge, Ontar io) or C 0 2 f o l l owed by cervical d is locat ion. A var ie ty of t issues w e re obta ined as descr ibed be low. 2 . 4 . 1 Peripheral b lood Blood w a s removed by cardiac puncture f r o m anaesthet ized mice using a 1 ml syr inge f i t w i t h either a 5 / 8 " or 7 / 8 " 25 gauge needle. Blood hematocr i ts we re de termined by immediate ly t ransfer r ing co l lected blood to a capi l lary tube and cent r i fug ing at 1 0 , 0 0 0 x g for 5 minutes on a min i -cent r i fuge equipped w i t h a capi l lary tube ro tor . The cellular por t ion of the blood w a s measured by means of a hematocr i t scale. Blood hematocr i t is g iven as the percentage of the to ta l vo lume (cells plus p lasma) . To determine peripheral b lood e ry th rocy te concen t ra t ions , 0.1 ml of co l lected b lood w a s immediate ly t ransfer red into 0 .4 ml of bu f fe red-sod ium ci t rate (Becton and Dickinson) to p revent c lo t t ing . The b lood mix ture w a s fur ther di luted 1:40 w i t h PBS and red cells enumerated in a hemacy tomete r chamber . Total b lood leukocy te concen t ra t ions were determined by immediate ly d i lu t ing non-hepar in ized blood 1/6 in 2 % acet ic acid conta in ing 0.1 % methy lene blue and enumerat ing cells w i t h the h e m a c y t o m e t e r . Serum w a s prepared by permi t t ing the b lood to c lo t for approx imate ly one hour at room tempera tu re in 1 ml po lypropy lene mic ro fuge tubes . Tubes were 4 0 cent r i fuged at 1 4 , 0 0 0 x g for 10 minutes at 4 ° C . Serum w a s p ipet ted of f , t ransfer red to cryov ia ls and mainta ined f rozen at - 2 0 ° C unt i l requi red. Serum samples obta ined f r o m normal mice up to 7 2 hours fo l low ing the admin is t ra t ion of Photofrin® or the cont ro l so lvent were analyzed for their con ten t of IL-1 a (Genzyme Corpora t ion , Cambr idge M A . 1 9 0 0 - 0 1 ) , IL-6 (Endogen Inc. , Boston M A , EM-IL-6) , and GM-CSF (Endogen, EM-GMCSF) by ELISA. Assays were per fo rmed as out l ined in the kit ins t ruc t ions . Lower de tec t ion l imits for these assays cor responded to 15 pg/ml for IL-1 a and IL-6 and 5 pg /ml for GM-CSF. 2 . 4 . 2 Spleen Spleens of euthanized mice were removed under asept ic condi t ions using steri l ized ins t ruments . Mice were d ipped in 7 0 % ethanol and spleens were removed t h r o u g h a t ransverse incision across the a b d o m e n . Organs were t ransfer red to 1 5 ml po lypropy lene cu l ture tubes conta in ing 9 ml of RPMI 1 6 4 0 (StemCell Techno log ies , Vancouver , B.C. or Gibco BRL, Cambr idge, Ontario) or Iscove 's modi f ied Du lbecco 's med ium ( I M D M , StemCel l or Gibco BRL) and 1 ml of 5 % heat - inact iva ted ( 5 6 ° C , 3 0 minutes) fetal calf serum (FCS, Gibco BRL or Hyc lone, Logan UT). Single cell suspensions were made by pressing the t issue t h r o u g h a stainless steel tea strainer using a 3 ml syr inge plunger. In some exper iments , to ta l spleen red cell numbers were determined f r o m these cell suspens ions. Cells w e re cent r i fuged for 5 minutes at 3 0 0 x g and the supernatant decan ted . Ery th rocy tes w e re lysed by adding 5 ml of Tr is-buf fered 0 . 1 4 M NH 4 CI . Cells we re again cen t r i f uged , washed w i t h 12 .5 ml m e d i u m and made up to 5-10 41 ml w i t h m e d i u m . Viable cell numbers were determined by T rypan blue dye exc lus ion . Spleen cell v iabi l i ty w a s character is t ical ly 8 5 - 9 0 % . Spleen cell numbers are repor ted as viable cells normal ized to a b o d y w e i g h t of 2 0 g rams. 2 . 4 . 3 Bone m a r r o w (BM) Excised femurs , free of skin and musc le , had their cellular con ten ts f lushed into steri le 15 ml po lypropy lene cu l ture tubes w i t h 5 -10 ml of I M D M conta in ing 1 0 % FCS f r o m a 5 ml syr inge f i t ted w i t h a 5 / 8 " 2 5 gauge needle. Viable cell coun ts w e re determined by Trypan blue dye exc lus ion . Cell v iabi l i ty typ ica l ly exceeded 9 5 % for BM cell preparat ions. In exper iments in w h i c h cells were rout inely obta ined f r o m in tac t femurs , BM cell y ields are repor ted as to ta l viable cells per femur . 2 . 4 . 4 T h y m u s Thymic lobes were removed and r insed in PBS to minimize con tamina t ion w i t h b lood cel ls. Single cell suspensions in I M D M , 1 0 % FCS were prepared by pressing the t issue th rough a stainless steel mesh . Cell v iabi l i ty w a s assessed by T rypan blue dye exc lus ion and character is t ica l ly exceeded 9 7 % . 2 . 4 . 5 Peri toneal cells A t var ious t imes fo l l ow ing the admin is t ra t ion of 5 % dext rose or Photofrin® (25 m g / k g ) , mice were euthanized and in jected i.p. w i t h 8 ml of I M D M , 1 0 % FCS f r o m a 10 ml syr inge f i t ted w i t h a 1 " 2 0 gauge needle conta in ing 10 ml 4 2 of the so lu t ion . The abdomen w a s gent ly massaged, the mouse placed on its side to fac i l i ta te access to the m e d i u m , and approx imate ly 6 ml of the injected med ium w a s w i t h d r a w n . Cells w e re col lected by cent r i fugat ion and viable (character ist ical ly > 9 5 % ) cell numbers were determined by T rypan blue dye exc lus ion . Samples conta in ing red cel ls, indicat ing peripheral b lood con tamina t i on , were exc luded f r o m s tudy . 2 . 4 . 6 L y m p h node cells L ymp h nodes (peri-aort ic, inguinal , popli teal) we re removed f r o m normal mice or mice immunized 10 days previously w i t h prote in ant igen emulsi f ied in ad juvant (sect ion 2 . 1 2 . 1 ) . Single cell suspensions for indiv idual animals were prepared by pressing l ymph nodes t h r o u g h stainless steel screens as descr ibed above for spleen cel ls, excep t the red cell lysis step w a s not requi red. Cells were col lected by cen t r i fuga t ion and viable (character ist ical ly > 9 8 % ) cell numbers were determined by T rypan blue dye exc lus ion. 2 .5 Hematopo ie t ic progeni tor assays 2 . 5 . 1 Granu locy te -macrophage progeni tor assay Levels of splenic and BM c o m m i t t e d progeni tors of the granu locyte and macrophage l ineages (colony fo rming uni ts g ranu locy te -macrophage , CFU-GM) were de termined by in vitro co lony assays. Cells f r o m individual mice were plated at 5 -10 x 10 5 / p la te (spleen cells) or 5 x 10 4 /p la te (BM cells) in dupl icate or t r ip l icate 10 x 35 m m petr i d ishes w i th in a 1 ml mix ture conta in ing I M D M , 2 0 % FCS, 10 4 3 mg/ml BSA, 0 . 3 % Bacto-agar (Difco 0 1 - 0 1 4 0 , Detro i t , Mich igan) and 3 % serum-free mur ine spleen ce l l -pokeweed-mi togen-cond i t ioned-med ium ( P W M - S C C M , StemCell) as a source of g r o w t h fac tors including IL-1 a (2 .7 pg /m l ) , GM-CSF (75 pg/ml) and IL-3 (1 .5 ng /ml ) . Levels of IL-1 a (Genzyme Corpora t ion , Cambr idge, M A ) , and GM-CSF (Endogen Inc., Bos ton , MA) were determined by ELISA whi le IL-3 levels w e re determined by the suppl ier. Fo l lowing 7 days at 3 7 ° C in 5 % C 0 2 , 9 5 % air in a humid i f ied incubator , plates were examined under an Ax iove r t 35 inver ted mic roscope (Carl Weiss , Inc.) and c lusters > 5 0 cells were scored as a co lony (CFU-GM). To determine if Photofrin® migh t in f luence CFU-GM generat ion in vitro, spleen cells obta ined f r o m naive mice were incubated w i t h Photofr in® (0.5 ng-25 / /g/ml) w i t h or w i t h o u t P W M - S C C M as descr ibed above for the CFU-GM co lony assay. 2 . 5 . 2 Erythro id progeni tor assays Levels of e ry thro id co lony- fo rming uni ts (CFU-E) were determined by plat ing 1 0 6 spleen cells in dupl icate 10 x 35 m m petr i d ishes in I M D M conta in ing 3 0 % FCS, 1 0 % BSA, 1 x 10~4 M 2-mercaptoethano l (2-ME), recombinant murine ery th ropo ie t in (3 U / m l ; 1 U = 10 ng) and 1 % methy lce l lu lose (MethoCu l t M 3 3 3 0 , StemCell) in a f inal vo lume of 1 ml . These dishes were placed in 10 x 100 m m petri d ishes along w i t h a th i rd plate w h i c h conta ined 3 ml of dist i l led wa te r in order to mainta in max imal levels of humid i ty w i th in the cu l tures . CFU-E (clusters of > 8 refract i le , hemoglob in ized cells) were enumerated under the inver ted microscope 4 4 f o l l o w i n g 2 days at 37 °C in 5 % C 0 2 . Erythro id burs t - fo rming uni ts (BFU-E) were determined by cu l tur ing 1 0 6 spleen cells or 5 x 1 0 4 BM cells under the same cond i t ions descr ibed for the CFU-E assay excep t the mix ture also conta ined 1 % P W M - S C C M (MethoCu l t M 3 4 3 0 , StemCel l ) . BFU-E (large hemoglobin ized colonies occasional ly visible to the naked eye) w i t h i n these cu l tures were scored under the inver ted mic roscope after 12 -14 days of cu l tu re . A l te rna t ive ly , hemoglob in-conta in ing splenic BFU-E colonies were stained w i t h benzidine. This reagent w a s prepared by d isso lv ing benzidine d ihydroch lor ide (Sigma B0386) at a concent ra t ion of 0 . 2 % (w/v) in 3 % acetic acid (Palma e r a / . , 1 9 9 4 ) . Immediate ly before use, one part of 3 % H 2 0 2 w a s added to 25 parts of the benzidine s tock so lu t ion . Approx imate ly 0 .8 ml of th is mix ture w a s carefu l ly layered on to the methylce l lu lose gel . Erythroid colonies ( > 5 0 cells) were easily enumera ted t h r o u g h the inver ted microscope by their intense blue-black colour w h i c h w a s ev ident af ter a 15 -30 minute t rea tmen t w i t h benzidine. 2.6 Prol i ferat ion assays In order to gauge spleen cell prol i ferat ive responses, the M T T co lor imetr ic assay ( M o s m a n n , 1983) w a s per fo rmed. The M T T me thod has been uti l ized to determine cellular responses to mi togens ( M o s m a n n , 1 9 8 3 ; Chen et al., 1 9 9 0 ) , prote in ant igens (Wooley et al., 1 9 9 3 ) , hematopo ie t ic g r o w t h fac tors ( M o s m a n n , 1 9 8 3 ) , and minor l ymphocy te -s t imu la t ing ant igen (Mls-1) (Blackman et al., 1 9 9 0 ) . The M T T assay measures the reduct ion of a ye l l ow te t razo l ium salt to a b lue-co loured fo rmazan p roduc t by reducing enzymes present in cells w i t h act ive 4 5 mi tochondr ia . The assay result (spec t rophotomet r ic absorbance) c losely parallels 3 H - t h y m i d i n e incorpora t ion by cells cu l tured under ident ical cond i t ions (Mosmann , 1 9 8 3 ; Chen et al., 1 9 9 0 ) . 2 . 6 . 1 Responses to mi togens To measure responses to the mi togens concanava l in A (Con A , Sigma C 2 0 1 0 ) , Salmonella typhosa l ipopolysacchar ide A (LPS, Sigma L-7895) , spleen cells (1 x 10 6 /m l ) w e re cu l tured in RPMI 1 6 4 0 med ium conta in ing s t rep tomyc in (100 / /g /m l ) , penici l l in ( 1 0 0 U/ml) 25 m M HEPES, 2 m M L-glutamine, 5 x 10" 5 M 2-ME and 5 % FCS in steri le 96-we l l f l a t -bo t tom microt i ter plates (Falcon, Becton Dick inson) . Cells (0 .2 ml /wel l ) in repl icates of 4 -6 were cu l tured for 72 hours at 3 7 ° C , 5 % C 0 2 before 10 //I of a 5 mg/ml so lut ion of the M T T reagent (3 - [4 ,5 -d imethy l th iazo l -2 -y l ] -2 ,5 -d ipheny l te t razo l ium bromide , Sigma M-2128) w a s added to each w e l l . Plates were returned to the incubator for an addi t ional 2-4 hours. To te rminate co lour deve lopment , 100 //I of supernatant w a s r e m o v e d , and 1 5 0 //I of acidi f ied isopropanol added to each wel l and then tho rough ly mixed w i t h a mul t i -channel p ipet te . To determine w h e t h e r Photofrin® or BPD migh t a f fec t the prol i ferat ive response to a m i t o g e n , normal DBA/2 spleen cells were cu l tured w i t h concent ra t ion gradients of Photofr in® or BPD w i t h or w i t h o u t Con A (2 .5 / /g/ml) for 7 2 hours in the s tandard M T T prol i ferat ion assay. 4 6 2 . 6 . 2 Responses t o hematopoie t ic cy tok ines The M T T assay techn ique w a s modi f ied to assess spleen cell pro l i ferat ive responses to d i f fe rent hematopoie t ic cy tok ines (Table 2 .1 ) . Sp lenocy tes , obta ined f r o m mice injected 7 2 hours prev iously w i t h 5 % dext rose or Photofr in® (25 mg/kg) were cu l tured in the presence of concen t ra t ion gradients of rGM-CSF, rG-CSF, rM-CSF, r lL-3, or P W M - S C C M as descr ibed be low. In separate sets of exper imen ts , normal DBA/2 sp lenocytes (1 x 1 0 5 cells per wel l) were cu l tured at 3 7 ° C , 5 % C 0 2 in the presence of var ious indiv idual or combina t ions of g r o w t h fac to rs (0 .2 ml /wel l ) w i t h or w i t h o u t Photofrin® ( 0 . 0 2 - 2 5 / /g/ml) for 9 6 hours in I M D M conta in ing 25 m M HEPES, 2 m M L-glutamine, s t rep tomyc in (100 / /g /m l ) , penici l l in ( 1 0 0 U/ml), 2 0 % FCS and bovine serum a lbumin (BSA, 10 m g / m l , Boehringer M a n n h e i m , Dorva l , Quebec) . Colour deve lopment w a s te rminated 4 hours f o l l o w i n g the addi t ion of M T T and 150 //I of the cu l ture supernatant w a s removed and replaced w i t h 100 //I of PBS. Ac id i f ied isopropanol w a s added and mixed as before . Removal of a large propor t ion of the supernatant and di lut ion of the remain ing m e d i u m w i t h PBS minimizes the risk of serum prote in precip i tat ion w h i c h can occur f o l l ow ing the addi t ion of acidi f ied isopropano l , wh i le retaining the fo rmazan dye crysta ls w i t h i n the we l l . In order to de tec t prol i ferat ive responses to rEpo by sp lenocytes f r o m t reated animals, it w a s necessary to increase the cell concen t ra t ion to 4 x 1 0 5 / w e l l . In add i t ion , these cells were cu l tured w i t h th is fac tor for only 4 8 hours prior to the addi t ion of the M T T reagent . The colour react ion w a s te rmina ted after a 3 hour incubat ion . The high cell dens i ty and shor t incubat ion period w a s associated w i t h 4 7 elevated levels of M T T act iv i ty in the cont ro l cu l tu res , but speci f ic responses to rEpo above these levels were still demonst rab le . To measure spleen cell responses to recombinant human inter leukin 2 ( r lL-2, A m g e n , Thousand Oaks CA) spleen cells (1 x 10 6 /m l ) we re cu l tured in RPMI 1 6 4 0 m e d i u m (StemCell) conta in ing s t rep tomyc in ( 1 0 0 / / g / m l ) , penici l l in (100 U/ml) 25 m M HEPES, 2 m M L-glutamine, 5 x 1 0 " 5 M 2-ME and 5 % FCS in steri le 96 -we l l f l a t -bo t tom.mic ro t i te r plates (Falcon, Becton Dick inson) . Cells (0 .2 ml /wel l ) in repl icates of 4 -6 were cu l tured for 9 6 hours at 3 7 " C , 5 % C 0 2 before the M T T reagent w a s added. 2 . 6 . 3 Evaluat ion of rest ing cellular metabol ic ac t iv i ty To assess " spon taneous " pro l i ferat ion, f reshly prepared spleen cells (4 x 10 5 ) w e re added in I M D M , 2 0 % FCS, 1 0 % BSA in repl icates of 4 -6 into 9 6 -wel l micro t i te r plates at 0 .2 m l /we l l . Cells were a l lowed to sett le and equi l ibrate for one hour at 3 7 °C , 5 % C 0 2 . The M T T reagent w a s then added to each wel l for 3 hours . Colour deve lopment w a s te rminated as descr ibed above for cul tures conta in ing h igh levels of p ro te in . 2 . 6 . 4 Prol i ferat ive responses to a protein ant igen To assess the inf luence of Photofrin® on speci f ic immune respons iveness, ova lbumin (OVA) w a s either co-admin is tered w i t h Photofrin® (25 m g / k g , i.p.) or g iven 7 days after the admin is t ra t ion of the porphyr in preparat ion (Sect ion 2 . 1 2 . 1 ) . Contro l mice received the same ant igen preparat ion but were 4 8 T a b l e 2.1. R e c o m b i n a n t m u r i n e a n d h u m a n c y t o k i n e s u t i l i z e d in t h e s e s t u d i e s . # Recombinant cytokine Species Expression vector Cellular source of natural protein Commercial supplier catalogue # 1. Interleukin-10 (rll_-1£) Mouse E. coli monocytes, keratinocytes Upstate Biotechnology, Inc. 01-173 2. lnterleukin-2 (rlL-2) Human E. coli activated T cells Amgen 3. lnterleukin-3 (rlL-3) Mouse E. coli activated T cells Upstate Biotechnology, Inc. 01-374 4. lnterleukin-6 (rlL-6) Mouse E. coli T cells, B cells, monocytes, macrophages, endothelial cells, keratinocytes Upstate Biotechnology, Inc. 01-156 5. lnterleukin-7 (rlL-7) Mouse E. coli bone marrow stromal cells, Upstate Biotechnology, Inc. 01-180 6. Granulocyte colony stimulating factor (rG-CSF) Human E. coli monocytes, macrophages, bone marrow stromal cells, fibroblasts Sigma Chemical Co. G-6652 7. Macrophage colony stimulating factor (rM-CSF) Human E. coli monocytes, fibroblasts, endothelial cells Sigma Chemical Co. M-9667 8. Granulocyte-macrophage colony stimulating factor (rGM-CSF) Mouse E.coli activated T cells, B cells, macrophages, endothelial cells, fibroblasts Upstate Biotechnology, Inc. 01-167 9. Erythropoietin (rEPO) Mouse Chinese hamster ovary cells kidney cells, macrophages Boehringer Mannheim 1276 964 10. Stem cell factor, c-kit ligand (rSCF) Mouse Yeast bone marrow stromal cells, fibroblasts, activated T cells Sigma Chemical Co. S-8028 4 9 given 5 % dext rose i.p. One-hal f of the mice f r o m each group were sacr i f iced 10 days f o l l o w i n g O V A immunizat ion and single cell suspensions f r o m individual spleens and l ymph nodes ( inguinal and peri-aort ic) were prepared. Prol i ferative responses to a concen t ra t ion range of O V A (0, 1 2 . 5 , 2 5 , 5 0 and 1 0 0 / / g / m l ) were determined by the M T T color imetr ic assay. Prol i ferat ive responses to Con A (2.5 / /g/ml) p roduced by each cell preparat ion were evaluated in parallel to evaluate overal l immune c o m p e t e n c e . Al l tes ts were per fo rmed in quadrup l ica te we l ls . Cells we re cu l tured w i t h the s t imulants for 9 6 hours at 37 °C , 5 % C 0 2 . Cells prepared f r o m un immunized male DBA/2 mice were cu l tured under the same condi t ions to determine baseline responsiveness to O V A . 2 . 6 . 5 M ixed leukocy te react ion In order to character ize their immunos t imu la to ry ac t iv i t y , splenic dendr i t ic cells (DC) were assessed in vitro by the mixed leukocy te react ion (MLR). Purif ied splenic DC (Sect ion 2 . 1 3 . 2 . 2 ) were pre- t reated w i t h the ant i -prol i ferat ive agent m i t o m y c i n C (Sigma M-0503) at 5 0 / / g / m l , 3 7 °C for 2 0 minu tes , washed t w i c e w i t h RPMI 1 6 4 0 m e d i u m , 2 % FCS and once w i t h m e d i u m alone. T i t rated (2 x 1 0 2 - 1 x 10 5 /we l l ) DBA/2 DC were co-cu l tu red w i t h 4 x 1 0 5 ny lon woo l -enr i ched , al logeneic DBA/1 T cells (Sect ion 2 . 1 3 . 2 . 4 ) . The MLR w a s per fo rmed w i th in t r ip l icate r o u n d - b o t t o m e d 96-we l l Falcon microt i ter plates (0 .2 ml /wel l ) conta in ing RPMI 1 6 4 0 m e d i u m and 1 0 % FCS. Contro l cu l tures inc luded T cells alone or DC alone. A f te r 3 days at 3 7 ° C , 5 % C 0 2 , the M T T reagent w a s added to each we l l . Four hours later, plates w e re cent r i fuged for 10 minutes and the supernatant 5 0 discarded. PBS (50 //I) w a s then added to each wel l and cells were lysed and the fo rmazan p roduc t solubi l ized w i t h the addi t ion 1 5 0 //I acidi f ied isopropanol . Finally, wel l con ten ts w e re t ransfer red to f l a t -bo t tom microt i ter plates in order to accurate ly quan t i f y co lour deve lopment w i t h a dens i tometer . For all M T T assays, co lour in tensi ty w a s measured w i t h an au tomated dens i tometer (Dynatech Laborator ies, A lexandr ia VA) at a w a v e l e n g t h of 5 9 0 n m . Data are presented as the absorbance value obta ined in wel ls conta in ing the tes t art icle minus the mean absorbance obta ined in wel ls conta in ing the cu l ture med ium alone. 2 .7 Cell l ines The DBA/2 m a s t o c y t o m a (Mor i etal., 1979) (CD45 + , HSA", C D 7 1 + , F 4 / 8 0 + ) P81 5 cell line has been mainta ined in the laboratory for a number of years. The Friend v i rus- induced mur ine ery thro leukemia (MEL) cell l ine, D1B (Chesebro et al., 1976) (CD45", HSA + , CD71 + ) of DBA/2 origin w a s obta ined f r o m the Amer ican Type Cul ture Col lec t ion, Rockvi l le, M D (ATCC # TIB 56 ) . Both cell lines were cu l tured in RPMI 1 6 4 0 med ium conta in ing 1 0 % FCS, 5 x 10" 5 M 2-ME, penicil l in ( 1 0 0 U/ml) , s t rep tomyc in ( 1 0 0 / / g / m l ) and 2 m M L-glutamine. Cells were passaged t w i c e w e e k l y by reseeding in f resh cul ture med ium at approx imate ly 1 x 1 0 4 ce l ls /ml . The M 1 / 7 5 . 1 4 . 4 hybr idoma (ATCC # TIB 1 2 7 ) , p roduc ing a rat l g G 2 c ant ibody react ive to mouse ery th rocy te- res t r i c ted heat stable ant igen (HSA, Springer et al., 1 9 7 8 ) , w a s mainta ined in Du lbecco 's modi f ied Eagle med ium 51 conta in ing 1 0 % FCS, ant ib io t ics and L-glutamine. Cul tures were passaged every 3-4 days by re-seeding the cells at approx imate ly 1 x 1 0 5 / m l . Cul ture supernatants obta ined after a 9 6 hour cu l ture were uti l ized for cell label l ing. 2 . 8 F low c y t o m e t r y Leukocy tes were assessed for their expression of var ious cell sur face markers by f luorescence-ac t iva ted cell sor t ing (FACS) analysis. Cells (5 -10 x 1 0 5 per tube) w e re placed in ice-cold 0 . 1 4 M phosphate-buf fe red sal ine, pH 7.3 (PBS) conta in ing 2 - 5 % FCS and 0 . 0 3 % N a N 3 . For DC, 1 05 ce l ls / tube were subjected to label l ing. Saturat ing concent ra t ions of unlabel led or f luorescein isoth iocyanate (FITC-) or phycoery th r in (PE-) label led, or rat or hamster monoc lona l ant ibodies to mouse leukocy te ant igens (Table 2 .2 ) , or the matched isotype cont ro l (Table 2.3) were added for approx imate ly one hour on ice. Cells were then w a s h e d t w i c e w i t h the assay buf fer . Where requi red, an ant i -rat IgG or ant i - rat IgM PE- or FITC-labelled secondary reagent (Table 2.4) w a s added for an addi t ional hour . In prel iminary exper iments , con t ro l cells (background) were not t reated w i t h iso typ ica l ly -matched ant ibodies but w e re exposed to the identical w a s h procedure and the same FITC-labelled an t ibody reagent as the cells stained w i t h leukocyte-spec i f ic monoclonal ant ibodies. A f te r s ta in ing , cells were washed t w i c e w i t h the FACS buf fer and f ixed in 1 % p - fo rma ldehyde in PBS. Cell sur face f luorescence w a s analyzed immediate ly a f te rward or w i th in 2 4 hours fo l l ow ing the labell ing procedure w i t h ei ther a Coul ter Epics C or Profile (Depar tment of Clinical Immuno logy , Brit ish Columbia Chi ldren 's Hospital) 52 or an XL 2 6 0 0 (QLT PhotoTherapeut ics , Inc.) f l o w c y t o m e t e r (Coulter Corporat ion Inc., Hialeah, FL). Dead cells and debris were gated ou t . F luorescence signals were co l lected on a logar i thmic scale. The cell gat ing procedure w a s ver i f ied by p rop id ium iodide (PI) s ta in ing. PI accumula tes in non-viable cells but is exc luded f r o m l iv ing cel ls. For single-label s tudies ( 5 , 0 0 0 - 1 0 , 0 0 0 cells analyzed) the background sta in ing w a s arbi trar i ly set to approx imate ly 2 - 5 % for the isotype cont ro l samples. Speci f ic s ta in ing w a s determined by sub t rac t ion of the backg round result f r o m the percent staining result obta ined for each ant i -mouse leukocy te monoc lona l an t ibody . For double- label s tudies ( 1 0 , 0 0 0 - 2 5 , 0 0 0 cel ls analyzed) , the signal w i t h i n the FITC (FL1) and PE (FL2) channels of the XL f l o w cy tomete r were adjusted to minimize inter-channel "c ross- ta lk " . This procedure w a s ver i f ied by analyzing mouse spleen cells stained w i t h FITC-ant i -CD8 and PE-anti-CD4 monoc lona l ant ibod ies, g iv ing a mutua l ly exc lus ive sta in ing pat tern for T cells f r o m th is t issue (Figure 2 .1 ) . T h y m o c y t e s exhib i ted a preponderance of C D 4 + / C D 8 + cells wh i le the BM conta ined f e w cells w i th in ei ther T cell subset . Cell labell ing results obta ined w i t h the Coul ter XL f l o w cy tomete r were saved in a l is tmode fo rmat , w h i c h w a s ut i l ized to per fo rm more detai led analyses of the data at a later t ime . 2 .9 Cell cyc le analysis Nuclear DNA con ten t of cells w a s assessed by means of a prop id ium iodide (PI) s ta in ing techn ique (Fleming etal., 1 9 9 3 ) . Freshly prepared, e ry th rocy te -free sp lenocy tes ( 1 x 1 0 6 / t u b e ) were washed t w i c e w i t h PBS and then t reated w i t h 53 Bone Marrow Thymus Spleen Figure 2.2 Ver i f i ca t ion of the f l o w c y t o m e t r y colour compensa t ion procedure. BM cel ls, t h y m o c y t e s , and sp lenocytes were prepared f r o m normal DBA/2 mice and subjected to sur face labell ing w i t h the PE-conjugated ant i -CD4 monoc lona l an t ibody RM4-5 in con junc t ion w i t h the FITC-conjugated ant i -CD8 monoc lona l an t ibody 5 3 - 6 . 7 . Cell f luorescence w a s analyzed w i t h the Coulter XL f l o w cy tomete r . The labell ing pat terns for the indiv idual t issues displayed an expec ted C D 4 / C D 8 co-d is t r ibu t ion , in tha t CD4 and CD8 were present on f e w BM cel ls, wh i le the vas t major i ty ( > 8 0 % ) of t h y m o c y t e s we re C D 4 + C D 8 + , and a mutua l ly exc lus ive ( < 0 . 5 % C D 4 + C D 8 + cells) expression pat tern w a s ev ident for these ant igens on sp lenocy tes . 5 4 Table 2.2. Monoc lona l ant ibodies used for leukocy te sur face analysis by f l o w c y t o m e t r y . Affini ty-pur i f ied ant ibodies were used excep t where noted as either t issue cul ture supernatants (s/n) or clarified asc i tes . # Cell surface antigen Cellular distr ibution Monoclonal antibody clone Isotype Fo rma t b Source -cata logue* Reference 1. CD3 T KT3 Rat lgG 2 a U (s/n) Serotec MCA 5 0 0 Tomonari, 1988 2. CD3 T 145-2C11 Hamster IgG FITC Pharmingen 0 1 0 8 4 D Leo et al., 1987 3. CD4 Thym. , T KT174 Rat lgG 2 c U (s/n) Serotec MCA 6 1 2 n/a 4 . CD4 Thym. , T RM4-4 Rat lgGb 2 b , K FITC Pharmingen 0 1 0 7 4 D n/a 5. CD4 Thym. , T RM4-5 Rat lgG 2 a , K PE Pharmingen 0 1 0 6 5 B n/a 6. CD8 cytotoxic T KT15 Rat lgG 2 a U (s/n) Serotec MCA 6 0 9 Tomonari and Lovering, 1988 7. CD8a cytotoxic T 53-6.7 Rat lgG 2 a , K FITC Pharmingen 0 1 0 4 4 D Ledbetter ef al., 1980 8. CD8a cytotoxic T 53-6.7 Rat l gG 2 a i K PE Pharmingen 0 1 0 4 5 A Ledbetter ef al., 1980 9. CD11b (Mac-1) m<5>, G M1/70 .15 .1 Rat lgG 2 b , K U (s/n) Serotec MCA 74 Springer et al., 1978; Springer etal., 1979 10. CD24 (HSA) splenic B, E, most thym. , most BM J11d Rat IgM U Pharmingen 0 1 2 5 1 D Bruce et al,. 1981 1 1 . CD24 (HSA) splenic B, E, most thym. , most BM M 1 / 6 9 . 1 6 Rat lgG 2 s , K FITC Dr. Suzanne Chappel, Terry Fox Laboratories Springer et al., 1978 12. LR-1 (likely HSA, CD24) splenic B, E, most thym. , most BM LR6 Rat IgM U ascites Serotec MCA 4 5 0 Hutchings ef al., 1985 13. HSA erythroid M 1 / 7 5 . 1 6 . 4 Rat l gG 2 c U (s/n) American Type Culture Collection T1B 127 Springer et al., 1978; Miller ef al., 1985 14. CD25 (IL-2Ra) activated T, B A M T 13 Rat lgG 2 a U Boehringer Mannheim 881 155 Osawa and Diamenstein, 1984 15. CD25 (IL-2Ra) activated T, B 3C7 Rat l g G 2 b U Pharmingen 0 1 1 0 1 D Ortega et al., 1984 55 # Cell surface antigen (cont.) Cellular distr ibution Monoclonal antibody Isotype F o r m a t b Source -catalogue # Reference 16. CD45 pan-leukocyte, 30F11.1 Rat l g G 2 b U Pharmingen 0 1 1 1 1 D Ledbetter et al., 1979 17. CD45R (B220) pre-B, B RA3-6B2 Rat lgG 2 a , K U Pharmingen 0 1 1 2 1 D Coffman, 1982 18. CD45R (B220) pre-B, B RA3-6B2 Rat lgG 2 a , K FITC Pharmingen 0 1 1 2 4 D Coffman, 1982 19. CD45R (B220) pre-B, B RA3-6B2 Rat lgG 2 a , K PE Pharmingen 0 1 1 2 5 B Coffman, 1982 2 0 . CD54 (ICAM-1) endothelial, activated leukocytes, DC 3E2 Hamster IgG FITC Pharmingen 0 1 5 4 4 D Schynius et at., 1993 2 1 . CD71 (TrfR) proliferating cells, E progenitors C2F2 Rat lgG 2 a PE Pharmingen 0 1 5 9 5 A Kemp et al., 1987 22 . CD71 (TrfR) proliferating cells, E progenitors ER-MP 21 Rat l gG 2 a U Serotec MCA 767 Leenen et al., 1990 23 . CD71 (TrfR) proliferating cells, E progenitors R17 2 1 7 . 1 . 3 Rat l gG 2 a PE Sigma P-6318 Lesley et al., 1984 2 4 . CD80 (B7-1) activated B, m<£>; DC 1G10 Rat lgG 2 a FITC Pharmingen 0 1 9 4 4 D Powers et al., 1994 2 5 . CD90 (Thy1.2) Thym. , T 53-2.1 Rat lgG 2 a,/r U Pharmingen 0 1 0 1 1 D Ledbetter et al., 1980 26 . CD90 (Thy1.2) Thym. , T 53-2.1 Rat l gG 2 a , K FITC Pharmingen 0 1 0 0 4 D Ledbetter et al., 1980 27 . F4/80 mature m<5 F4/80 Rat l g G 2 b U (s/n) Serotec MCA 4 9 7 Austyn and Gordon, 1981 28 . Gr-1 G RB6-8C5 Rat lgG 2 b , K U Pharmingen 0 1 2 1 1 D Holmes ef al., 1986; Lewinsohn et al., 1987 29 . Gr-1 G RB6-8C5 Rat lgG 2 b , K FITC Pharmingen 0 1 2 1 4 D Holmes et al., 1986; Lewinsohn et al.., 1987 3 0 . IgM B LO-MM-9 Rat lgG 2 a U Serotec MCA 199 n/a 3 1 . IgM B R6-60.2 Rat lgG 2 a FITC Pharmingen 0 2 0 8 4 D n/a 3 2 . MHC Class I all nucleated cells 34-2-12 Mouse (C3H) lgG 2 a , K FITC Pharmingen 0 6 1 3 4 D n/a 3 3 . MHC Class II B, m$>, DC P7/7 Rat l g G 2 b U (s/n) Serotec MCA 09 Momburg et al., 1986 5 6 # Cell surface antigen (cont.) Cellular d is t r ibut ion 8 Monoclonal antibody Isotype Format' ' Source -catalogue # Reference 3 4 . MHC Class II (l-Ad) B, m<5>, DC 39-10-8 Mouse (C3H) lgG 3 , K FITC Pharmingen 0 6 2 7 4 D n/a 3 5 . MOMA-2 m<S> MOMA-2 Rat l g G 2 b U (s/n) Serotec MCA 519 Kraal etal., 1989 3 6 . DEC-205 DC NLDC-145 Rat lgG 2 a U (s/n) Serotec MCA 949 Kraal et al.,, 1986 Jiang et al., 1995 3 7 . TER-119 E progenitors., mature E TER-119 Rat lgG2 6,/c Biotin Pharmingen 0 9 0 8 2 D Ikuta et al.,, 1990 a. B: B l y m p h o c y t e s ; B M : bone m a r r o w ; DC: dendr i t ic cel ls; E: e ry th ro id ; G: g ranu locy tes ; HSA: heat stable ant igen; rv&: m o n o c y t e / m a c r o p h a g e ; T: T l y m p h o c y t e s ; t h y m . : t h y m o c y t e s ; b. b io t in : b iot in- label led; FITC: f luoresce in- iso th iocyanate-con jugated ; PE: phycoery th r in -con juga ted ; U: unlabel led; n/a: in fo rmat ion not avai lable. Table 2 . 3 Pur i f ied, unlabel led and con jugated iso typ ic con t ro l ant ibodies emp loyed in th is s tudy . # Isotype Clone Format Commercial source Catalogue # 1. Rat lgG 2 a, K R35-95 Unlabelled Pharmingen 11021D 2. Rat lgG 2 a, K n/a FITC Pharmingen 11014C 3. Rat lgG 2 a, K n/a PE Pharmingen 11025A 4. Rat lgG2b,/f R35-38 Unlabelled Pharmingen 11031D 5. Rat lgG 2 b, K n/a FITC Pharmingen 11034C 6. Rat lgG 2 b, K n/a Biotin Pharmingen 11032C 7. Rat lgG 2 c, K IR304 Unlabelled Serotec PRP 06 8. Rat IgM G42-6.1 Unlabelled Pharmingen 11061D 9. Hamster IgG UC8-4B3 FITC Pharmingen 11094C 57 Table 2 . 4 F luorochrome-con jugated secondary monoc lona l and polyc lonal an t ibody reagents . # Reactivity Host species Antibody format Conjugate label Supplier -catalogue # Use 1. Rat IgG Donkey Polyclonal F(ab')2 fragment FITC Jackson Immunoresearch 712-096-153 FACS analysis 2. Rat IgM Mouse IgG, monoclonal (MARM-4) FITC Serotec MCA 189F FACS analysis 3. Rat IgM Mouse IgG, monoclonal (clone G53-238) PE Pharmingen 10065B FACS analysis 4. Rat IgM Goat Polyclonal {fj chain -specific) Alkaline Phosphatase Biodesign International W99224A Western blot analysis 5. Mouse IgG heavy and light chains Goat Polyclonal (whole molecule) Alkaline Phosphatase Jackson Immunoresearch 115-055-062 ELISA 1 ml of PBS conta in ing 1 % Tr i ton X - 1 0 0 (Sigma X - 1 0 0 ) , 10 / /g /ml PI (Sigma P 4 1 7 0 ) , and 1 0 0 / / g / m l r ibonuclease A (Sigma) for 3 0 minutes at 4 ° C in the dark. The ex t rac ted cell nuclei (n = 1 0 , 0 0 0 ) were analyzed for red f luorescence at 6 2 0 n m (FL3) w i t h the Coulter XL f l o w cy tomete r . Fluorescence signals were col lected w i t h linear sca l ing. The DNA h is togram resul t ing f r o m the f l o w cy tomet r i c w a s d iv ided into three major g roup ings : 1) to ta l DNA (cells in G 0 , G 1 f G 2 , S, or M phases of the cell cyc le ) ; 2) DNA f r o m " res t i ng " cells (cells in G 0 or G, phases of the cell cyc le ) , and 3) DNA in cells synthesiz ing DNA or conta in ing double the normal a l lo tment o f DNA (cells in G 2 , S, or M phases of the cell cyc le ) . A n index of cell d iv is ion w a s obta ined by d iv id ing the to ta l number of cells in the G 2 , S, or M 58 phases of the cell cyc le by the to ta l number of cells analyzed. Cell nuclei conta in ing less than the normal levels ( < 2N) of DNA may represent cells undergoing apopto t ic cell death or matur ing ery thro id cells losing nuclear mater ia l . 2 . 1 0 E r y t h r o i d d i f f e r e n t i a t i o n o f m u r i n e e r y t h r o l e u k e m i a ( M E L ) c e l l s To s tudy the capac i ty of d i f fe rent agents to invoke ery thro id d i f fe ren t ia t ion , MEL cells were plated into 35 x 100 m m petr i d ishes (20 ml per dish) at 1 x 1 0 5 / m l in RPMI 1 6 4 0 conta in ing 1 0 % FCS, ant ib io t ics and 5 x 10" 5 M 2-ME. Cells w e re cu l tured at 3 7 °C , 5 % C 0 2 for 7 2 hours in m e d i u m alone or w i t h 0.1 m M hemin (Sassa, 1 9 8 8 ) , 1 .5% W/v) DMSO (Chan etal., 1 9 9 3 ) , or Photofrin® (2 , 10 or 2 5 / /g /m l ) . If the d ihematoporphyr in c o m p o n e n t of Photofr in® is assigned an approx imate molecular w e i g h t of 1 2 0 0 , the above cu l ture concen t ra t ions w o u l d cor respond to molar equiva lents of approx imate ly 2 0 , 8, and 1.6 / / M , respect ive ly . Individual plates were harvested daily and cell v iabi l i ty and numbers determined by T rypan blue dye exc lus ion . Erythroid d i f ferent ia t ion w a s mon i to red by staining for hemoglob in using an acidic benzidine so lu t ion . This reagent w a s prepared by d issolv ing benzidine d ihydroch lor ide (Sigma B-0386) at a concen t ra t ion of 0 . 2 % (w/v) in 3 % acet ic acid (Palma et al., 1 9 9 5 ) . To t reat cel ls, 10 //I of th is solut ion w a s added to a 0.1 ml vo lume of cells suspended in PBS. Cell s ta in ing was evaluated af ter a 2 hour incubat ion at room tempera tu re . Cells conta in ing hemoglob in exh ib i ted a cy top lasmic b lue-b rown s ta in ing. To establ ish the cell speci f ic i ty of the response to Photofrin® on CD71 express ion , th is exper iment w a s per fo rmed w i t h the P815 m a s t o c y t o m a cell line 59 but at on ly one concen t ra t ion (10 / /g/ml) of the c o m p o u n d . MEL and P815 cell express ion of C D 4 5 , HSA, or CD71 sur face ant igens w a s mon i to red using the monoc lona l ant ibodies 3 0 F 1 1 . 1 (Ledbetter et al., 1 9 7 9 ) , LR-1 (Hutch ings et al., 1 9 8 5 ) , and C2F2 (Kemp etal., 1 9 8 7 ) , respect ive ly . The in f luence of BPD on MEL cell sur face express ion of CD71 w a s also eva luated. 2 . 1 1 Transfer r in b ind ing To assess t ransferr in receptor l igand b inding ac t iv i ty by spleen cel ls, FITC-conjugated human holo- t ransferr in (Trf-F, Boehringer M a n n h e i m 1 5 4 4 543) w a s ut i l ized. Human Trf b inds to the TrfR of all mammal ian species. Spleen cells (5 x 10 5 ) w e re incubated w i t h 1 jjg of Trf-F in PBS conta in ing 0 . 2 % N a N 3 and 0 . 1 % BSA. Non-speci f ic b inding w a s determined by the addi t ion of a 200- fo ld excess of unlabel led human holo- t ransferr in (Sigma T -2158) to the cont ro l tubes for 10 minutes prior to the addi t ion of Tr f -F. A f te r 3 0 minutes on ice, cells were washe d t w i c e w i t h FACS buf fer , f ixed in 1 % p- fo rma ldehyde and cell f luorescence analyzed on the Coul ter XL f l o w cy tomete r . To assess Trf-F b inding by MEL cel ls, t reated cells were f i rst washed t w i c e w i t h serum-f ree RPMI 1 6 4 0 m e d i u m , re turned to the incubator in serum-free m e d i u m , for one hour at 37 ° C, 5 % C 0 2 in order to a l low ut i l izat ion of Tr fR-bound Tr f , w h i c h w o u l d l imit b inding of Trf-F to the Tr fR. 2 . 1 2 Ana lys is o f per i toneal cell hematopo ie t ic accessory ac t iv i ty 2 . 1 2 . 1 Granu locy te -macrophage progeni tor assay and de terminat ion of per i toneal cell co lony s t imula t ing ac t iv i ty 6 0 A set of exper iments w a s per formed to assess the hematopoie t ic accessory ac t iv i ty of per i toneal cells obta ined at var ious t imes fo l low ing the admin is t ra t ion of Photofr in®. Peritoneal cells were plated at 1 - 4 0 x 10 5 /m l in dupl icate or t r ip l icate 10 x 3 5 m m petr i d ishes in I M D M , 2 0 % FCS, BSA at 10 m g / m l , 0 . 3 % Bacto-agar (Difco 0 1 - 0 1 4 0 , Detro i t , M ich igan) , w i t h or w i t h o u t 3 % serum-f ree P W M - S C C M , StemCell) as a source of g r o w t h fac to rs including IL-1 a (2 .7 pg /m l ) , GM-CSF (75 pg/ml) and IL-3 (1.5 ng/ml) in the presence of 5 x 1 0 4 normal bone m a r r o w cel ls. Contro l cu l tures conta ined per i toneal cells alone, per i toneal cells plus P W M - S C C M , bone m a r r o w cells alone or bone m a r r o w cells plus P W M - S C C M . Fo l lowing 7 days at 3 7 ° C in 5 % C 0 2 , 9 5 % air in a humidi f ied incubator , plates w e re examined under an inver ted mic roscope and clusters > 50 cells we re scored as a co lony (CFU-GM). Peritoneal cell supernatants were prepared by cu l tu r ing these cells at 1 x 1 0 6 / m l in I M D M , 1 0 % FCS for 2 4 hours at 37 °C , 5 % C 0 2 . Cell-free supernatan ts w e re obta ined by cent r i fug ing the cu l tures for 10 minutes at 4 0 0 x g and then passing the f lu id t h o u g h a 0 .22 / / f i l ter. Supernatants we re evaluated for GM-CSF c o n t e n t by ELISA (Endogen). In add i t ion , per i toneal cell supernatants were added at a f inal concen t ra t ion of 2 0 % to the rout ine CFU-GM co lony assay conta in ing normal mouse bone m a r r o w cel ls, either in the presence or absence of an opt imal concen t ra t ion of P W M - S C C M . Cul tures we re evaluated after 7 days at 3 7 ° C , 5 % C 0 2 in air. 61 2 . 1 2 . 2 Peri toneal cell FACS analysis Peritoneal cells obta ined f r o m 0 - 3 days af ter the i.p. in ject ion of 5 % dext rose or Photofr in® were assessed for their expression of var ious cell sur face markers by FACS analysis. Cells (5 -10 x 1 0 5 / tube) were placed in ice-cold 0 . 1 4 M phosphate-bu f fe red sal ine, pH 7.3 (PBS) conta in ing 5 % FCS and 0 . 0 3 % N a N 3 Saturat ing concen t ra t ions of unlabel led rat monoc lona l ant ibodies to mouse leukocy te ant igens (Table 2.1) were added for approx imate ly 4 5 minutes on ice. Cells we re w a s h e d t w i c e w i t h the assay buf fer and FITC-conjugated donkey F (ab ' ) 2 ant i - rat IgG (Table 2.3) w a s added for 4 5 minu tes . Cells were again washed w i t h the FACS buf fer and f ixed in 1 % p- fo rmaldehyde in PBS. Cell sur face f luorescence w a s measured w i t h i n 2 4 hours using a Coulter Epics C f l o w c y t o m e t e r (Depar tment of Clinical Immuno logy , Brit ish Columbia Chi ldren 's Hospi ta l ) . Dead cells and debris were gated ou t . Speci f ic an t ibody staining w a s est imated by subt rac t ion of the background result f r o m the percent staining result obta ined for each ant i -mouse leukocy te monoc lona l an t ibody . 2 . 1 2 . 3 Determinat ion of ni tr ic ox ide (NO) p roduc t ion To assess the impac t of Photofrin® on NO fo rmat ion by per i toneal cells or MEL cel ls, supernatant levels of NO were measured by the Griess react ion (Doher ty et al., 1 9 9 3 ) . Peritoneal cells were harvested f r o m mice injected i.p. 2 4 hours prev ious ly w i t h 5 % dext rose or Photofrin® and cu l tured in 24-we l l Falcon plates (1 ml /wel l ) conta in ing phenol red-free I M D M (Gibco BRL), 1 0 % FCS and ant ib io t ics at 1 0 6 ce l ls /we l l . Supernatants were prepared 2 4 hours later by 62 cent r i fuga t ion for 10 minutes at 6 0 0 x g. Fi f ty fj\ w a s p ipet ted in quadrupl icate wel ls of 96 -we l l microt i ter plates (Falcon) and 5 0 JJ\ of the Griess reagent conta in ing 1 .5% sul fani lamide (Sigma S-9251) , 0 . 1 % naphthy le thy lened iamine d ihydroch lor ide (Sigma N-9125) in H 3 P 0 4 w a s added. Na ni tr i te (Sigma S-2252) w a s used to generate the s tandard curve . Plates were placed in the dark for 10 minutes and the co lour in tens i ty Was measured w i t h the Dyna tech microt i ter plate reader set at a w a v e l e n g t h of 5 5 0 n m . 2 . 1 3 Evaluat ion of the inf luence of Photofrin® and BPD on the immune response 2 . 1 3 . 1 A n t i g e n sensi t izat ion To determine whe the r Photofrin® migh t a f fec t the in vivo response to a complex fore ign p ro te in , 9 9 % pure ch icken ova lbumin (OVA, Sigma A - 7 6 4 1 ) w a s solubi l ized in PBS at 2 m g / m l , steri l ized by passage t h r o u g h a 0.22/y f i l ter and emuls i f ied w i t h an equal vo lume of comple te Freund 's ad juvant (CFA) [Mycobacterium tuberculosis Ra37 (Difco) at 4 mg /m l in incomple te Freund's ad juvant (D i fco) ] . The mix ture w a s emulsi f ied by a cont inua l mix ing b e t w e e n 10 ml syr inges connec ted by a leur lock on ice for 10 -12 minu tes . Mice were immunized on each side of the tai l base w i t h 5 0 //I of the mix ture ( total dose of O V A = 100 //g) using a 1 ml syr inge f i t w i t h a 1/2" 2 6 gauge needle. O V A w a s either co-admin is tered w i t h Photofr in® (25 m g / k g , i.p.) or g iven 7 days a f te rwards the admin is t ra t ion of the porphyr in preparat ion. Contro l mice received the same ant igen preparat ion but were g iven 5 % dext rose i.p. One-half the mice f r o m each group were sacr i f iced 10 days fo l l ow ing O V A immunizat ion 6 3 and single cell suspens ions f r o m individual spleens and l ymph nodes (inguinal and peri-aort ic) w e re prepared. Prol i ferat ive responses to a concen t ra t ion range of O V A (0, 1 2 . 5 , 2 5 , 5 0 and 1 0 0 pg /ml ) were determined by the M T T co lourmet r ic assay (Sect ion 2 . 6 . 4 ) . Prol i ferat ive responses to Con A (2 .5 pg /ml ) generated by each each cell preparat ion were evaluated in paral lel. Al l tes ts were per formed in quadrup l ica te we l l s . Cells were cu l tured w i t h these mater ials for 9 6 hours at 37 ° C, 5 % C 0 2 . Cells prepared f r o m naive male DBA/2 mice were cu l tured under the same cond i t ions to determine baseline react iv i ty to O V A . 2 . 1 3 . 2 Determinat ion of serum an t ibody levels Mice (4 per group) were sacr i f iced after a period of 21 days fo l low ing immunizat ion w i t h O V A . Blood w a s removed by cardiac punc ture and serum prepared. Serum w a s analyzed for to ta l IgG ant i -OVA ant ibodies by enzyme- l inked immunoso rben t assay (ELISA). The cent re 6 0 wel ls of 96 -we l l lmmu lon-2 microt i ter plates (Dynatech Laborator ies, Inc. , Chant i l ly VA) were coated overn igh t w i t h a 0.1 ml so lu t ion of O V A (50 pg /ml ) in PBS at 4 ° C. Plates were w a s h e d three t imes w i t h PBS conta in ing 0 . 0 5 % T w e e n 2 0 (BDH) conta in ing 2 % FCS and 0 . 0 2 % N a N 3 (assay bu f fe r ) . The remainder of the assay w a s per fo rmed at r o o m tempera ture . Serial d i lu t ions of each serum sample (0.1 ml) were added into dupl icate OVA-coated we l ls and incubated for 2 hours . Plates were washed 3 t imes w i t h the assay buf fer and 0.1 ml of a goat ant i -mouse IgG alkaline phosphatase-con jugated polyc lonal an t ibody (Table 2.4) d i luted 1 :1000 in the assay buf fer w a s added to each wel l fo r a to ta l of 2 hours . Plates were again washed three t imes and 0.1 ml 6 4 of 1 0 % d ie thano lamine, 0 . 0 2 % N a N 3 , 0 . 0 0 1 M M g C I 2 conta in ing p-n i t ropheny l phosphate (Sigma 1 0 4 - 1 0 5 ) at 1 m g / m l . Colour deve lopment w a s te rminated by the addi t ion of 0.1 ml of 1 M NaOH 15 minutes later. Plates were read on the Dynatech microt i ter plate reader f i t ted w i t h a 4 0 5 nm absorbance f i l ter. Serum prepared f r o m age-matched , un immunized male DBA/2 mice served as negat ive con t ro ls . 2 . 1 3 . 3 Evaluat ion of an t i -OVA IgG ant ibody spec i f ic i ty To assess the speci f ic i ty of the serum IgG ant ibodies raised against O V A , an t ibody inhib i t ion studies were pe r fo rmed. Pooled sera (final d i lut ion = 1:100) prepared f r o m cont ro l or Photofr in®-treated mice were incubated overn ight at 4 ° C in PBS conta in ing 2 % FCS, 0 . 0 5 % T w e e n 2 0 in the presence of d i f ferent concen t ra t ions ( 0 - 1 0 0 / /g/ml) of either O V A or human serum a lbumin (HSAlb, Sigma A - 8 7 6 3 ) as compet i to r prote ins. These serum mix tu res were added to microt i ter plates pre-coated w i t h O V A and levels of unbound IgG ant i -OVA ant ibodies de termined by the regular ELISA. Results are expressed as the % inhib i t ion of the result obta ined in the cont ro l assay wel ls conta in ing ant iserum but w i t h no added compet i to r p ro te in . 2 . 1 4 Character izat ion of the LR-1 ant igen 2 . 1 4 . 1 Tissue d is t r ibut ion and co-express ion studies Single and double- label an t ibody studies of normal t h y m u s , BM and spleen cells w e re per fo rmed to determine the d is t r ibut ion of the LR-1 ant igen w i th in 65 the immunohematopo ie t i c s y s t e m . Co-expression of the LR-1 ant igen w i t h CD3, C D 4 , C D 8 , T h y - 1 . 2 , C D 4 5 R - B 2 2 0 , I g M , C D 1 1 b , G r - 1 , C D 7 1 , and TER-119 ant igens w a s evaluated by using d i rect ly labelled FITC- or PE-linked monoclonal ant ibodies (Table 2 .1 ) . Either PE- or FITC labelled ant i - rat IgM (Table 2.2) were used to counter- label cell sur face bound L R - 1 . Cells were analyzed by t w o - c o l o u r FACS analysis on the Coul ter XL f l o w cy tomete r . On the basis of a t issue d is t r ibut ion suggest ive of t ha t for heat-stable ant igen (HSA) , normal spleen cells were double-labelled w i t h d i f fe rent leukocy te ant igen-speci f ic ant ibodies in parallel w i t h either LR-1 or the HSA-speci f ic monoc lona l an t ibody J 1 1 d and co-express ion of these markers w a s analyzed by t w o colour f l o w cy tomet r i c analysis. Double- label l ing studies of spleen cells obta ined f r o m solvent and Photofr in®-injected mice w e re also per formed using the LR-1 monoc lona l ant ibody and a panel of an t i - leukocyte reagents. In add i t ion , the express ion of the LR-1 and HSA (J 11 d and M 1 / 6 9 ) ant igens on pur i f ied, normal DBA/2 peripheral blood e ry th rocy tes w a s eva lua ted . Fur thermore, the sur face express ion of the LR-1 ant igen on normal D B A / 2 splenic DC, and rest ing and act iva ted splenic B cells was assessed. To assess the speci f ic i t ies of the d i f fe rent ant i -HSA monoc lona l ant ibodies for spleen cell ant igenic de te rminants , an t ibody inhib i t ion studies were pe r fo rmed . Normal spleen DBA/2 spleen cells were incubated w i t h 10 fjg of unlabel led rat IgM or the rat IgM ant ibodies LR-1 or J 1 1 d for one hour on ice. Cells were w a s h e d w i t h FACS buf fer and either M1 /69 -F ITC or an FITC-labelled ant i -M H C Class I monoc lona l an t ibody added for an addi t ional 3 0 minu tes . Levels of cell 6 6 sur face an t ibody w e re determined by f l o w c y t o m e t r y . To fu r ther invest igate the target speci f ic i t ies of these three ant i -HSA monoc lona l ant ibod ies, normal spleen cells we re labelled w i t h LR-1 in the presence or absence of ei ther J 1 1 d or M 1 / 6 9 and the cell f luorescence profi le de te rmined. 2 . 1 4 . 2 Pur i f icat ion of d i f ferent leukocytes for LR-1 ant igen express ion studies 2 . 1 4 . 2 . 1 Peripheral b lood e ry th rocy tes To prepare blood e ry th rocy tes , 1 ml of b lood obta ined f r o m naive male DBA/2 mice w a s added to 0 . 4 ml of sod ium ci t rate and then fu r ther di luted to 10 ml w i t h RPMI 1 6 4 0 med ium (Gibco BRL) conta in ing 5 % FCS. The mix ture was carefu l ly layered over Lympholy te®-M and cent r i fuged for 10 minu tes , as descr ibed above. The superna tan t , mononuc lear cell in ter face and granu locy t ic bu f fy coat were p ipet ted o f f . The red cell pellet w a s washed three t imes w i t h med ium and then examined under the mic roscope. The preparat ion conta ined on ly e ry th rocy tes . Cells prepared in th is fash ion were subsequent ly stained for their sur face expression of HSA. 2 . 1 4 . 2 . 2 Splenic dendr i t ic cells (DC) To fur ther survey the d is t r ibut ion of the ant igen recognized by the LR-1 monoc lona l an t ibody , DC were isolated f r o m the spleens of normal male DBA/2 mice using an establ ished techn ique (Swiggard et al., 1 9 9 2 ) . This method util izes col lagenase (Sigma C-0130) d igest ion to opt imize release of DC f r o m splenic t issue and cent r i fuga l f rac t ionat ion in dense BSA (Sigma A - 9 4 1 8 ) to enr ich for low 67 buoyan t dens i ty cel ls, shor t t e r m adherence on plast ic to remove contaminat ing l y m p h o c y t e s , and an overn igh t cul ture in order to separate prev iously adherent DC f r o m adherent macrophages . DC and macrophages are init ial ly p last ic-adherent but DC become non-adherent upon overn igh t cu l ture . DC were character ized by f l o w cy tomet r i c analysis for their sur face expression of M H C Class I, M H C Class II, C D 8 0 (B7-1) , DEC-205 , heat stable ant igen (HSA) , CD45R (B220) , C D 1 1 b , and C D 5 4 ( intercel lular adhesion m o l e c u l e - 1 , ICAM-1) (Table 2 .2 ) . In add i t ion , the abi l i ty of these cells to s t imulate al logeneic (DBA/1) mouse T cells w a s assessed in vitro by the mixed leukocy te react ion (MLR). 2 . 1 4 . 2 . 3 Splenic B cells B l y m p h o c y t e s were part ial ly pur i f ied f r o m ery th rocy te -dep le ted DAB/2 spleen cell suspens ions. Adheren t cells were removed by a one hour incubat ion on plastic at 3 7 °C . T l ymphocy tes were removed by t reat ing the cells w i t h the rat an t i -Thy -1 .2 monoc lona l an t ibody 3 0 - H 1 2 (Ledbetter et al., 1 9 8 0 ) , suppl ied by Pharmingen ( # 0 1 0 1 1 D ) at 2 5 / /g /ml for 3 0 minutes on ice. L o w - t o x guinea pig comp lemen t (0 .5 ml) (Cedarlane CL4051) w a s added for an addi t ional 2 hours. Cells were then w a sh e d extens ive ly w i t h med ium. The resul tant cell preparat ion w a s ~ 8 0 % B cells as indicated by their labell ing w i t h the an t i -B220 ant ibody RA3-6B2 . A c t i v a t i o n of the B cel l -enr iched popula t ion w a s execu ted by cu l tur ing these cells at 1 x 1 0 6 / m l in RPMI 1 6 4 0 , 5 % FCS, and 5 x 10" 5 M 2-ME in the presence of LPS (5 / /g /ml) for 3 days at 3 7 ° C , 5 % C 0 2 in 1 0 0 x 3 3 m m po lys ty rene petr i d ishes. A f te r 3 days , v i r tual ly all of the cu l tured cells were B 2 2 0 + . Cells were 6 8 evaluated for their express ion of M H C Class II, C D 2 5 , C D 8 0 markers and the LR-1 ant igen on days 0 and 3 by f l o w cy tomet r i c analysis 2 . 1 4 . 2 . 4 Splenic T cells T l y m p h o c y t e s were isolated by passing e ry th rocy te -dep le ted spleen cell suspens ions prepared f r o m normal DAB/1 mice over Type 2 0 0 L ny lon scrubbed w o o l (Dupont Canada) co lumns . Co lumns were washed and equi l ibrated w i t h w a r m (37 °C) RPMI 1 6 4 0 med ium conta in ing 5 % FCS for 9 0 minu tes . Spleen cells were in t roduced into the co lumn and a l lowed to enter the w o o l . The co lumn w a s topped o f f w i t h m e d i u m and the lower out le t c losed. Fo l lowing one hour at 3 7 ° C , cells were s low ly e luted w i t h med ium and the T cel l -enr iched f rac t ion ( 1 5 - 2 0 ml) co l lec ted . The cells w h i c h passed over t h rough the co lumn were ~ 8 5 % C D 3 + and ~ 1 0 % B 2 2 0 + cells (B l ymphocy tes ) . T cells prepared in th is fash ion were used as responders to al logenic DC in MLR cu l tures . 2 . 1 4 . 3 Evaluat ion of g lycosy l -phosphat idy l inos i to l (GPI)-l inkage The nature of the l inkage of the LR-1 ant igen to the cell sur face w a s assessed by incubat ing 1 0 7 spleen cel ls, obta ined f r o m normal mice, or mice injected w i t h 5 % dext rose or Photofr in®, in serum-f ree I M D M w i t h o u t or w i t h phosphoinos i to l -spec i f ic phosphol ipase C f r o m Bacillus thuringiensis (PI-PLC, ICN Biochemicals 1 5 2 3 5 4 , Cleveland OH) at either a 1:50 or 1 :100 d i lu t ion ( 0 . 1 3 3 or 0 . 2 6 6 U/ml) for one hour in a 3 7 ° C wa te r bath (Liu et al., 1 9 9 2 ) . Cells were 69 w a s h e d t w i c e w i t h PBS and then stained w i t h monoc lona l ant ibodies react ive w i t h L R - 1 , HSA, C D 4 , T h y - 1 . 2 or I g M . HSA (Pierres et al., 1987) and Thy-1 (Tse etal., 1 9 8 5 ; L o w and Kincade, 1985) are GPI-l inked pro te ins , whereas CD4 (Tourviel le et al., 1986) and IgM (Reth, 1992) associate w i t h the cell membrane th rough prote in t rans -membrane domains . 2 . 1 4 . 4 W e s t e r n b lot analysis Membrane ex t rac ts of t h y m u s , BM and spleen cells were prepared by lysing single cell suspens ions at 1 x 1 0 7 / m l in a solubi l izat ion buf fer consis t ing of 1 % Nonidet P-40 (BDH), 2 0 m M Tr is-HCl , pH 7 .4 , 1 5 0 m M NaCl, 2 m M EDTA and 1 m M pheny lme thy l ysu l fony l f luor ide (PMSF, Sigma P7626) on ice (Food et al., 1 9 9 4 ) . Cells w e re cent r i fuged at 1 4 , 0 0 0 x g for 3 0 minutes at 4 ° C . Supernatants were co l lec ted and stored at - 7 0 ° C unt i l required. Protein con ten t of each sample w a s de termined w i t h the B C A ™ Protein Assay Reagent (Pierce, Rock fo rd , IL). Cell ex t rac ts (4 -10 fjg protein per lane) were f rac t ionated by sod ium dodecy l su lphate po lyacry lamide gel e lect rophoresis (SDS-PAGE) on 8 . 5 % minigels under reduc ing cond i t ions (Laemml i , 1970) using a Biorad 2 5 0 / 2 . 5 power supply . Molecular w e i g h t s tandards (BSA, 6 6 kDa: A - 7 5 1 7 ; O V A , 4 3 . 5 kDa: A - 7 6 4 2 ; carbonic anhydrase, 2 9 kDa: C -2273 and RNAse, 14.1 kDa, Sigma) were run (10 /jg per marker) in parallel in the same gel . Gels were run at 2 2 m A for 9 0 minutes and then immersed in the t ransfer buf fer (25 m M Tr is-HCl ; pH 8 . 3 , 192 m M g lyc ine, 2 0 % methanol ) for 3 0 minutes at room tempera tu re . Separated proteins were e lec t rob lo t ted (75 V) on to 0 .2 fj pore size n i t rocel luose paper (TM-NC2-RL-15 ; 7 0 Hoeffer Scient i f ic Ins t ruments , San Francisco, CA) for 3 .5 hours at 4 ° C in t ransfer buf fer . Lanes conta in ing the molecular w e i g h t s tandards were cu t o f f and these prote ins visual ized by sta in ing the str ips in a so lut ion of 1 0 % acet ic acid and 4 5 % methano l conta in ing 0 . 1 % (w/v) amido black (BDH). A f te r 6 0 minutes the str ips were desta ined in a so lut ion of 9 0 % methano l , 2 % acet ic acid and then placed in dist i l led wa te r . For immunos ta in ing , ni t rocel lu lose w a s b locked in a solut ion of PBS conta in ing 2 % sk im milk p o w d e r overn igh t at 4 ° C . Monoc lona l ant ibodies L R - 1 , J 1 1 d or the IgM isotype cont ro l (6 / /g/ml) in PBS, 2 % sk im milk p o w d e r were added for 3 .5 hours at 4 ° C on an orbi tal shaker. Str ips were washed t w i c e w i t h dist i l led wa te r and goat alkaline phosphatase-con jugated ant i -rat IgM ant ibody (final concen t ra t ion = 1.2 / /g/ml) w a s added for an addi t ional 1.5 hours at room tempera tu re w i t h shak ing . Blots were washed w i t h dist i l led wa te r four t imes and placed in a so lu t ion conta in ing 100 m M Tris (pH 9 .5 ) , 1 0 0 m M NaCl, 5 m M M g C I 2 , 5 -bromo-4-ch loro-3- indo ly l phosphate (BCIP, 165 / / g / m l ; UBI) and nitro blue te t razo l ium ( 3 3 0 / / g / m l ; UBI) for approx imate ly 3 0 minutes at room tempera ture . Colour deve lopmen t w a s te rminated by wash ing the str ips in dist i l led wate r . 2 . 1 5 Data presenta t ion and stat is t ical analysis Exper imental results are g iven as ar i thmet ic means w i t h s tandard dev ia t ions (SD) of the mean for each t rea tmen t g roup . Sample var iances and means were stat is t ica l ly compared using the F-test and S tuden t ' s f - test , respect ive ly , using the CoStat stat is t ical analysis p rogram (CoHort S o f t w a r e , Berkeley, CA) . 71 CHAPTER 3 RESPONSE OF THE INTACT MURINE HEMATOPOIETIC SYSTEM TO PHOTOSENSITIZING PORPHYRINS 3.1 In t roduc t ion To establ ish w h e t h e r d i f ferent porphyr ins w i t h photosensi t iz ing act iv i ty migh t produce hematopo ie t ic e f fec ts in the absence of ac t iva t ing l ight , the spleen served as the pr imary indicator of th is ac t iv i ty . The normal mouse spleen has a hematopo ie t ic role in addi t ion to its immunolog ic func t ions . Splenic erythropoies is and myelopoies is are readily demonst rab le . In add i t ion , mos t cy tok ines w i t h hematopo ie t ic ac t iv i ty have been s h o w n to produce changes in the hematopoiet ic s ta tus of the sp leen. The organ can be easily exc ised, its w e i g h t and to ta l cell numbers rel iably de te rm ined , and hematopo ie t ic progeni tor ac t iv i ty assessed. There fore , as a measure of the act ion of a porphyr in , changes in the w e i g h t and cel lular i ty of the spleen 7 days after a single in ject ion of the preparat ion served to ident i fy hematos t imu la to ry c o m p o u n d s . To fur ther evaluate these changes, hematopo ie t ic progeni tor (CFU-GM, BFU-E, and CFU-E) assays and FACS analyses were p e r f o r m e d . To more broadly def ine the hematopo ie t ic ac t iv i ty o f cer ta in porphyr ins , the BM w a s also eva luated. 3 .2 Results 3 .2 .1 Splenic response to photosensi t iz ing porphyr ins A range of doses (10 - 5 0 mg/kg) o f the porphyr in photosensi t izer Photofrin® adminis tered i.p. s igni f icant ly elevated spleen w e i g h t and cel lular i ty 7 2 above t h a t o f t he so lvent - in jected cont ro ls by 7 days post- in ject ion (Table 3.11). A t th is t i m e , the spleens of Photofr in®-treated mice were v is ib ly enlarged and deep red in co lour (Figure 3 .1 ) . A t lower doses (2 .5 or 5 m g / k g ) , Photofrin® did no t produce s ign i f icant changes in the spleen parameters examined . W h e n Photofr in® (10 or 25 mg/kg) w a s g iven i.v., a s igni f icant increase in splenic cel lular i ty w a s observed , a l though the increase in spleen w e i g h t p roduced by these doses w a s not s tat is t ica l ly s ign i f icant (Table 3.1 II). In cont ras t , w h e n examined at the day 7 sampl ing t ime , nei ther BPD nor Hp had s igni f icant ly altered spleen w e i g h t or cel lu lar i ty (Table 3 . I l l , Iv) . In add i t ion , no change in spleen we igh t occur red in mice g iven d i f fe ren t doses of PPIX (Table 3 . 1 V ) . Figure 3.1 Inf luence of Photofrin® on spleen size and appearance. Spleens obta ined f r o m mice administered either 5 % dex t rose (left) or Photofr in® (25 mg/kg ) (r ight) seven days previously are dep ic ted . The w e i g h t o f the por t rayed organs w e re 9 2 mg and 1 3 0 mg for the so lvent and Photofr in®-injected mice respect ive ly . Sp leen /bodywe igh t ratios (x 100) determined for these mice cor responded to values of 0 . 3 8 2 and 0 . 5 0 2 . 73 Table 3 .1 Spleen parameters of mice adminis tered d i f fe rent photosensi t iz ing porphyr ins . Spleen w e i g h t s and nucleated cell numbers we re determined for male DBA/2 mice admin is tered the appropr iate cont ro l so lvent , Photofr in®, BPD, Hp, or PPIX 7 days prev ious ly . Mean values ± SD are s h o w n . Results obta ined w i t h porphyr in- in jec ted mice w e re compared stat is t ical ly w i t h the resul ts obta ined w i t h the so lvent - in jec ted con t ro ls . Test Series Preparation Dose (mg/kg) Route n Relative spleen weight (x 1 0 0 ) Total nucleated spleen cells (x 1 0 6 / 2 0 g) I. 5% dextrose 0 i.p. 63 0.374 ± 0.069 39.74 ± 7.54 Photofrin® 2.5 i.p. 3 0.401 + 0.037 45.44 ± 10.20 Photofrin® 5.0 i.p. 3 0.395 ± 0.048 43.76 ± 4.36 Photofrin® 10 i.p. 11 0.516 ± 0.126* 60.34 ± 1.16** Photofrin® 25 i.p. 62 0.563 + 0.116** 65.48 ± 26.41** Photofrin® 50 i.p. 4 0.579 ± 0.104** 67.52 ± 12.72** II. 5% dextrose 0 i.v. 4 0.419 + 0.045 43.43 ± 6.20 Photofrin® 10 i.v. 4 0.474 ± 0.061 60.50 ± 9.94 + Photofrin® 25 i.v. 4 0.499 + 0.097 61.42 ± 7.80 + III. 5% DMSO/PBS 0 i.v. 4 0.331 ± 0.060 39.62 ± 2.38 BPD 10 i.v. 4 0.405 ± 0.051 45.64 ± 7.00 IV. Solvent B 0 i.p. 3 0.343 ± 0.025 30.88 ± 5.45 HP 25 i.p. 3 0.369 ± 0.028 32.45 ± 2.56 V. Solvent C 0 i.p. 3 0.334 + 0.048 nd PPIX 2.5 i.p. 3 0.382 ± 0.076 nd PPIX 5 i.p. 3 0.362 ± 0.045 nd PPIX 10 i.p. 3 0.425 ± 0.103 nd PPIX 25 i.p. 3 0.372 ± 0.022 nd , P< 0 . 0 5 ; * , P< 0 . 0 1 ; * * , P < 0 . 0 0 1 compared to the value for spleen cells obta ined f r o m so lvent - in jected cont ro l mice, nd = not done. 7 4 Mice mon i to red over a 7 day period after a single i.p. in ject ion of Photofrin® (25 m g / k g ) , exh ib i ted s igni f icant increases in spleen we igh t and cel lu lar i ty , t h a t w e re f i rs t d iscernable by day 2 post - in jec t ion . These at t r ibutes increased fu r ther unt i l day 7 post - in ject ion (Figure 3 . 2 A , B). A s igni f icant increase in spjenic CFU-GM levels w a s noted by day 2 post - in ject ion and th is ac t iv i ty w a s max imal at day 4 , then tapered s o m e w h a t , but remained s ign i f icant ly elevated above con t ro l va lues at day 7 post - in ject ion (Figure 3 .2C) . Concomi tan t w i t h these changes w a s an e levat ion in spleen metabol ic ac t iv i ty by day 4 post - in ject ion as assessed by the M T T color imetr ic assay. Spleen cell M T T dye reduct ion act iv i ty w a s also s ign i f icant ly e levated above cont ro l values at day 7 post - in ject ion in 2 of 3 exper iments , but w h e n the data f r o m these 3 exper iments w a s combined th is overal l level w a s not stat is t ical ly d i f ferent f r o m tha t of the cont ro ls (Figure 3 .2D) . Spleen e ry th rocy te numbers were also increased by Photofr in®, produc ing an approx imate doub l ing in the number of these cells by day 4 post - in ject ion and cell numbers remained s igni f icant ly e levated at the day 7 sampl ing t ime (Figure 3.2E). W h e n the impact of Photofrin® at doses of 10 or 25 m g / k g on myelo id progeni tors w a s c o m p a r e d , a stat is t ical ly s igni f icant 2-3 fo ld increase in levels of splenic CFU-GM w a s ev ident w i t h bo th doses at the day 3 and 7 sampl ing t imes, a l though the 2 5 m g / k g dose produced a s t ronger response at bo th of these days (Figure 3 .3 ) . The character is t ic appearance of splenic CFU-GM w h i c h f o r m in vitro is s h o w n in Figure 3 . 4 . In unt reated and solvent- in jected cont ro l mice, CFU-GM occur red w i t h an approx imate f requency of of 1 CFU per 1 0 , 0 0 0 spleen cells p la ted. 7 5 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 01 2 3 4 5 6 7 Days post-injection Figure 3 .2 T ime course of the splenic response to Photofrin®. Spleen parameters of male DBA/2 mice fo l l ow ing the in ject ion of 5 % dext rose ( • ) or Photofr in® (25 m g / k g , O ) are summar ized . The presented data we re comb ined f r o m a large number of indiv idual exper iments . Changes in (A) relat ive spleen w e i g h t (n = 15 -63 per g roup/sampl ing t ime) , (B) spleen nucleated cell numbers (n = 15 -63 per g roup/sampl ing t ime) , and (C) levels of CFU-GM (n = 7-15 per g roup /sampl ing t ime) are s h o w n . Spleen cell M T T act iv i ty (D) and e ry th rocy te numbers (E) were assessed in 3 m ice /g roup /day except on days 4 and 7 post - in jec t ion at w h i c h t ime 9 mice /group were analyzed. Day 0 values for spleen w e i g h t and spleen cel lular i ty were determined for 2 4 unt reated male DBA/2 mice mainta ined under cond i t ions ident ical t o those of the exper imenta l animals. f P < 0 . 0 5 ; * P< 0 . 0 0 0 1 compared to the result obta ined w i t h solvent- in jected con t ro l m ice . 7 6 Figure 3 . 3 Compar ison of t w o d i f ferent Photofrin® doses on splenic CFU-GM levels. Tota l numbers of splenic CFU-GM were determined for mice injected w i t h 5 % dex t rose ( • ), or Photofrin® at either 10 (cross-hatched) or 25 ( • ) m g / k g either 3 or 7 days prev ious ly . Values have been normal ized to a b o d y w e i g h t of 2 0 g rams. T rea tment groups cons is ted of 12 -15 mice at each sampl ing t ime , except for the group g iven the 10 m g / k g dose of Photofrin® w h i c h conta ined either 3 or 5 mice. f P < 0 . 0 5 ; * P < 0 . 0 1 ; * * P < 0 . 0 0 5 compared to the result obta ined w i t h spleen cells prepared f r o m mice injected w i t h 5 % dex t rose . 7 7 Figure 3 .4 Morpho log i c appearance of splenic CFU-GM. Typical appearance of a splenic CFU-GM cell cluster which develops over a 7 day period after the introduction of spleen cells into a semi-solid agarose culture system containing serum-rich medium and PWM-SCCM as a source of growth factors (magnification x 250). 78 3 . 2 . 2 Evaluat ion of the in vitro CFU-GM progeni tor tes t sys tem A n exper iment w a s per formed in order to determine the ident i ty of the cells t ha t w e re synthes ized w i th in the CFU-GM tes t s y s t e m . Equal numbers of nuc leated spleen cells obta ined three days after the in ject ion of 5 % dext rose or Photofrin® (25 mg/kg) were cu l tured in bulk in the presence of P W M - S C C M for 7 days . Agar w a s omi t ted f r o m th is sys tem to ensure a comple te recovery of the cells af ter cu l tu re . FACS analysis per fo rmed of f reshly isolated cells and cells obta ined after the cu l ture indicated tha t the sys tem p romoted an expans ion in the number of cells w h i c h expressed the macrophage ant igen F 4 / 8 0 , the g ranu locy te marker G r - 1 , or the g ranu locy te -macrophage sur face receptor C D 1 1 b , but not the B cel l-rest r ic ted ant igen B 2 2 0 or M H C Class II molecules (Figure 3 .5 ) . Numbers of CD71 + cells also increased w i t h i n th is cul ture s y s t e m . Interest ingly , numbers of T h y - 1 . 2 + cells we re increased w i t h i n th is s y s t e m . Greater cell numbers and yields of F4 /80 + , Gr-1 + , CD11 b + , CD71 + , and T h y - 1 . 2 + cells were obta ined in cu l tures ini t iated w i t h spleen cells f r o m Photofr in®-injected mice. However , w i t h the except ion of Thy-1 . 2 + cel ls, none of these d i f ferences were stat is t ical ly s ign i f icant . W h e n the percentage increase (cell yield / cells added x 100) in cell numbers w a s cons idered, spleen cell numbers f r o m Photofr in®-treated animals were increased 170 .8 ± 3 9 . 8 % as compared to 1 1 9 . 8 ± 1 9 . 4 % for the cu l tured cells of the cont ro l mice. Thus , the cu l ture of cells f r o m cont ro l and Photofr in®-injected mice in the presence of PWM-cond i t i oned med ium promoted the expans ion of cells bearing myelo id ant igens. T h e expans ion in the number of cu l tured cells w h i c h expressed Thy-7 9 1.2, an ant igen character is t ica l ly present on t h y m o c y t e s and mature T cel ls, is unl ikely to be associated w i t h lymphopoies is . It has been s h o w n tha t Thy-1 appears early in cu l tures ini t iated by normal mouse bone m a r r o w cells depleted of T cells and conta in ing m e d i u m cond i t ioned by Con A-ac t i va ted T cells or a mye lomonocy t i c cell line (Schrader et al., 1 9 8 2 ) . These f ind ings indicated tha t Thy-1 w a s present on d i f fe rent hematopo ie t ic p rogen i to rs , s tem cells and deve lop ing myelo id cells (Schrader et al., 1 9 8 2 ) . There fore , the increased expression of the T h y - 1 . 2 ant igen w i th in the l iquid cu l ture sys tem w a s likely related to myelo id cell d i f fe rent ia t ion . 3 . 2 . 3 In f luence of Photofr in® and Hp on splenic ery thropo ie t ic ac t iv i ty Evaluat ion of splenic ery thro id progeni tors ind icated tha t levels of CFU-E were increased above cont ro l values in Photofr in®-treated mice over 30- fo ld at day 4 and by 20- fo ld at day 7 post - in ject ion (Figure 3 . 6 A ) . Morpholog ica l appearance of CFU-E fo rmed in vitro using spleen cells f r o m cont ro l or Photofrin®-injected mice are s h o w n in Figure 3 . 7 A and exhib i t the increased f requency of CFU-E in cu l tures ini t iated by spleen cells f r o m Photofr in®-treated animals. Levels of splenic BFU-E w e re e levated 5-fold at day 4 and 10- fo ld at day 7 post- in ject ion w i t h Photofr in®, a l though these increases were stat is t ical ly s ign i f icant only for the day 7 sampl ing t ime (Figure 3 .6B) . Hp w a s evaluated in parallel w i t h Photofrin® but in con t ras t , p roduced no change in levels of splenic CFU-E or BFU-E (Figure 3.6 A , B). The appearance of splenic BFU-E w h i c h f o r m in vitro and stained w i t h acidic benzidine for the presence of hemoglob in is s h o w n in Figure 3 .7B. 8 0 CD a* CD o a* co ~CD O Total Cells Thy1.2 CD45R (B220) MHC Class II F4/80 Gr-1 (Ly-6G) CD11b CD71 U UJ H TTTTTT te LTJJH r -I I I l-H 0 10 15 20 25 30 35 40 45 Cell yield (x 10°) Figure 3 . 5 Expansion of spleen cells w i t h P W M - S C C M in a l iquid cu l ture sys tem. Est imates of to ta l cell numbers and express ion of var ious leukocyte ant igens before and after a 7 day cul ture of 2 0 x 1 0 6 sp lenocy tes in the presence of P W M - S C C M are presented. Spleen cells obta ined f r o m mice (n = 4 per group) t ha t had been in jected 3 days before w i t h 5 % dext rose ( • ) or Photofrin® ( • ) were seeded at 5 x 1 0 5 / m l in 4 0 ml of m e d i u m . The cu l ture med ium w a s identical to t ha t emp loyed in the CFU-GM progeni tor assay, excep t for the omiss ion of agar. F low c y t o m e t r i c analyses were per formed on f reshly isolated spleen cells and on the cells w h i c h w e re recovered f r o m the f lasks af ter the 7 day cu l tu re . Post-cul ture resul ts are presented as c ross-hatched bars for so lvent - in jected and vert ical line bars for Photofr in®-treated mice. 81 A 60 B 50 CVJ X CO 50 40 30 20 10 0 40 30 x -3 20 u_ O 10 0 Days post-injection Figure 3.6 In f luence of Photofr in® and Hp on splenic e ry thro id progeni tor levels. Levels of ( A ) BFU-E and (B) CFU-E were determined for spleen cells obta ined 4 and 7 days fo l l ow ing the i.p. in ject ion of ei ther 5 % dext rose ( • ) , Photofr in® (25 m g / k g , • ) , so lvent B ( • ) or Hp ( • , 25 m g / k g ) . Three mice per group w e re evaluated at each sampl ing t ime . In the spleens of so lvent- in jected cont ro l m ice , CFU-E occur red w i t h an approx imate f requency of 1 per 2 , 8 0 0 spleen cells plated wh i le splenic BFU-E were present at an approx imate f requency of 1 per 7 4 , 0 0 0 spleen cells p la ted. * P < 0 . 0 5 ; * * P < 0 .01 compared to the result obta ined for spleen cells obta ined f r o m mice g iven the appropr ia te ly -matched so lvent . 8 2 Figure 3.7 Morpho log ic appearance of splenic CFU-E and BFU-E. The appearance of a number of CFU-E w h i c h fo rmed in vitro w i th in 4 8 hours in methy lce l lu lose-based co lony assays in the presence of rEpo (3 U/ml) is dep ic ted (magni f ica t ion x 2 5 0 ) . Cul tures were ini t iated w i t h spleen cells obta ined f r o m mice g iven (A) 5 % dext rose or (B) Photofrin® 4 days prev ious ly . Appearance of a splenic BFU-E (C) w h i c h fo rmed in vitro over a 10 day cu l ture period in the presence of P W M - S C C M and rEpo is also s h o w n . BFU-E were stained for the presence of hemog lob in w i t h an acidic benzidine solut ion (magni f ica t ion x 100) . 8 3 Figure 3.7 A. CFU-E (Control) B. CFU-E (Photofrin®) 3 . 2 . 4 F low cy tomet r i c analysis of spleen cell ant igens Evaluat ion of a w ide var ie ty of sur face ant igens present on spleen cells obta ined 7 days post - in ject ion indicated tha t Photofrin® did not produce any s ign i f icant change in the number of cells bearing T l y m p h o c y t e (CD3, CD4, CD8) or B l y m p h o c y t e (CD45R-B220 , lgM)-rest r ic ted markers , or the l ymphocy te ac t iva t ion ant igen C D 2 5 (Figure 3.8 A ) . In add i t ion , no s ign i f icant d i f ference b e t w e e n the number of spleen cells w h i c h expressed macrophage-assoc ia ted F 4 / 8 0 , CD11 b, or M O M A - 2 prote ins, the granu locy te marker Gr-1 (Ly-6G) or MHC Class II ant igens w a s observed for cont ro l and Photofr in®-treated mice at th is sampl ing t ime . H o w e v e r , s igni f icant increases in the number of spleen cells w h i c h expressed the B cell ant igen L R - 1 , HSA (as recognized by the monoclonal ant ibodies J 1 1 d and M 1 / 6 9 ) , the ery th rocy te- res t r i c ted var iant of HSA (as labelled by M 1 / 7 5 ) , the ery thro id TER-119 marker , the t ransfer r in receptor CD71 (as recognized by the monoc lona l ant ibodies C2F2 and R17 2 1 7 . 1 . 3 ) , and C D 4 5 , were ev ident af ter Photofr in® admin is t ra t ion . Photofr in® s igni f icant ly increased the toal number and overall percentage of spleen cells w h i c h labelled w i t h the LR-1 monoc lona l ant ibody [cont ro ls = 5 9 . 7 ± 9 . 9 % LR-1 + (n = 2 2 ) ; Photofr in®-injected = 6 7 . 3 ± 7 . 8 % LR-1 + (n = 2 0 ) ; P < 0 . 0 1 ] . Converse ly , the t rea tmen t decreased the propor t ion of nuc leated spleen cells w h i c h expressed the leukocy te c o m m o n ant igen CD45 [cont ro ls = 8 8 . 7 ± 5 . 7 % C D 4 5 + (n = 3 ) ; Photofr in®-injected = 7 4 . 7 ± 3 . 5 % C D 4 5 + (n = 3 ) ; P< 0 . 0 2 5 ] or M H C Class I ant igens [ (contro ls = 9 3 . 8 ± 1 . 1 % M H C Class l + (n = 3 ) ; Photofr in®-injected = 7 9 . 9 ± 7 . 6 % M H C Class l + (n = 3 ) ; 8 5 P< 0 . 0 5 ] con t r ibu t ing to a s ign i f icant expans ion in the size of the CD45" 0 and MHC Class l " / l 0 spleen cell popula t ions (Figure 3 .8B) . FACS labell ing prof i les for LR-1 , M H C Class I and C D 4 5 ant igens are presented in Figure 3 .8C) . The LR-1 labell ing prof i le of spleen cells f r o m cont ro l mice character is t ica l ly compr ised of a single LR-1 + peak w i t h a minor , more br ight ly staining shoulder , whereas spleen cells f r o m Photofr in®-injected mice exhib i ted a d is t inc t b imodal LR-1 sta in ing pat tern (Figure 3 .8C) . In an exper iment per formed to evaluate the impac t of d i f ferent Photofr in® doses on the character of the splenic response, the preparat ion was adminis tered i.p. at ei ther 10 , 2 5 , or 5 0 m g / k g . FACS analysis w a s per formed on spleen cells prepared on day 7 post - in jec t ion. Each Photofrin® dose produced s ign i f icant increases in spleen w e i g h t and cel lu lar i ty, and highly similar monoclonal an t ibody sta in ing pat terns (Table 3 .2 ) . Spleen cells f r o m Photofr in®-injected mice were compr ised of propor t ional ly f e w e r CD3 + , C D 4 + , CD8 + , and C D 4 5 R - B 2 2 0 \ CD25 + , and F 4 / 8 0 + cel ls, bu t greater numbers of LR-1 + cells than those seen in the so lvent - in jec ted mice. These studies suggested tha t the spleen has a l imited capac i ty to respond to Photofrin® and the h ighest dose (50 mg/kg) g iven did not produce a splenic hematopo ie t ic response any greater than tha t p roduced by a dosage 5 fo ld lower . In add i t ion , none of these t rea tments increased the relat ive f requency of l ympho id l ineage cells w i th in the spleen. 8 6 Figure 3 .8 Summar ized spleen cell sur face ant igen express ion s tudies. ( A ) Expression of d i f fe rent leukocyte d i f fe rent ia t ion ant igens was determined for nucleated spleen cells obta ined 7 days fo l l ow ing the adminis t rat ion of ei ther 5 % dext rose ( • ) or Photofrin® (25 m g / k g , • ) by single label FACS analysis. For presentat ion purposes, cell sur face markers have been al located to d i f fe rent leukocy te g roups , a l though some markers may not be restr ic ted to a single l ineage, as wel l as some ant igens may be present on cells of non-hematopo ie t ic or ig in . W h e r e more than one monoc lona l an t ibody w a s uti l ized for a marker, the c lone des ignat ion is g iven in parentheses. Results s h o w n for each sur face ant igen were obta ined w i t h spleen cells f r o m 3-22 mice per t rea tmen t g roup . Mean ( + SD) numbers of ant igen posi t ive spleen cells (normalized to a b o d y w e i g h t of 2 0 grams) are s h o w n . (B) Est imates of numbers of nucleated spleen cells w h i c h express l ow or undetec tab le (designated -/lo) amounts of the leukocy te c o m m o n ant igen (CD45) or M H C Class I ant igens are g iven . (C) F low cy tomet r i c prof i les for C D 4 5 , M H C Class I, and LR-1 ant igens as expressed by nucleated spleen cells prepared 7 days after the admin is t ra t ion of the con t ro l so lvent or Photofrin® are s h o w n . Thin t rac ings cor respond to results obta ined w i t h iso typ ica l ly -matched cont ro l ant ibodies or cells alone (MHC Class I resul ts) . r P < 0 . 0 5 ; * P < 0 . 0 2 5 ; * * P < 0 . 0 0 1 by S tuden t ' s t - tes t as compared to the result obta ined for spleen cells f r o m solvent- in jected cont ro l mice 8 7 Figure 3.8 A ca S O C D C D as C D o C O " C D o B CD o O Q • o " o <D ca o ca CD45 MHC Class I Thy 1.2 CD3 CD4 . CD8 CD45R/B220 IgM MHC Class II HSA(J11d) HSA(M1/69) LR-1 Gr-1 CD11b F4/80 TER-119 HSA(M1/75) CD25 CD71 (ER-MP 21) CD71 (C2F2) CD71 (R17217) CD45*W MHC Class I ** 10 20 30 40 50 Total cells (x 60 100 1000 -1 MHC Class 10 100 1000 .1 LR-1 Control Photofrin .1 1 10 100 1000 -1 1 10 100 1000 .1 1 10 100 1000 Fluorescence Intensity 8 8 Table 3 .2 Compar ison of 3 d i f ferent Photofrin® doses on spleen parameters. Spleen w e i g h t , cel lular i ty and expression of var ious leukocy te ant igens were de termined for spleen cells obta ined 7 days after the i.p. admin is t ra t ion of a range of Photofr in® doses. Each t rea tment group cons is ted of four mice. Spleen cells f r o m indiv idual mice in each group were pooled for these cell sur face labell ing s tud ies. Photofrin® dose (mg/kg) Spleen parameter 0 10 2 5 5 0 Relative weight (x 100) 0.338 ± 0.064 0.678 ± 0.084 0.583 ± 0.184 0.579 ± 0.104 t-value (6 df) Significance - 6.44 P< 0.001 2.51 P< 0.05 3.94 P< 0.01 Nucleated cells (x 106/20 g) t-value (6 df) Significance 36.1 ± 9.30 70.53 ± 3.97 6.81 P< 0.001 53.95 ± 8.06 2.90 P< 0.05 67.45 ± 12.70 3.98 P< 0.01 Cell sur face ant igen An t i gen posi t ive spleen cells (%) C D 3 17 .3 10 .2 11.1 8 .2 C D 4 15 .2 6.5 7.1 10 .2 C D 8 3.6 3 .2 2.5 1.1 C D 2 5 2.1 2 .0 0 .3 0 . 4 C D 4 5 R - B 2 2 0 4 1 . 0 3 1 . 5 2 4 . 3 2 6 . 4 F 4 / 8 0 6.6 7.1 3.7 2 .8 LR-1 6 8 . 2 7 3 . 6 6 9 . 9 7 4 . 6 8 9 3 . 2 . 5 Cell cyc le analysis The evaluat ion of spleen cell nuclei for DNA con ten t by PI staining 4 and 7 days af ter a single in ject ion of Photofrin® s h o w e d tha t a s igni f icant ly greater p ropor t ion o f spleen cells exhib i ted > 2 n amoun ts of DNA than mice g iven Hp or either con t ro l so lvent at bo th sampl ing t imes (Figure 3 .9 ) . This suggested tha t the spleens of Photofr in®-treated mice conta in increassed numbers of cells in the S, M or G 2 phases of the cell cyc le . Representat ive f l o w cy tomet r i c prof i les of cell cyc le analyses are s h o w n in Figure 3 . 1 0 . Highly similar results we re obta ined w h e n intact spleen cells obta ined at the above sampl ing t imes were analyzed for DNA con ten t using a p - fo rma ldehyde cell f i xa t ion method rather than the nuclear ex t rac t ion techn ique descr ibed herein (data not s h o w n ) . 3 . 2 . 6 Spleen cell responses to d i f ferent hematopo ie t ic g r o w t h fac tors Spleen cells obta ined 72 hours af ter the in ject ion of Photofrin® or the cont ro l so lvent w e re cu l tured in the presence of a spleen cell condi t ioned superna tan t (PWM-SCCM) or concent ra t ion gradients of a var ie ty of speci f ic g r o w t h fac to rs . Cellular responses to these fac tors were evaluated by the M T T color imetr ic assay. Spleen cells obta ined f r o m the Photofr in®-treated mice were more responsive over a t i t ra t ion range of P W M - S C C M than spleen cells f r o m the solvent- in jected cont ro ls . In add i t ion , spleen cells obta ined f r o m Photofr in®-injected mice at th is t ime responded more s t rong ly to the myelo id g r o w t h fac tors r lL-3, rG-CSF, rM-CSF, and rGM-CSF in vitro than spleen cells obta ined f r o m the cont ro l animals (Figure 3 .11 ) . In con t ras t , there w a s no d i f ference in the response of spleen cells f r o m cont ro l and 9 0 CVI A C D <_> C_> CO " C D CJ> C D C D C O 4 7 Days post-injection Figure 3.9 Cell cyc le analys is. Cell cyc le analyses were per formed on spleen cell nuclei prepared 4 or 7 days af ter the adminst ra t ion of 5 % dext rose ( • ), Photofr in® (25 m g / k g , • ), so lvent B ( • ), or Hp (25 m g / k g , • ). Percentages (%) refer to the propor t ion of cell nuclei w h i c h conta in greater than 2n amoun t of DNA and indicates tha t these cells w e re in the S, M or G 2 phases of the cell cyc le . Three mice per group were analyzed on each sampl ing day . * P < 0 . 0 2 5 ; * * P < 0 .01 compared to the result obta ined w i t h spleen cells obta ined f r o m so lvent - in jected mice. 91 100 C D CJ> o o S£ 0 . A _ B - Go/G, I i 6 ? i Go/G, 1 13'1I° I - - r H v A . ' . I , J I I i ' r"*r*r i T 1024 0 1024 100 C D T 3 CD OC c Go/G, I 5.7% | ^^^^^^ i — r — i — r 100 CJ> C D o . A " Gj/G, C D 0 ^ 100 I 0 r Y^v 'Hf . r i i i ' 1024 B - Gj/G, I 11.9% j S^ f^ i' ' T — i — i — i — r 1024 0 1024 C D C D DC . c _ D - Gj/G, I 4 ' 5 % I G„/G, i 4 , 0 % i I I i i i i i i i ^•-rS i i — I I i 1024 0 Fluorescence intensity (channel number) 1024 Figure 3 . 1 0 Cell cyc le analysis - f l o w cy tomet r i c prof i les. Cell cyc le analyses were per formed on spleen cell nuclei prepared f rom mice g iven (A) 5 % dex t rose , (B) Photofrin® (25 m g / k g ) , (C) so lvent B, or (D) Hp (25 mg/kg) ei ther (I.) 4 or (II.) 7 days before. Representat ive f l o w cy tomet r i c prof i les are s h o w n . Percentages (%) g iven w i th in each f igure refer to the propor t ion of cell nuclei w h i c h conta ined a greater than a 2n a l lo tment of DNA and hence in the S, M, or G 2 phases of the cell cyc le . The scale of the y-axis w a s reduced in order to emphasize the relat ive sizes of the > 2 n DNA por t ion of the h is tograms and cor responds to the area designated by brackets . 92 C D L O CD O CT3 O C O O crj O O " O CD CD 0.00 0.2 1 2 10 20 PWM-SCCM (%) 0.1 1.0 10 100 rlL-3 (ng/ml) 0.1 1 10 100 rM-CSF (ng/ml) 10'1 10° 101 102 103 104 rlL-2 (U/ml) 0.1 1.0 10 100 rG-CSF (ng/ml) 0.1 1.0 10 100 rGM-CSF (ng/ml) Growth factor Figure 3 . 1 1 Spleen cell responses to d i f ferent hematopo ie t ic g r o w t h fac tors . Prol i ferat ive responses of spleen cells obta ined f r o m mice injected 3 days prev ious ly w i t h 5 % dext rose ( • ) or Photofrin® (25 m g / k g , O ) cu l tured in the presence of concen t ra t ion gradients of d i f fe rent recombinant (r) g r o w t h fac tors or mur ine spleen cell p o k e w e e d mi togen-cond i t ioned m e d i u m (PWM-SCCM) are s h o w n . For r lL -2 , an ac t iv i ty of 100 uni ts /ml cor responds to a fac to r concent ra t ion of 12 .7 n g / m l . Three to four mice per tes t group were analyzed. Assay background M T T act iv i ty (cells alone) w a s subt rac ted f r o m each tes t result in order to obtain a measure of the fac tor -spec i f ic response. Contro l spec t ropho tomet r i c absorbance ( 5 9 0 nm) va lues ranged f r o m 0 . 1 0 8 to 0 . 2 5 9 and 0 . 1 0 9 to 0 . 3 4 7 for spleen cells f r o m cont ro l and Photofr in®-injected mice, respect ive ly . 93 0.10 L O rEpo (U/ml) Figure 3 . 1 2 Spleen cell responses to rEpo. Prol i ferat ive responses produced by spleen cells obta ined 4 days after the admin is t ra t ion of ei ther 5 % dext rose ( • ) or Photofrin® (25 m g / k g , O) and cu l tured for 4 8 hours w i t h a concent ra t ion gradient of rEpo are s h o w n . Three mice per t rea tmen t g roup were analyzed. Background M T T act iv i ty (cells alone) w a s subt rac ted f r o m the result obta ined w i t h rEpo in order to obta in a measure of the speci f ic cy tok ine response. M T T act iv i ty in the cont ro l cu l tures cor responded to absorbances ( 5 9 0 nm) values of 0 . 3 2 2 ± 0 . 0 7 7 and 0 . 3 2 1 ± 0 . 0 5 7 for the so lvent - in jec ted and Photofr in®-injected mice, respect ive ly . 9 4 Photofr in®-treated mice to the l y m p h o c y t e g r o w t h fac to r , r lL -2 . Spleen cells obta ined f r o m mice 4 days after the admin is t ra t ion of Photofrin® were much more responsive to rEpo than spleen cells prepared f r o m so lvent - in jected mice (Figure 3 . 1 2 ) . 3.2.7 Spleen cell expression of activation antigens Evaluat ion of spleen cells for ac t ivat ion ant igens by f l o w cy tomet r i c analysis revealed tha t the relat ive expression of the l y m p h o c y t e ac t iva t ion ant igen IL-2RCT (CD25) remained at background levels in bo th cont ro l and Photofr in®-treated mice f r o m 6-96 hours post - in ject ion (Figure 3 . 1 3 ) . H o w e v e r , expression of the receptor for t ransfer r in (CD71) as recognized by the monoc lona l an t ibody ER-MP-21 (Leenan et al., 1 9 9 0 ) , w a s s igni f icant ly increased by 7 2 hours and remained elevated at 9 6 hours af ter the in ject ion of Photofrin® (Figure 3 . 1 3 ) . CD25 and CD71 express ion prof i les for spleen cells obta ined 9 6 hours post- in ject ion are s h o w n in Figure 3 . 1 4 . The abi l i ty of Photofrin® to increase splenic CD71 expression was con f i rmed w i t h the independent ly der ived rat ant i -mouse CD71 monoclonal ant ibodies C2F2 (Kemp etal., 1987) and R17 2 1 7 (Lesley et al., 1 9 8 4 ) . As w i t h E R - M P - 2 1 , CD71 express ion w a s found to be elevated above cont ro l levels w i th in 2-3 days pos t - in jec t ion . CD71 expression remained s ign i f icant ly e levated in the spleen unt i l 7 days after Photofrin® w a s adminis tered (Figure 3.1 5A , B). Flow cy tomet r i c prof i les of spleen cells obta ined at the day 4 sampl ing t ime and labelled w i t h the rat ant i -mouse CD71 monoc lona l ant ibodies C2F2 or R17 2 1 7 are s h o w n 95 in Figure 3.1 6. A series of double label FACS analyses per fo rmed on spleen cells obta ined 9 6 hours af ter Photofrin® in ject ion, indicated tha t h ighest levels of CD71 were present on cells w h i c h also expressed the LR-1 or TER-119 markers . Modes t levels of CD71 were present on splenic l g M + cells (B l ymphocy tes ) in bo th cont ro l and Photofr in®-injected mice. CD71 expression w a s at background levels on spleen cells t ha t expressed T h y - 1 . 2 , C D 4 , CD8 , or Gr-1 sur face ant igens in bo th cont ro l and Photofr in®-injected mice. These double label results are inc luded in Table 3 .4 and representat ive dual co lour f l o w cy tomet r i c analysis of CD71 co-express ion pat terns are presented in Figure 3 . 1 7 . In an exper iment per formed to determine w h e t h e r the natural ly-occur r ing porphyr in PPIX migh t also a f fec t spleen express ion of C D 7 1 , the c o m p o u n d w a s adminis tered to normal DBA/2 mice. In con t ras t to Photofrin®, PPIX did not alter spleen w e i g h t or the propor t ion of cells w h i c h expressed CD71 at the day 4 post - in jec t ion sampl ing t ime (Figure 3 . 1 8 ) . H o w e v e r , PPIX s igni f icant ly decreased the cel lular i ty of the o rgan , and a l though not stat is t ical ly s igni f icant , a t rend t o w a r d s a lower expression of CD71 in the spleens o f these mice was indicated by these studies (Figure 3 . 1 8 ) . In th is exper iment , spleens of Photofrin®-t reated mice d isplayed the character is t ic increase in numbers of C D 7 1 b n g h t cells wh i le the sta in ing pat tern of spleen cells f r o m PPIX-injected mice w a s no d i f ferent f r o m tha t of the cont ro ls (Figure 3 . 1 9 ) . 9 6 CO C D C D Q _ C O C D CO O C D CJ> C D 0 24 48 72 96 Time post-injection (hours) Figure 3 . 1 3 Spleen cell expression of C D 2 5 and C D 7 1 ant igens fo l low ing Photofr in® admin is t ra t ion . Spleen cells were evaluated for their expression of CD25 (IL-2Ra), as recognized by the rat monoc lona l an t ibody 3C7 (Ortega et al., 1 9 8 4 ) , and C D 7 1 , as labelled w i t h the rat monoc lona l an t ibody ER-MP-21 (Leenan et al., 1990) f r o m 4 to 9 6 hours fo l l ow ing the in ject ion of 5 % dext rose or Photofrin® (25 mg /kg ) . Day 0 values represent results obta ined w i t h naive, sex- and age-matched DBA/2 mice. A t each sampl ing t ime , sp lenocytes f r o m 3 mice per g roup were analyzed for expression of C D 2 5 and C D 7 1 , except at 7 2 hours w h e n 11 mice per group were tes ted for C D 7 1 and at 9 6 hours post - in ject ion w h e n 7 mice per g roup were evaluated for CD71 express ion. * * P< 0 . 0 0 1 compared to the result obta ined w i t h cells obta ined f r o m solvent-in jected con t ro l mice. 97 5 % Dextrose Photofrin® Fluorescence Intensity Figure 3 . 1 4 Spleen cell express ion of C D 2 5 and C D 7 1 sur face ant igens - FACS prof i les. Spleen cells were obta ined f r o m mice g iven 5 % dext rose or Photofrin® (25 mg/kg) and t reated w i t h either the (A) an t i -CD25 ant ibody 3 C 7 , (B) the ant i -CD71 an t ibody E R - M P - 2 1 , or the appropr iate rat immunog lobu l in isotype con t ro l . Representat ive sta in ing prof i les obta ined w i t h spleen cells prepared 9 6 hours post-in ject ion are s h o w n . 98 C2F2 FM7217 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Days post-injection Figure 3.15 Spleen cell expression of CD71 as recognized by the monoclonal antibodies C2F2 and R17 217. Spleen cell expression of CD71, as labelled by the rat anti-mouse TrfR-specific monoclonal antibodies (A) C2F2 or (B) R17 217, was evaluated from 1-7 days after the administration of 5% dextrose (•) or Photofrin® (25 mg/kg, O) . Each treatment group consisted of three mice per sampling time. Day 0 values were determined for spleen cells prepared from untreated male DBA/2 mice (n = 3). t P< 0.05; * P< 0.025; * * P< 0.001 compared to the result obtained with spleen cells obtained from solvent-injected control 99 Figure 3.16 Spleen cell CD71 express ion - FACS prof i les. Representat ive f l o w cy tomet r i c prof i les for spleen cells obta ined 4 days after the in ject ion of 5 % dext rose or Photofrin® (25 mg/kg) and t reated w i t h the PE-conjugated ant i -CD71 monoc lona l ant ibodies C2F2 or R17 2 1 7 (bold lines) or a PE-conjugated l g G 2 a isotypic cont ro l an t ibody (thin t rac ings) are s h o w n . The vas t major i ty of cells express ing l ow levels of CD71 are B l y m p h o c y t e s . Spleen cells deemed CD71 are represented by the labelled cells w h i c h fall w i th in the range of the cursor . 100 Figure 3 . 1 7 Spleen cell C D 7 1 co-express ion pat terns. Spleen cells obta ined 4 days fo l l ow ing the admin is t ra t ion of 5 % dext rose or Photofr in® (25 mg/kg) were labelled w i t h the PE-conjugated ant i -CD71 ant ibody C2F2 in con junc t ion w i t h a select ion of ant ibodies react ive against d i f fe rent leukocy te ant igens. A n t i b o d y combinat ions were tes ted 2-8 t imes. 101 102 Treatment group Figure 3 . 1 8 Inf luence of Photofrin® and PPIX on spleen w e i g h t , cel lulari ty and express ion of the t ransferr in receptor (CD71) . The e f fec t of 5 % dex t rose , Photofrin® (25 m g / k g ) , Solvent C or PPIX (25 mg/kg) admin is t ra t ion on d i f fe rent spleen parameters inc luding (A) spleen w e i g h t , (B) spleen nucleated cell numbers , (C) % CD71 + spleen cel ls, and (D) to ta l numbers of C D 7 1 + spleen cells w a s determined for DBA/2 mice 4 days post-in ject ion. Each t rea tmen t group consis ted of four animals. P < 0 . 0 5 compared to the result obta ined for spleen cells obta ined f r o m mice g iven the cont ro l so lvent . 103 Fluorescence Intensity Figure 3 . 1 9 In f luence o f Photofr in® and PPIX on spleen cell C D 7 1 express ion -FACS prof i les. Spleen cells obta ined 4 days fo l low ing the admins t ra t ion of Photofrin® (25 m g / k g ) , PPIX (25 m g / k g ) , or the porphyr in -matched con t ro l so lven t we re labelled w i t h t he an t i -CD71 monoc lona l ant ibody ER-MP-21 (bold l ines). Staining profi le obta ined w i t h the l g G 2 a i so type cont ro l is indicated w i t h th in l ines. 1 0 4 3 . 2 . 8 F low cy tomet r i c ev idence of a splenic e ry thro id progeni tor response t o Photofr in® The ery thro id nature of the splenic response to Photofrin® w a s indicated by single label FACS analysis employ ing the ery thro id l ineage-specif ic monoc lona l an t ibody TER-119 (Ikuta etal, 1 9 9 1 ) . A t the day 4 sampl ing t ime , 1.80 ± 0 . 6 9 % of spleen cells f r o m solvent- in jected (n = 6) and 4 . 9 ± 0 . 7 2 % of spleen cells f r o m Photofr in®-injected (n = 7) mice labelled w i t h TER-119 ( t - test value = 7 . 9 5 ; P< 0 . 0 0 0 1 , 11 d f ) . Spleen cell TER-119 FACS stain ing pat terns are s h o w n in Figure 3 . 2 0 . W h e n the cel lular i ty of the organ at th is sampl ing t ime w a s taken into accoun t , to ta l T E R - 1 1 9 + spleen cells numbered 6 .2 ± 1.7 x 1 0 5 / 2 0 g in cont ro l mice and 3 3 . 4 ± 1 1 . 4 x 1 0 5 / 2 0 g in Photofr in®-injected mice, a greater than 5-fold increase ( t - test value = 5 . 7 5 , P< 0 . 0 0 0 1 , 11 d f ) . Single label FACS analysis w a s uti l ized to d i rect ly compare the express ion of HSA, an ant igen k n o w n to be present on by deve lop ing and mature ery thro id cells (Bruce et al., 1 9 8 1 ; Mil ler etal., 1 9 8 5 ) , w i t h the ant igen recognized by the LR-1 reagent . By 4 days post - in jec t ion, Photofrin® had produced a s igni f icant increase in the percentage and to ta l number of LR-1 + spleen cells and the to ta l number of spleen cells w h i c h labelled w i t h the ant i -HSA ant ibody J 1 1 d (Table 3 .3) . F low cy tomet r i c prof i les of spleen cells labelled w i t h LR-1 or J 1 1 d are s h o w n in Figure 3 . 2 1 . These f ind ings indicated tha t Photofrin® had p romoted splenic express ion of HSA and the ant igen recognized by the LR-1 ant ibody to a similar degree. A more detai led examinat ion of th is relat ionship is d iscussed in Chapter 7. 105 Table 3.3 C o m p a r i s o n of spleen cell expression of H S A with that of the LR-1 marker. Expression of the LR-1 ant igen and HSA (as recognized by the monoc lona l an t ibody J 1 1 d) w a s determined for spleen cells f r o m in tac t DBA/2 mice g iven ei ther 5 % dex t rose or Photofrin® (25 mg/kg) 4 days prev ious ly . Four mice per group w e re analyzed. In th is exper iment , to ta l nuc leated spleen cells cor responded to 3 3 . 3 1 ± 3 . 4 3 x 1 0 6 / 2 0 g and 5 9 . 9 1 ± 6 .30 x 1 0 6 / 2 0 g for the cont ro l and Photofr in®-injected mice, respect ive ly . Trea tment LR-1 + cel ls (%) Tota l LR-1 + spleen cells (x 106/20 g) J11d+ cel ls (%) Tota l J11d+ spleen cells (x 106/20 g) 5% Dext rose (n = 4) 68.35 ± 2.06 22.74 ± 2.57 60.88 ± 1.99 21.08 ± 2.43 Photofr in® (n = 4) 74.53 ± 1.42 44.72 ± 5.03 63.75 ± 3.07 38.70 ± 3.09 t -value (6 df) S ign i f i cance 4.94 P < 0.005 7.78 P < 0.0001 1.57 ns 8.97 P < 0.0001 Results f r o m an exper iment , w h i c h mon i to red the co-express ion of C D 7 1 , as recognized by the ant i -CD71 monoc lona l ant ibodies C2F2 and R17 2 1 7 , w i t h the LR-1 ant igen up to 7 days after Photofrin® in ject ion demons t ra ted tha t the propor t ion of cells w h i c h expressed high levels of bo th ant igens w a s increased above con t ro l levels as early as 2 days post - in jec t ion , and increased fur ther up to 7 days post - in jec t ion (Figure 3 . 2 2 A , B). Character is t ic CD71/LR-1 co-express ion labell ing prof i les for spleen cells obta ined at day 7 post - in ject ion are presented in Figure 3 . 2 3 . The evaluat ion of L R - 1 / C D 7 1 , LR-1/TER-119 TER-119 /CD71 ant igen 106 co-express ion pat te rns on spleen cells f r o m cont ro l and Photofr in®-injected mice indicated tha t the spleens of mice w i th in each group conta ined cells w i t h pheno types similar to those present in normal B M , albeit at a lower f requencies (Figure 3 . 2 4 ) . H o w e v e r , numbers of cells w i t h an LR-1 V C D 7 1 + , LR-1 + / T E R - 1 1 9 + , or T E R - 1 1 9 V C D 7 1 + pheno type were 3-4 t imes greater w i t h i n the spleens of Photofr in®-treated animals than in the cont ro ls (Table 3 .4 ) . Phenotyp ic evaluat ion of spleen cells obta ined 4 days after the in ject ion of Photofr in® also revealed tha t a small sub-popu la t ion w i t h a LR-1 + /CD45" / | 0 pheno type w a s propor t ional ly and absolute ly increased by the t rea tmen t (Figure 3 . 2 5 ) . This popula t ion represented 5.0 ± 1.4% of the nucleated spleen cell popu la t ion in the cont ro l mice (n = 3 ) . T rea tment w i t h Photofrin® s ign i f icant ly ( t-value = 5 . 3 5 ; P = 0 . 0 0 6 , 4 df) increased the percentage of spleen cells w i t h th is pheno type to 10 .4 ± 1.0% (n = 3 ) . Tak ing the overal l cel lular i ty of the organ into accoun t at th is sampl ing t ime , cells w i t h an LR-1 + / C D 4 5 " / | 0 pheno type numbered 2 . 3 0 ± 0 . 9 7 x 1 0 6 and 7 .83 ± 0 . 6 9 x 1 0 6 in the spleens of the cont ro l and Photofr in®-injected mice respect ive ly , a h ighly s ign i f icant d i f ference (t-value = 8 . 0 5 ; P = 0 . 0 0 1 , 4 d f ) . 107 Figure 3 . 2 0 Spleen cell expression of the ery thro id TER-119 an t igen . Spleen cells obta ined 4 days after the admin is t ra t ion of 5 % dext rose or Photofr in® (25 mg/kg) were subjected to sur face labell ing w i t h the b iot iny lated ant i -ery thro id monoc lona l an t ibody TER-119 or a b io t iny la ted rat l g G 2 b isotype cont ro l an t ibody . FACS staining prof i les obta ined w i t h these reagents are s h o w n . Summar ized resul ts for splenic TER-119 expression studies are g iven w i th in the t e x t of Sect ion 3 . 2 . 8 . 108 Fluorescence intensity Figure 3 . 2 1 Spleen cell HSA ( J 1 1 d ant igen) and LR-1 ant igen express ion. Spleen cells obta ined 4 days after the admin is t ra t ion of 5 % dext rose or Photofr in® (25 mg/kg) were incubated w i t h the ant i -HSA monoc lona l ant ibody J 1 1 d , the monoc lona l an t ibody LR-1 or a rat IgM isotype con t ro l . Cell sur face-associated rat IgM w a s revealed w i t h a FITC-conjugated ant i - rat IgM monoclonal an t ibody ( M A R M - 4 ) . FACS stain ing pat terns obta ined w i t h these ant ibodies in th is exper iment are s h o w n . Summar ized results f r o m these analyses are presented in Table 3 .3 . 109 C O " C D o C D C D Q _ C O Q O 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Days post-injection Figure 3 . 2 2 Inf luence of Photofrin® on levels of C D 7 1 + / L R - 1 + spleen cells. The percentage of spleen cells w h i c h co-expressed CD71 and the ant igen recognized by the LR-1 an t ibody w a s determined at d i f fe rent t imes up unti l 7 days af ter the admin is t ra t ion of 5 % dext rose ( • ) or Photofrin® ( O , 25 mg /kg ) . Cell sur face CD71 w a s labelled w i t h the rat l g G 2 a monoc lona l ant ibodies ( A ) C2F2 or (B) R17 2 1 7 . * P< 0 . 0 5 ; * P< 0 . 0 1 ; * * P< 0 . 0 0 1 compared to the result obta ined w i t h spleen cells prepared f r o m the solvent- in jected cont ro l mice. 110 A 5% Dextrose Photofrin® HI -I Q . : <o ' CN 3: CD I O) 1 . 2 3 1 ^ 4 B CN L L CM 0 LU Q. 1 r> Q O 3 8: 1 2 3 4 Rat IgM / FITC 1 2 i 3 Jut .1 1 10 LR-1 / FITC iee 1088 . .1 LR-1 / FITC ioe looe 1 ': 2 3 • & w 1 2 0$ Figure 3 . 2 3 Spleen cell co-express ion o f C D 7 1 w i t h the LR-1 ant igen - FACS prof i les. Spleen cells obta ined f r o m mice in jected 4 days prev iously w i t h 5 % dext rose or Photofr in® w e re t reated w i t h (A) isotype cont ro l ant ibodies or the LR-1 monoc lona l an t ibody in comb ina t ion w i t h the PE-conjugated ant i -CD71 ant ibodies (B) C2F2 or (C) R17 2 1 7 . 1 . 3 . Cell sur face-associated LR-1 w a s counter- label led w i t h the FITC-conjugated ant i - rat IgM monoc lona l an t ibody ( M A R M - 4 ) . 111 Control spleen Photofrin® spleen Normal bone marrow TER-119 - Log Fluorescence (FITC) L U LR-1 - Log Fluorescence (FITC) Figure 3 . 2 4 Spleen and BM erythro id-assoc iated ant igen express ion prof i les. Spleen cells prepared 4 days after the in ject ion of 5 % dext rose or Photofrin® (25 mg/kg) were assessed for their co-express ion of C D 7 1 / L R - 1 , TER-1 1 9 / C D 7 1 , or TER-119/LR-1 ant igens. For compara t i ve purposes, the cor respond ing sta in ing pat terns obta ined w i t h normal DBA/2 BM cells are also s h o w n . 112 Table 3.4 Spleen and BM cell e ry thro id l ineage-associated ant igen express ion. Co-express ion pat terns for the L R - 1 , C D 7 1 , and TER-119 ant igens were de termined for spleen cells obta ined f r o m mice g iven 5 % dext rose or Photofr in® (25 mg/kg) 4 days prev ious ly . For compara t ive purposes, data obta ined w i t h normal BM cells has been inc luded. CD71 express ion w a s present at background levels on spleen cells t h a t expressed T h y - 1 . 2 , C D 4 , C D 8 , I g M , or Gr-1 sur face ant igens in bo th cont ro l and Photofr in®-injected mice . F low cy tomet r i c prof i les of the labell ing pat terns of these cells are presented in Figure 3 . 2 3 . Cell sur face ant igen co-express ion pat te rn Cell Origin LR-1+/CD71 + LR-1+/TER-119 + TER-119 + /CD71 + Control spleen number of analyses double positive cells (%) total double positive cells (x lO"6) 6 6.0 ± 2.7 2.46 ± 1.25 2 5.4 ± 1.1 2.23 ± 0.19 3 2.6 ± 0.5 1.24 ± 0.32 Photofrin® spleen number of analyses double positive cells (%) total double positive cells (x 1CT6) 7 11.9 ± 2.1 * 6.62 ± 1.78* 2 11.1 ± 0 .28* 8.77 ± 1.87* 3 5.1 ± 1.5* 3.87 ± 1.38* Normal BM number of analyses double positive cells (%) 2 22.1 ± 6.6 1 13.3 1 5.5 * P < 0 . 0 5 compared to the result obta ined w i t h spleen cells f r o m cont ro l mice. 113 5% Dextrose Photofrin CD45 Fluorescence Intensity - FITC Figure 3 . 2 5 Numbers of L R - 1 + / C D 4 5 / l 0 cells are increased in the spleens of Photofr in®-treated mice. Spleen cells obta ined 4 days after the admin is t ra t ion of 5 % dext rose or Photofr in® (25 mg/kg) w e r e co- label led w i t h t he rat an t i -CD45 monoc lona l an t ibody 3 0 F 1 1 . 1 and the LR-1 reagent . Representat ive FACS stain ing pat terns obta ined w i t h these ant ibodies and the appropr iate pair of iso type cont ro l ant ibodies are s h o w n . The area del ineated w i th in the rectangle inside each f igure represents the spleen cell popu la t ion exhib i t ing an LR-1 " 7 C D 4 5 " 0 p h e n o t y p e . 1 1 4 3 . 2 . 9 Splenic response to Photofr in® by DBA/1 mice W h e n the splenic response to Photofrin® by normal male DBA/1 mice w a s eva lua ted , s ign i f icant increases in spleen w e i g h t (Figure 3 .26A) and cel lular i ty (Figure 3 .26B) and in levels of BFU-E (Figure 3 .26C) and CFU-E (Figure 3 .26D) were observed at days 4 and 7 post - in jec t ion, in relat ion to the solvent- in jected cont ro l mice. By day 7 post - in jec t ion , there, w a s a s ign i f icant increase in the propor t ion of spleen cells f r o m Photofr in®-injected mice w h i c h labelled w i t h the LR-1 monoc lona l an t ibody (Figure 3 .26E) . A t 4 days pos t - in jec t ion , there w a s a modes t increase in the number of cells w h i c h mos t s t rong ly expressed the LR-1 ant igen (Figure 3 . 2 7 A ) . By day 7 post - in jec t ion, the LR-1 sta in ing profi le of spleen cells f r o m Photofr in®-injected mice exhib i ted a fur ther enhancement of the br ight ly labell ing second c o m p o n e n t of the LR-1 + peak (Figure 3 .27B) . W h e n the binding of Trf-F w a s eva lua ted , spleen cells f r o m Photofr in®-injected DBA/1 mice bound s igni f icant ly more of the fac tor than spleen cells f r o m cont ro l mice at bo th sampl ing t imes (Figure 3 .26F) . Spleen cell Trf-F binding prof i les are presented in Figure 3 . 2 8 . 115 Days post-injection Figure 3 . 2 6 Spleen parameters o f Photofr in®-injected D B A / 1 mice . (A) Relative spleen w e i g h t , (B) spleen cel lu lar i ty , levels of splenic (C) BFU-E, (D) CFU-E, (E) the percentage of spleen cells w h i c h labelled w i t h the monoc lona l an t ibody L R - 1 , and (F) the percentage of cells t ha t bound Trf-F were evaluated 4 or 7 days fo l l ow ing the i.p. admin is t ra t ion of 5 % dext rose ( • ) or Photofrin® (25 m g / k g , • ) t o normal male DBA/1 mice. Four mice per group were analyzed at each sampl ing t ime . For cont ro l DBA/1 mice, CFU-E were present w i t h an approx imate f requency of 1 per 7 5 0 0 spleen cells p la ted, wh i le BFU-E occurred at an es t imated f requency of 1 per 1 6 0 , 0 0 0 spleen cells p la ted. + P< 0 . 0 5 ; * P< 0 . 0 1 ; * * P< 0 . 0 0 1 compared to the result obta ined w i t h spleen cells f r o m solvent - in jected cont ro l mice. 116 Figure 3 . 2 7 LR-1 ant igen express ion by DBA/1 mouse spleen cel ls. DBA/1 mouse spleen cells obta ined either (A) 4 or (B) 7 days after the in ject ion of 5 % dext rose or Photofrin® (25 mg/kg) were labelled w i t h the LR-1 monoc lona l an t ibody (bold t rac ings) . The result obta ined w i t h the rat IgM isotype cont ro l an t ibody is represented by th in t rac ings . Cel l -associated rat IgM was de tec ted w i t h a PE-conjugated mouse ant i -rat IgM monoc lona l an t ibody . 117 Fluorescence Intensity Figure 3 . 2 8 Trf-F b ind ing by D B A / 1 spleen cel ls. DBA/1 mouse spleen cells obta ined 7 days after the in ject ion of (A) 5 % dex t rose or (B) Photofrin® (25 mg/kg) were incubated w i t h f luoresceinated-human holo-Tr f in the absence (bold lines) or presence (l ight lines) of a 200- fo ld excess of unlabel led human holo-Trf . Spleen cell b inding of the Trf-F probe w a s assessed by f l o w c y t o m e t r y . 118 3 . 2 . 1 0 Regulatory aspects o f the splenic hematopo ie t i c response to Photofrin® In order to evaluate h o w the splenic myelopo ie t ic response to Photofrin® migh t be con t ro l led , the fo l l ow ing exper iment w a s pe r fo rmed . DBA/2 mice w e re admin is tered the ant i - in f lammatory agent indomethac in ( 1 0 0 //g) orally by gavage tube on days - 1 , 1 , and 3 of a trial in w h i c h Photofrin® (25 mg/kg) w a s g iven i.p. on day 0. Contro l mice received the oral del ivery vehic le (0.1 % T w e e n 20) in paral lel . A t 7 days post - in jec t ion, Photofrin® had produced the expected increases in spleen w e i g h t , cel lular i ty and levels of CFU-GM as compared to the cont ro l mice g iven 5 % dext rose (Figure 3 .29 A-C) . Mice g iven 5 % dext rose on day 0 and three doses of indomethac in exhib i ted a modes t , bu t not s ign i f icant , increase in these spleen parameters compared to mice g iven 5 % dext rose and 0.1 % T w e e n 2 0 . H o w e v e r , mice g iven Photofrin®, in addi t ion to three doses of indomethac in , exhib i ted fu r ther enlarged spleens, greater spleen cell numbers and higher levels of splenic CFU-GM at the day 7 sampl ing t ime than mice g iven Photofr in® and three oral doses of 0.1 % T w e e n 2 0 . 3 . 2 . 1 1 In f luence of BPD on splenic hematopo ie t ic ac t i v i t y W h e n the spleen w a s mon i to red over a 4 day per iod after a single i.v. in ject ion o f BPD (10 m g / k g ) , no s igni f icant change in spleen w e i g h t , spleen cel lular i ty or CFU-GM levels in the organ w a s observed (Figure 3 . 3 0 A-C) . 119 cETO.6 Treatment group Figure 3 . 2 9 Inf luence of indomethac in on the splenic response to Photofrin®. D B A / 2 mice (5 animals per t rea tment group) were t reated w i t h either 0.1 % T w e e n 2 0 and 5 % dex t rose , 0.1 % T w e e n 2 0 and Photofr in®, indomethac in and 5 % dex t rose , or indomethac in and Photofrin®. T w e e n 2 0 and indomethac in were admin is tered oral ly in three 100 jjg doses on days - 1 , 1 , and 3 whi le 5 % dext rose and Photofrin® (25 mg/kg) were g iven as a single i.p. dose on day 0. (A) Spleen w e i g h t s , (B) cel lu lar i ty , (C) and CFU-GM levels were determined for spleens obta ined 7 days post - in jec t ion. P < 0 .01 compared to the result obta ined w i t h spleen cells f r o m mice g iven 5 % dext rose and 0 . 1 % T w e e n 2 0 . * P < 0 . 0 1 ; * * P < 0 . 0 0 0 1 compared to the result obta ined w i t h spleen cells f r o m mice g iven 5 % dext rose in addi t ion to 0.1 % T w e e n 2 0 or indomethac in . + P < 0 . 0 5 ; + +P < 0 .01 compared to the result obta ined w i t h spleen cells f r o m mice g iven 0 . 1 % T w e e n 2 0 and Photofrin®. 120 3 . 2 . 1 2 Inf luence of Photofr in® and BPD on BM hematopo ie t ic ac t iv i ty Photofr in® at 2 5 m g / k g caused a s ign i f icant e levat ion in the f requency of BM C F U - G M , rising to levels 3 0 - 4 0 % above cont ro l levels f r o m 2 4 - 7 2 hours post - in ject ion (Figure 3 . 3 1 ) . In con t ras t , no s ign i f icant change in the f requency of BM CFU-GM occur red up to 9 6 hours fo l l ow ing the admin is t ra t ion of BPD (10 mg/kg) (Figure 3 . 3 2 ) . In unt reated and so lvent - in jected cont ro l m ice , CFU-GM were present at 1 per 3 3 0 bone m a r r o w cells p la ted, a f requency approx imate ly 3 0 t imes greater t han tha t for spleen cel ls. There w a s no change in the relat ive f requency of BM BFU-E w i th in the f i rst 3 days f o l l o w i n g Photofrin® admin is t ra t ion (Figure 3 . 3 3 A ) . Morpho log ica l appearance of BM BFU-E w h i c h develop in vitro are s h o w n in Figure 3 .33B . Levels of BM CFU-E w e re not evaluated in these mice. 3 . 2 . 1 3 Inf luence of Photofrin® on C D 7 1 express ion in the m a r r o w BM cells were evaluated for CD71 expression f r o m 2 4 - 7 2 hours after the admin is t ra t ion of Photofrin®. Labell ing w i t h the ant i -CD71 an t ibody ER-MP-21 indicated t h a t Photofr in® caused a small but s igni f icant increase in the propor t ion of BM cells t h a t expressed CD71 by 4 8 hours post - in jec t ion (Figure 3 .34 ) . H o w e v e r , by 7 2 hours post - in ject ion the relat ive express ion of th is ant igen was s ign i f icant ly lower in the BM of Photofr in®-treated mice wh i le at th is t ime , expression of CD71 w a s s igni f icant ly e levated in the spleens of the Photofrin®-t reated mice (Figure 3 . 3 5 ) . Further evaluat ion w i t h the ant i -Tr fR monoc lona l an t ibody C2F2 also demons t ra ted tha t CD71 w a s s igni f icant ly e levated w i th in the 121 Figure 3 . 3 0 Inf luence of BPD on d i f fe rent spleen parameters . (A) Relative spleen w e i g h t s , (B) spleen cell numbers , and (C) levels of splenic CFU-GM were determined for DBA/2 mice adminis tered either 5 % DMSO in PBS ( • ) or BPD (10 m g / k g , • ) i.v. on day 0. Each data po in t represents the mean result ± the s tandard dev iat ion for 3 mice per t rea tmen t g roup . 122 CO CD O O CD • u_ O o as CD O CD 160 140 120 100 80 60 40 20 0 * ^ 6 * * / 6 ' cb-J l I I L -0 J L 0 1 2 3 4 5 6 7 Days post-injection Figure 3 .31 Inf luence of Photofrin® on levels of BM CFU-GM. Relative f requencies of CFU-GM in the BM of normal DBA/2 mice injected i.p. w i t h ei ther 5 % dext rose ( • ) or Photofrin® at 2 5 m g / k g (O) were de te rmined . Data is presented as the mean percentage (%) of the result obta ined w i t h the so lvent - in jec ted mice sampled in parallel. Each data po in t represents the mean percentage (%) ± SD for 6 -16 animals. Mean number of CFU-GM for cont ro l cu l tures ranged f r o m 1 3 6 - 1 4 6 / 5 x 1 0 4 cells p la ted. Few (0-2) colonies fo rmed in the absence of P W M - S C C M in the cul ture s y s t e m . * * * P< 0 . 0 2 5 ; P< 0 .01 compared to the result obta ined w i t h BM cells f r o m so lvent - in jected cont ro l mice. 123 ^ 120 1 2 3 4 Days post-injection Figure 3 . 3 2 Inf luence of BPD on BM CFU-GM levels. Relative f requencies of BM CFU-GM were determined for mice admin is tered either 5 % DMSO in PBS ( • ) or BPD (10 m g / k g , • ) on day 0. Each data po in t represents t he resul t obta ined w i t h cells f r o m 3 mice . Data is presented as the mean percentage (%) of the result obta ined w i t h the so lvent - in jected mice sampled in paral lel . Mean number of CFU-GM for cont ro l cu l tures ranged f r o m 1 54-171 / 5 x 1 0 4 cells p la ted. Few (0-2) colonies fo rmed in the absence of P W M -SCCM in the cu l ture s y s t e m . 124 F i g u r e 3 . 3 3 I n f l u e n c e o f P h o t o f r i n ® o n B M l e v e l s o f B F U - E . (A) Relative frequencies of BFU-E were determined for BM cells obtained from mice given 5% dextrose ( • ) or Photofrin® (25 mg/kg, O ). Data is presented as the mean percentage (%) of the result obtained with the solvent-injected mice sampled in parallel. Each data point represents the mean percentage (%) ± SD for 3 animals per group. Mean number of BFU-E for BM cells from control animals ranged between 21-35 / 1 x 10 5 cells plated. (B) Appearance of BM BFU-E which form in vitro in semi-solid methylcellulose cultures within 10 days of culture (magnification x 50). 125 m a r r o w 2 days fo l l ow ing Photofrin® admin is t ra t ion (Figure 3 . 3 6 ) . However , at the day 4 sampl ing t ime relat ive expression of CD71 w a s s igni f icant ly lower in the m a r r o w of Photofr in®-treated mice, fal l ing to approx imate ly one-hal f of cont ro l va lues (Figure 3 . 3 6 ) . In the same mice the relat ive express ion of CD71 in the spleen w a s s t rong ly elevated in the Photofr in®-treated animals 4 days post- in ject ion (Figure 3 . 3 7 ) . 3 . 2 . 1 4 In f luence of Photofr in® on m a r r o w cel lular i ty and express ion o f d i f fe ren t leukocy te ant igens A t day 4 post - in jec t ion , numbers of nuc leated BM cells were comparab le for un t rea ted , so lvent - in jec ted, and Photofr in®-injected mice a l though these values d isp layed a modes t degree of inter-animal var iabi l i ty (Figure 3 .38 ) . F low c y t o m e t r i c analysis revealed tha t the porphyr in preparat ion caused a dramat ic , h ighly cons is ten t decrease in BM expression of the B cel l - restr ic ted ant igen B220 and CD71 and a s t rong increase in the propor t ion of cells w h i c h expressed the myelo id ant igens C D 1 1 b or Gr-1 (Figure 3 . 3 9 ) . In bo th t rea tmen t g roups , v i r tual ly all ( > 9 5 % ) BM cells expressed the ant igen recognized by the LR-1 reagent. Flow cy tomet r i c prof i les obta ined in these labell ing studies are s h o w n in Figure 3 . 4 0 . A l t h o u g h Photofr in® did not change the propor t ion of BM cells w h i c h labelled w i t h the LR-1 monoc lona l an t ibody , there w a s s igni f icant d im in ishment in the intensi ty of cell sur face labell ing w h e n cells f r o m these mice were analyzed w i t h this reagent . BM cells f r o m the cont ro ls exhib i ted a b imodal LR-1 expression profi le whereas BM cells f r o m Photofr in®-treated mice displayed a marked p ronouncement 126 C O ~ C D o o cB C D O + O 1 2 3 Days post-injection Figure 3 . 3 4 Inf luence of Photofrin® on BM cell C D 7 1 express ion as recognized by the monoc lona l an t ibody ER-MP-21 . BM cells were prepared at d i f fe rent sampl ing t imes f r o m mice injected w i t h 5 % dex t rose ( • ) or Photofrin® (25 m g / k g , O ) and labelled w i t h the rat ant i -mouse CD71 monoc lona l an t ibody ER-MP-21 (Leenan et al., 1 9 9 0 ) . Three mice per t rea tmen t g roup w e re analyzed. * P< 0 . 0 5 compared to the result obta ined w i t h BM cells f r o m the solvent- in jected cont ro ls . 127 5% Dextrose Photofrin® A 81 I 81 : Log Fluorescence Intensity (FITC) Figure 3 . 3 5 In f luence of Photofr in® on BM CD71 expression as recognized by monoc lona l an t ibody ER-MP-21 - FACS prof i les. F low c y t o m e t r i c prof i les of CD71 expression on BM cells prepared either (A) 2 or (B) 3 days af ter the in ject ion of 5 % dext rose or Photofr in® (25 mg/kg) are s h o w n . Cell sur face CD71 was detected w i t h the rat ant i -Tr fR monoclonal an t ibody C 2 F 2 . For compara t ive purposes, (C) representat ive CD71 expression pat te rns for spleen cells obta ined f rom the mice sacr i f iced on day 3 of the exper iment are also s h o w n . Labell ing w i t h an l g G 2 a isotype con t ro l an t ibody is also exhib i ted (upper panels) . For the cont ro ls (n = 3 ) , 5.3 ± 2 . 2 % of spleen cells were C D 7 1 + whereas 14 .9 ± 4 . 7 % of spleen cells f r o m the Photofr in®-treated mice (n = 3) w e r e CD71 + ( t -value =• 3 . 1 6 ; P< 0 .05 ) . Cells deemed CD71 + are represented by the labelled cells w h i c h fall w i th in the range of the cursor . 128 20 C O " C D o 15 O o C D O CD 10 0 2 4 Days post-injection Figure 3 . 3 6 Inf luence of Photofrin® on BM cell CD71 expression as labelled by the monoc lona l an t ibody C2F2. BM cells were prepared f r o m mice injected w i t h 5 % dext rose ( • ) or Photofrin® (25 m g / k g , • ) and labelled w i t h the PE-conjugated ant i -mouse CD71 monoc lona l an t ibody G2F2 or PE-conjugated rat l g G 2 a i so type-matched ant ibody. On day 2 pos t - in jec t ion , 3 mice per tes t group were analyzed whereas on day 4 pos t - in jec t ion , 7 mice per tes t group were tes ted . * P< 0 . 0 5 compared to the result for the so lvent- in jected cont ro ls . P< 0 . 0 0 0 1 compared to result for the solvent- in jected cont ro ls . 129 Figure 3 . 3 7 In f luence of Photofr in® on BM CD71 express ion as recognized by the C 2 F 2 monoc lona l an t ibody - FACS prof i les. F low cy tomet r i c prof i les of CD71 expression for BM cells prepared either (A) 2 or (B) 4 days after the in ject ion of 5 % dext rose or Photofrin® (25 mg/kg) are s h o w n . Cell sur face CD71 w a s de tec ted w i t h the monoc lona l ant ibody C2F2 (bold l ines). Labell ing w i t h a PE-conjugated rat l g G 2 a i so type cont ro l an t ibody is also s h o w n (thin l ines). For compara t ive purposes, representat ive CD71 express ion pat te rns for spleen cells obta ined f r o m the mice sacr i f iced on day 4 of the exper iment are also s h o w n (C). In th is exper iment , 5 .2 ± 3 . 7 % of the spleen cells f r o m cont ro l mice (n = 3) were CD71 + whereas 16 .3 ± 3 . 0 % of the spleen cells f r o m the Photofr in®-treated mice (n = 3) were CD71 + ( t -value = 3 . 1 6 ; P< 0 . 0 5 ) . CD71 + cells fall w i t h i n the range denoted by the cursor . 130 15 Figure 3 . 3 8 Inf luence of Photofrin® on m a r r o w cel lu lar i ty. BM cells were obta ined f r o m unt reated DBA/2 mice (cross-hatched) , or animals g iven 5 % dext rose ( • ), or Photofrin® (25 m g / k g , • ) 4 days prev ious ly . Viable BM cell numbers were determined by T rypan blue dye exc lus ion. Each tes t g roup cons is ted of 7 mice. 131 I 1 1 1 1 1 1 1 1 I 1 I I I I I I I I I I 0 20 40 60 80 100 % positive cells Figure 3.39 In f luence of Photofrin® on BM leukocy te ant igen express ion. BM cells obta ined 4 days fo l l ow ing the admin is t ra t ion of 5 % dext rose ( • ) or Photofr in® (25 m g / k g , • ) were sur face labelled w i t h rat monoc lona l ant ibodies react ive w i t h the mur ine B cel l - restr ic ted iso form of CD45 (B220) , the myelo id integr in C D 1 1 b (Mac-1 ) , the granu locyte- res t r ic ted Gr-1 an t igen, the LR-1 marker , or the TrfR (CD71) . For each leukocy te an t igen, BM cells f r o m 4 mice were analyzed, excep t for the CD71 and LR-1 sur face ant igens w h e n 7 mice per t rea tmen t g roup w e re eva luated. P< 0 . 0 0 0 1 compared to BM cells obta ined f r o m so lvent - in jected mice. 132 5% Dextrose Photofrin Log Fluorescence Intensity Figure 3 . 4 0 BM cell express ion of C D 4 5 R - B 2 2 0 , C D 1 1 b , G r - 1 , and LR-1 ant igens af ter Photofr in® admin is t ra t ion - FACS prof i les. BM cells obta ined 4 days fo l l ow ing the admin is t ra t ion of 5 % dext rose or Photofr in® (25 mg/kg) were labelled w i t h rat monoc lona l ant ibodies react ive w i t h the mur ine B cel l - restr ic ted iso form of CD45 (B220) , the myelo id C D 1 1 b (Mac-1) an t igen, the granu locy te- res t r ic ted ant igen G r - 1 , the LR-1 marker , or CD71 (bold t rac ings) . F low cy tomet r i c prof i les obta ined w i t h iso typ ica l ly -matched cont ro l ant ibodies are s h o w n as th in t rac ings . 133 of the lower in tens i ty LR-1 + peak and a d imin ishment of the higher in tensi ty LR-1 + peak (Figure 3 . 4 0 ) . 3 . 2 . 1 5 Peripheral b lood parameters af ter Photofrin® admin is t ra t ion Analys is of peripheral b lood 7 days after the admin is t ra t ion of Photofr in® (25 mg/kg) revealed tha t s igni f icant increases in to ta l leukocyte and l y m p h o c y t e numbers had occur red (Figure 3 . 4 1 ) . H o w e v e r , no al terat ion in levels of b lood neut roph i ls , m o n o c y t e s or eosinophi ls w a s observed . The blood hematocr i t of Photofr in®-treated mice (mean = 4 5 . 3 ± 2 . 0 % , n = 8) w a s greater than tha t of the so lvent - in jec ted cont ro ls (mean = 4 3 . 6 ± 2 . 4 % , n = 8 ) , a l though th is d i f ference w a s no t stat is t ical ly s igni f icant (t-value = 1.54; P = 0 . 1 5 , 14 d f ) . In add i t ion , there w a s no s igni f icant d i f ference b e t w e e n the concent ra t ion of peripheral b lood e ry th rocy tes in mice g iven the cont ro l so lvent (mean = 1.53 ± 0 . 2 2 x 1 0 1 0 / m l ; n = 3) or Photofrin® (mean = 1.59 ± 0 . 3 4 x 1 0 1 0 / m l ; n = 3 ) . Measurement of serum levels of IL-1 or, GM-CSF and IL-6 were un in fo rmat i ve . Neither IL-1 a or GM-CSF were detectable in serum f r o m cont ro l or Photofr in®-injected mice at 2 4 , 4 8 or 7 2 hours post - in ject ion (3 mice per group analyzed) by ELISA. IL-6 w a s de tec ted in serum samples f r o m bo th so lvent and Photofr in®-injected mice as early as 6 hours and up to 4 8 hours post- in ject ion (Figure 3 . 4 2 ) . 1 3 4 o CD O O CD total white cel ls lymphocytes neutrophils monocytes eosinophi ls - f t Cells (x 107ml) Figure 3 .41 Peripheral b lood analysis. Tota l nuc leated cell numbers and leukocy te f rac t ion concent ra t ions were evaluated in normal DBA/2 mice 7 days fo l l ow ing the i.p. admin is t ra t ion of 5 % dext rose ( • ) or Photofrin® (25 m g / k g , • ). Di f ferent ial b lood cell analysis was per fo rmed by morpho log ica l analysis of peripheral b lood cells sta ined w i t h the Giemsa-Wr igh t reagent . Error bars cor respond to the s tandard dev ia t ion of the mean for 8 mice per t rea tmen t g roup . * P< 0 . 0 5 compared to the result obta ined for mice g iven the con t ro l so lvent . 135 50 40 2 30 c o CD C O 20 -10 0 0 24 48 72 Time post-injection (hours) Figure 3 . 4 2 Serum IL-6 levels of Photofr in®-treated mice. Serum levels of IL-6 were moni to red f r o m 0 -72 hours af ter the i.p. admin is t ra t ion of 5 % dext rose ( • ) or Photofrin® (25 m g / k g , O ) . IL-6 levels were determined by ELISA. Three mice per t rea tmen t g roup/sample day were analyzed. Day 0 values w e re obta ined w i t h sera obta ined f r o m 6 norma l , unt reated DBA/2 male mice. 136 3 .3 Discuss ion In the evaluat ion of four porphyr in photosens i t izers , Photofrin®, BPD, Hp, and PPIX, on ly Photofrin® had detectable hematopo ie t ic ac t iv i ty in normal mice. That Photofr in® can in f luence the immunohematopo ie t i c s y s t e m , in the absence of ac t iva t ing levels of l ight , is in agreement w i t h an earlier s tudy tha t s h o w e d tha t the c o m p o u n d p roduced sp lenomegaly in normal mice (Gomer et al., 1 9 8 8 ) . A s tudy by Cant i et al. (1989) indicated tha t Photofrin® could p romote the hematopoie t ic recovery of mice pre- t reated w i t h myelosuppress ive agents . H o w e v e r , in these t w o studies no analysis of these responses w a s per fo rmed beyond the determinat ion of spleen w e i g h t s and spleen and m a r r o w cel lu lar i ty. The in f luence of BPD, Hp, or PPIX on the ac t iv i ty of the immunohematopo ie t i c axis has no t been descr ibed prev ious ly . A l t h o u g h Photofrin® had a s t imula tory e f fec t on myelopoies is in the m a r r o w , the act ion of th is preparat ion w a s relat ively more p ronounced in the sp leen, modes t l y e levat ing myelopoiesis and and s t rong ly enhanc ing erythropoies is w i th in the o rgan . The hematos t imu la to ry act ion of Photofrin® in the spleen was character ized by increases in e ry th romye lo id progeni tor co lony - fo rm ing act iv i ty , prol i ferat ive responses to recombinant IL-3, G-CSF, M-CSF, GM-CSF, and Epo, increased metabo l i sm of the mi tochondr ia l dye M T T , and ev idence of e levated DNA synthes is . A single in ject ion of the c o m p o u n d el ici ted responses tha t were f i rst detectab le in the spleen w i th in 2-3 days post - in ject ion by progeni tor assays and by an increase in the w e i g h t and cel lular i ty of the o rgan. The hematopo ie t ic impact of a single in ject ion of Photofrin® w a s stil l h ighly ev ident w i th in the spleen at day 7 137 day pos t - in jec t ion . F low cy tomet r i c analysis ut i l iz ing a bat te ry of monoclonal ant ibodies d i rected against a w ide range of sur face ant igens, indicated tha t the porphyr in preparat ion e levated the number of spleen cells w h i c h expressed ery thro id-assoc ia ted (TER-119, C D 7 1 , HSA) ant igens but not T cell (CD3, CD4, CD8 , T h y - 1 . 2 , C D 2 5 ) , B cell ( IgM, CD45R-B220) , or myelo id (F4 /80 , CD11b) associated markers . In add i t ion , Photofrin® s t rongly increased the number and propor t ion o f spleen cells recognized by the monoc lona l an t ibody L R - 1 . It w a s or iginal ly descr ibed tha t LR-1 recognized mouse splenic B cells (Hutch ings et al., 1 9 8 5 ) . A l t h o u g h Photof r in increased the number of spleen cells t ha t possessed markers (MHC Class I, pan- leukocyte CD45) normal ly expressed on the vast major i ty of l eukocy tes , the drug also increased the number of nuc leated cells w h i c h expressed either l o w or undetectab le amoun ts of these ant igens. W h e n mice were t reated w i t h the an t i - in f lammatory agent i ndomethac in , an exaggerat ion of the splenic response to Photofr in® w a s observed, p roduc ing an enhancement of spleen w e i g h t , cel lular i ty and levels of CFU-GM. Indomethac in exer ts an inhib i tory e f fec t on the arachidonic acid metabol ic p a t h w a y , thereby reduc ing the in vivo f o rmat ion of prostaglandin E 2 (PGE 2 ) . PGE 2 has been s h o w n to exer t inh ib i tory e f fec ts on mur ine myelopoiesis w h e n g iven to normal mice (Pelus et al., 1 9 7 9 ) . Converse ly , PGE 2 has been s h o w n to inf luence the p roduc t ion of Epo by the k idney and enhance ery thropoies is by an d i rect e f fec t at the BFU-E level (Pelus, 1 9 8 7 ) . W h e n indomethac in w a s adminis tered to normal mice g iven an hematos t imu la to ry dosage of r lL -1a, spleen cel lular i ty and levels of splenic CFU-GM w e re fu r ther increased (Pelus, 1 9 8 9 ) . Since indomethac in 138 accentua ted the hematopo ie t ic response to Photofrin® by the sp leen, it suggested tha t the porphyr in preparat ion in teracts d i rect ly or indi rect ly w i t h the arachidonic acid p a t h w a y , the reby p romot ing PGE 2 f o r m a t i o n . A l te rna t ive ly , indomethac in might release progeni tor cells f r o m inhib i tory cont ro ls exer ted by endogenous levels of PGE 2 rather t han by b lock ing an aspect of the physio logical response to Photofrin®. Increased levels of splenic CFU-GM were present in con t ro l mice given indomethac in , prov id ing indirect ev idence tha t indomethac in may indeed dampen the endogenous fo rmat ion of PGE 2 . To fur ther invest igate th is ac t i v i t y , the inf luence of i ndomethac in upon ery thropoies is in Photofr in®-treated mice requires s tudy . In the m a r r o w , Photofrin® increased relat ive levels of CFU-GM up to 4 0 % at 4 8 hours pos t - in jec t ion , before return ing to normal levels by 9 6 hours post-in jec t ion. H o w e v e r , no change in m a r r o w BFU-E levels were noted up to 7 2 hours pos t - in jec t ion . The t rea tmen t increased expression of CD71 in the m a r r o w at 4 8 hours pos t - in jec t ion . However , CD71 expression in th is t issue fell wel l be low cont ro l levels by 9 6 hours post - in jec t ion. Since s t rong express ion of CD71 in mur ine ery thro id t issues is associated w i t h CFU-E, but not BFU-E act iv i ty (Lesley etal., 1 9 8 4 ) , it is conceivab le tha t Photofrin® migh t have d imin ished levels of these later e ry thro id progeni tor cells either by p romot ing their matura t ion or egress f r o m the t issue. The relat ive expression of the myelo id-associated ant igen C D 1 1 b and granu locy te marker Gr-1 in the m a r r o w were great ly p romoted by Photofrin® by day 4 pos t - in jec t ion . Concomi tan t l y , relat ive expression of the B lymphocy te - res t r i c ted i so fo rm of C D 4 5 (B220) w a s at a s igni f icant ly lower level in the m a r r o w of Photofr in®-treated mice. In the absence of any change in BM nucleated cell 139 numbers , it appears l ikely t ha t Photofrin® p romoted granulopoiet ic ac t iv i ty w i th in the reg ion. W h e t h e r th is occur red at the expense of m a r r o w B lymphopoies is or ery thropoies is requires e luc idat ion. It is unclear whe the r the d imin ishment in m a r r o w CD71 express ion w a s a result of mobi l izat ion of e ry thro id progeni tors out of th is reg ion, a lower demand for iron in the t issue, or as the consequence of the increased number of myelo id progeny in th is reg ion. In con t ras t to the spleen, Photofr in® did no t alter the overal l f requency of L R - 1 + cells in the m a r r o w , but there w a s a d o w n w a r d shi f t in the average dens i ty of the LR-1 ant igen upon cells w i th in the t issue. Presumably th is marker is expressed at lower levels on g ranu locy tes , cells w h o s e p roduc t ion w a s p romoted by Photofr in®. Tha t BPD did not alter myelopoie t ic ac t iv i ty in the spleen or m a r r o w , indicates tha t hematopo ie t ic s t imula t ion is not an act iv i ty shared by all porphyr in photosens i t izers . The rapid c learance of BPD in vivo (Richter et al., 1990) may l imit any abi l i ty of the c o m p o u n d to inf luence hematopo ie t ic progeni tor ac t iv i ty . Whi le , the monomer ic c o m p o n e n t s in Photofrin® are cleared rapidly in vivo (Bellnier et al., 1 9 8 9 ) , its o l igomers are detectable in var ious mur ine t issues for ex tended periods (4-8 weeks) f o l l ow ing its admin is t ra t ion (Bellnier et al., 1 9 8 9 ) . However , it is uncer ta in w h e t h e r " res idence t i m e " w i th in the organ ism is a cr i t ical fac tor whe the r or no t a porphyr in produces a hematos t imu la to ry e f fec t . In suppor t of th is c o n t e n t i o n , nei ther Hp (the monomer ic equivalent of Photofrin®) nor HVD (Chapter 5) or PPIX, all rapidly metabol ized monomer ic porphyr ins present in Photofrin®, had any s ign i f icant hematopo ie t ic ac t iv i ty in the mouse . A l t h o u g h , PPIX, the immediate precursor of heme, did not s igni f icant ly 1 4 0 af fec t spleen w e i g h t , mice g iven PPIX had lower to ta l and CD71 + nucleated spleen cell numbers than the cont ro ls . This suggests t ha t PPIX migh t have inhibi ted the splenic hematopo ie t ic apparatus in these mice. A f te r Photofrin® admin is t ra t ion , nei ther IL-1 a nor GM-CSF were detectab le in the sera of t reated or cont ro l mice. A l t h o u g h , these data were not in fo rmat ive in te rms of unders tand ing the mechan ism of Photofr in®-induced hematos t imu la t ion , t hey do indicate tha t the response w a s not comparab le to tha t el ici ted by endo tox in ( l ipopolysacchar ide) . This bacter ial cons t i tuen t may con tamina te biological or d rug preparat ions and cause sys temic immunolog ic ac t iva t ion (Morrs ion and Ryan, 1979) or hematopo ie t ic s t imula t ion ( A i n s w o r t h , 1989) w h i c h is marked by the appearance of co lony-s t imu la t ing act iv i ty in the c i rcu lat ion (Morr ison and Ryan, 1 9 7 9 ) . However , IL-6 w a s present in the serum of bo th con t ro l and Photofr in®-treated mice f r o m 6 to 4 8 hours post - in jec t ion . The appearance of IL-6 in the c i rcu lat ion w a s most l ikely a consequence of a stress-induced response el ici ted by the in ject ion procedure. A major ac t ion of IL-6 is to p romote the fo rma t ion and release of acute phase prote ins f r o m the liver (Van Snick, 1 9 9 0 ) . These fac to rs appear to buf fer s t ress- induced b iochemical changes w i th in the o rgans im. The earl iest phenotyp ic ev idence of the spleen cell ac t iva t ion caused by Photofr in® w a s an increase in the expression of the receptor for Trf (CD71) w i t h i n 2-3 days post - in jec t ion . The TrfR is s t rong ly upregulated on act ivated and pro l i ferat ing cells (Kemp etal., 1 9 8 7 ) . Since the mur ine spleen leukocy te populat ion is normal ly compr ised of 6 5 - 8 0 % l ymphocy tes , the mos t s t ra igh t fo rward 141 explanat ion for an increase in CD71 expression w o u l d be tha t an act ivat ion of splenic T and/or B cells had occur red . However , the baseline expression of the largely l ymphocy te - res t r i c ted IL-2 receptor a-chain (CD25) in the spleen at all sampl ing t imes examined and unal tered spleen cell responsiveness to the l y m p h o c y t e g r o w t h fac to r IL-2 suggested tha t l y m p h o c y t e ac t iva t ion had not occur red . Dual co lour f l o w cy tomet r i c analyses indicated tha t the greater splenic CD71 express ion in Photofr in®-treated mice w a s at t r ibutable to nucleated cells tha t also expressed the ery thro id-assoc ia ted TER-119 ant igen or the marker ident i f ied w i t h the monoc lona l an t ibody L R - 1 , but not those bearing l ympho id (Thy -1 .2 , CD4, CD8 , IgM) or myelo id (Gr-1) markers . Further analysis indicated tha t the LR-1 ant igen w a s also expressed by cells w h i c h also possessed the TER-119 marker. It w a s there fore not surpr is ing tha t CD71 and TER-119 also possessed an over lapping express ion pa t te rn . Overal l , these studies suggested tha t the LR-1 marker is s t rong ly expressed by ery thro id l ineage cells and tha t the major impact of Photofr in® w i t h i n the spleen w a s to increase the ery thropo ie t ic ac t iv i ty of the o rgan . This w a s borne ou t by progeni tor assays w h i c h demons t ra ted tha t Photofr in® s t rong ly increased splenic BFU-E and CFU-E progeni tor levels. In order to meet the h igh cellular demand for iron required in hemoblob in syn thes is , CD71 is expressed at h igh levels on develop ing ery thro id cells and are discernable on cells at the CFU-E stage and on ery throb las ts but at l ow levels on cells w i t h BFU-E potent ia l and absent f r o m mature red cells (Lesley et al., 1 9 8 4 ) . Thus , the increased numbers of cells w i t h CFU-E ac t iv i ty and ery throb las ts l ikely accoun t for 142 the e levated CD71 express ion in the spleens of Photofr in®-treated mice. The TER-119 ant igen w a s s h o w n to be expressed on ery throb las ts and on mature red cells but absent f r o m precursor cells w i t h BFU-E or CFU-E potent ia l ( Ikuta e f al., 1 9 9 1 ) . Spleen cells w i t h a CD71 + / T E R - 1 1 9 " expression pat tern may represent cells w i t h CFU-E poten t ia l , those w i t h a C D 7 1 + / T E R - 1 1 9 + pheno type are presumably ery throb las ts whereas cells w i t h a CD717TER-11 9 + pheno type may cor respond to cells at the normob las t s tage. H o w e v e r , the LR-1 ant igen is mainta ined at high levels t h r o u g h o u t these stages of ery thro id deve lopment . The observat ion tha t Photofrin® also s t rong ly p romoted splenic ery thropoies is in DBA/1 strain mice demons t ra ted the hematos t imu la to ry response to th is preparat ion is not s t ra in-re lated. In the spleens of these mice , the porphyr in mix ture e levated organ w e i g h t , cel lu lar i ty, levels of CFU-E and BFU-E, the relative express ion of the LR-1 ant igen and the binding of a f luoresceinated-Tr f probe. Of part icular note w a s the dramat ic exaggerat ion in the magn i tude of the br ight ly labell ing second LR-1 + peak w h e n spleen cells f r o m Photofr in®-injected DBA/1 mice were examined by f l o w cy tomet r i c analysis. This suggested the presence of large numbers of deve lop ing ery thro id cells in the o rgan. The elevated b inding of FITC-labelled Tr f t o spleen cells f r o m Photofr in®-injected DBA/1 mice fu r ther suppor ts the v i e w tha t Photofr in® p romotes mur ine splenic ery thropoies is . Iron is del ivered to cells by means of Tr f upon its b inding to its high af f in i ty cell sur face receptor C D 7 1 . In hematopo ie t ic t issues, the h ighest demand for iron is tha t by developing ery thro id cells w h i c h need large amoun ts of th is e lement in order to meet the requ i rements of hemoglob in synthes is (Ponka, 1 9 9 4 ) . 143 A s igni f icant increase in c i rculat ing levels of nuc leated cells was determined for mice g iven Photofrin® seven days prev ious ly . A n increase in blood l y m p h o c y t e numbers w a s largely responsible for the increased w h i t e cell coun t . No s ign i f icant change in the blood hematocr i t or in e ry th rocy te numbers w a s ev ident for these animals at th is sampl ing t ime . The explanat ion for the greater numbers of c i rcu la t ing l y m p h o c y t e s in the blood of Photofr in®-treated mice is not ev ident , a l though the enhanced splenic and m a r r o w myelopoie t ic ac t iv i ty may have p romoted the release of l ymphocy tes f r o m these sites into the c i rcu la t ion. The observat ion tha t Photofr in® produced a d imin ishment in the number of m a r r o w cells w h i c h expressed the B lymphocy te - res t r i c ted iso form of CD45 (B220) suggests tha t the c o m p o u n d may have acted in th is fash ion . In s u m m a r y , Photofrin® produced substant ia l , ye t d is t inct , hematopo ie t ic and phenotyp ic changes w i th in the m a r r o w and spleen of normal mice. In the m a r r o w , of Photofr in®-treated mice granulopoiesis became the p redominan t hematopo ie t ic ac t iv i t y , wh i le in the spleen the hematopo ie t ic response w a s represented by a large increase in ery thropoie t ic ac t iv i ty . It w i l l be impor tan t to determine w h e t h e r these represent t issue-speci f ic responses or whe the r the response inc ludes a mobi l izat ion of m a r r o w ery thro id progeni tors to the spleen due to the spacial l imi tat ions of the femora l cav i ty . The determinat ion of hematopoie t ic progeni tor levels w i t h i n the m a r r o w and peripheral b lood of Photofr in®-treated, sp lenectomized mice m igh t help reveal the ta rge t spec i f ic i ty of the c o m p o u n d . The possibi l i ty tha t l ight plays a role in the hematopo ie t ic response to Photofrin® has not been fu l ly exp lo red . If l ight is required in order to t r igger this 1 4 4 response, th is w o u l d imply t ha t l ight -act ivated Photofrin® may have a cell signal l ing role in the ac t iva t ion of hematopo ie t ic progeni tor cells or hematopo ie t ic accessory cel ls. Studies by Y a m a m t o et al. (1994) indicated tha t the phagocy t i c ac t iv i ty of mur ine per i toneal macrophages w a s s t imulated in mice g iven l o w amounts (15 pg /kg ) of Photofr in® in t ramuscular ly and mainta ined under ambient l ight. If mice were kept in the dark for up to 4 days after Photofrin® w a s g i ven , macrophage ac t iva t ion w a s no t demonst rab le . W h e n Photofrin® w a s g iven at doses of greater than 1 5 0 / / g / k g , macrophage phagocy t ic ac t iv i ty w a s suppressed (Yamamto et al. 1 9 9 4 ) . Monomer i c Hp w a s also s h o w n to s t imulate macrophage phagocytos is / /? vivo and ambient l ight w a s necessary for th is e f fec t (Yamamto et al., 1 9 9 4 ) . H o w e v e r , as the porphr in photosensi t izers PPIX, BPD and Hp are not hematos t imu la to ry in normal mice housed under cond i t ions ident ical t o those given Photofr in®, l ight may not be a requisi te fac tor for the hematopo ie t ic response to Photofr in®. It may be possible to examine th is relat ionship by expos ing Photofrin®-t reated mice to k n o w n amoun ts of ambient l ight and gaug ing the magn i tude of the hematopo ie t ic response. The inf luence of Photofrin® on mur ine peri toneal cell f unc t i on is examined in Chapter 5. A n al ternat ive explanat ion for the hematos t imu la to ry act ion of Photofrin® is t ha t the preparat ion is d i rect ly tox ic to hematopo ie t ic precursors or t h r o u g h in teract ion w i t h l ight the c o m p o u n d causes hematopo ie t ic damage w h i c h mus t be co u n te ra c te d . H o w e v e r , Photofrin® had no inhib i tory e f fec t upon spleen or m a r r o w CFU-GM levels at all sampl ing t imes after Photofr in® adminis t rat ion ind icat ing the absence of a general ized tox ic e f fec t against hematopoie t ic 145 progen i to rs . It is uncer ta in w h e t h e r the decrease in l ympho id ( C D 4 5 R - B 2 2 0 + ) and ery thro id (CD71 + ) cell levels in the BM is a result of speci f ic g r o w t h inhibi t ion of these cell l ineages or the consequence of increased myelo id cell fo rmat ion in the t issue. It has been observed tha t splenic eythropoies is is great ly increased in mice t reated w i t h pheny lhydraz ine , a c o m p o u n d tha t p roduces hemoly t i c anemia (Kay et al., 1 9 9 1 ) . H o w e v e r , it is unl ikely t ha t Photofrin® precip i tates a similar sequence of even ts . It w a s indicated tha t large doses ( < 1 0 0 mg/kg) of Photofrin® did not cause red cell hemolys is in the rat, even t h o u g h the mater ial w a s s h o w n to associate w i t h peripheral b lood e ry th rocy tes of these animals (Booth etal., 1986 ) . Thus , Photofr in® p romotes speci f ic progeni tor ac t iv i ty w i th in the spleen and m a r r o w of normal mice and these changes do not appear to be a response to hematopoiet ic damage p roduced by th is photosensi t izer . 146 CHAPTER FOUR INFLUENCE OF PORPHYRINS ON THE HEMATOPOIETIC RECOVERY OF MICE TREATED W I T H ^ R A D I A T I O N OR 5-FLUOROURACIL 4 . 1 . In t roduc t ion The studies descr ibed in th is chapter were per fo rmed in order to determine w h e t h e r Photofr in®, H V D , or FeHp migh t produce hematopoiet ic responses in mice g iven d i f fe rent myelosuppress ive t rea tmen ts . The major ant ic ipated cl inical use of agents w i t h hematos t imu la to ry ac t iv i ty wi l l be to accelerate the return of normal hematopo ie t ic ac t iv i ty in pat ients t reated w i t h chemotherapeut i c agents or o ther modal i t ies w h i c h are m a r r o w ablat ive. In order to reduce t u m o u r or leukemic cell bu rden , s t rong medical in te rvent ion is o f ten emp loyed . T rea tment w i t h ionizing ^-radiat ion or one or a comb ina t ion of a w ide spec t rum of chemotherapeut ic agents such as 5-f luorouraci l (5-FU) or me tho t rexa te , a lky lat ing agents such as c/s-p lat inum or cyc lophosphamide or ant ib io t ics represented by adr iamycin and m i t o m y c i n C may be required. Un fo r tuna te ly , many of these t rea tments exer t ca tas t roph ic e f fec ts upon the immunohematopo ie t i c ax is , reducing numbers of c i rcu lat ing leukocytes and BM c o m m i t t e d progeni tor cel ls, thereby immunocompromis ing the subject . However , the s low ly cyc l ing HSC may be largely spared after in tensive immunosuppress ive therapy (Harr ison and Lerner, 1 9 9 1 ) . The hematopo ie t ic recovery inst igated by the HSC or t ha t f o l l o w i n g auto logous BM t ransp lanta t ion may be insuf f ic ient to prevent oppor tun is t i c in fec t ions in the immunosuppressed host . There fore , it is of great impor tance to ident i fy fac tors w h i c h p romote s tem cell ent ry into the cell cyc le 147 and/or ampl i fy the g r o w t h of c o m m i t t e d hematopo ie t ic progeni tor cel ls. It has been clearly demons t ra ted tha t a number of recombinant cy tok ines inc luding IL-1 (Moore and W a r r e n , 1 9 8 7 : Gal l icchio, 1 9 8 8 ) , G-CSF or GM-CSF (Tan ikawa et al., 1989) enhance hematopo ie t ic recovery w h e n admin is tered to myelosuppressed mice and humans . Radioprotect ive e f fec ts have also been noted for some cy tok ines . For example , if r lL-1 is adminis tered to mice prior to exposure to a lethal dose of ^-radiat ion a s igni f icant improvemen t in survival rates are observed (Neta et al., 1 9 8 6 ) , either by b lock ing the ent ry of HSC into the cell cyc le and/or induc ing the fo rmat ion of rad ioprotec t ive fac to rs . In addi t ion, cer ta in microbia l s t ruc tura l c o m p o n e n t s such as g lucan act as hematopoiet ic s t imulants in mye losuppressed mice and indirect ly s t imulate hematopoie t ic precursor cells t h r o u g h an ac t iva t ion of macrophages . Cant i et al. (1984) s h o w e d tha t mul t ip le in ject ions of HpD could accelerate hematopo ie t ic recovery of mice t reated w i t h p-radiat ion, 5-FU or cyc lophosphamide . This g roup w e n t on to s h o w tha t Photofr in® posi t ive ly in f luenced hematopo ie t ic indices of mice exposed to a sub- lethal dose of p-radiation (Canti e r a / . , 1 9 8 9 ) . The ant i -metabol i te 5-FU (Figure 4 .1) drast ical ly reduces numbers of myelo id and l ympho id cells and their precursors (Vetv icka et al., 1 9 8 6 ; Van Zant , 1 9 8 4 ) . Rapidly pro l i ferat ing cells are mos t vulnerable to the e f fec ts of 5-FU (Lerner and Harr ison, 1 9 9 0 ) . A metabol ic der ivat ive of 5-FU acts by inhib i t ing the act iv i ty of t hym idy la te syn the tase , the enzyme w h i c h catalyzes the methy la t ion of deoxyur id ine monophospha te to deoxy thymid ine m o n o p h o s p h a t e , an obl igatory s tep in DNA synthes is . This produces an abnormal re lat ionship b e t w e e n normal 148 levels of RNA and prote in whi le DNA remains in a def ic ient s ta te , leading to a s i tuat ion of cellular imbalance and cell death (Lerner and Harr ison, 1 9 9 0 ) . However , the HSC appears largely resistant to 5-FU in t ha t the drug drast ical ly reduces levels of c o m m i t t e d progeni tors and c i rcu lat ing leukocytes but does not compromise the abi l i ty of HSC to execute long t e r m repopulat ion and in fac t enr iches for their ac t iv i ty in BM (Lerner and Harr ison, 1 9 9 0 ) . Thus , exper imenta l ly 5-FU t rea tment may be used to evaluate var ious therapies on their capac i ty to accelerate hematopo ie t ic recovery t h r o u g h an act ion w i th in the HSC c o m p a r t m e n t (Moore and W a r r e n , 1 9 8 7 ) . o II N C — F I II X CH H Figure 4 . 1 Chemica l s t ruc tu re of 5- f luorouraci l (5-FU). In the present set of exper iments , w e sough t to evaluate the impact of Photofr in® on the hematopo ie t ic recovery of mice t rea ted w i t h either a mye losuppress ive dosage of 5-FU ( 1 5 0 mg/kg) or sub- lethal doses (4-7 Gy) of ir-rad ia t ion. In add i t ion , H V D , a monomer ic cons t i tuen t of Photofr in®, w a s evaluated for its ac t iv i ty in H r r a d i a t e d mice. Finally, FeHp, a meta l loporphyr in w i t h o u t 149 photosens i t iz ing ac t iv i ty w a s evaluated for its hematopo ie t ic ac t ion in sub-lethal ly i r radiated animals. 4 . 2 RESULTS 4 . 2 . 1 Hematopo ie t ic recovery in sub- lethal ly i r radiated mice t reated w i t h Photofr in® (10 mg/kg) 1 and 4 days post- i r radiat ion Wi th in 2 4 hours fo l l ow ing who le body- i r rad iat ion (4 Gy) , relative spleen w e i g h t s fell to less than 5 0 % of normal and fell to less than 3 0 % of normal by day 4 post- i r radiat ion (Figure 4 . 2 A ) . Splenomegaly w a s f i rst no ted in Photofrin®-t reated mice by 11 days post- i r radiat ion and reached a m a x i m u m value (mean relat ive spleen w e i g h t x 1 0 0 = 0 . 5 3 7 ± 0 . 0 6 1 ) 15 days post - i r rad iat ion, s ign i f icant ly greater t han tha t observed in in tact ( 0 . 3 5 9 ± 0 . 0 1 2 ; P< 0 . 0 0 0 1 ) and irradiated con t ro l ( 0 . 3 3 2 ± 0 . 0 3 8 ; P< 0 .025 ) mice. Nucleated spleen cell numbers fell to approx imate ly 5 % of normal levels by 2 4 hours post- i r radiat ion (Figure 4.2B) and remained at less than 1 5 % of normal (Day 0) values for up to 10 days in bo th cont ro l and Photofr in®-treated mice. Spleen cell numbers were s igni f icant ly (P< 0 . 0 0 5 ) greater in Photofr in®-treated mice 6 and 7 days post - i r rad ia t ion. Spleen cel lu lar i ty increased dramat ica l ly in bo th groups by 11 days post - i r rad iat ion. For the f inal 10 days of the exper iments , spleen cell numbers in the solvent- in jected cont ro ls remained at approx imate ly 4 0 % of normal va lues. Over the same per iod, spleen cel lu lar i ty in Photofr in®-treated animals ranged b e t w e e n 5 5 - 9 0 % of normal va lues and w e re s igni f icant ly greater than those of the so lvent - in jected cont ro ls on all sampl ing days . 150 I rradiat ion decreased to ta l splenic CFU-GM to < 1 % of normal values f r o m days 1-7 post- i r radiat ion (Figure 4 .2C) . Splenic CFU-GM levels were greater in Photofr in®-treated mice than in the irradiated cont ro ls at all sample t imes over the f inal 1 5 days of the exper iment , r ising to a value greater than t w i c e tha t of normal mice 14 days post- i r radiat ion and achiev ing stat ist ical s igni f icance 7, 1 1 , 14 and 21 days post- i r radiat ion compared to the irradiated cont ro l mice. BM cell numbers in bo th g roups osci l lated s o m e w h a t dur ing the post- i r radiat ion per iod. BM cel lulari ty fell to 5 0 % of normal levels w i th in 3 days of i r radiat ion in bo th groups (Figure 4 . 2 D ) . BM cell numbers were increased by day 4 post- i r radiat ion and then fell to < 4 0 % of normal va lues in the solvent- in jected cont ro ls by 7 days post- i r radiat ion. BM cel lu lar i ty exceeded normal values 4 days post- i r radiat ion in mice given Photofr in® and ranged b e t w e e n 6 0 - 9 5 % of normal values in bo th t rea tmen t groups over the f inal 13 days of the exper iment . In each g roup , BM CFU-GM were < 4 % of normal va lues w i t h i n 2 4 hours and remained at baseline values up to 7 days post- i r radiat ion (Figure 4 .2E) . Mice adminis tered Photofrin® achieved normal levels of BM CFU-GM by day 10 post - i r rad ia t ion, t w o days earlier than the solvent-in jected con t ro ls . Peripheral b lood leukocyte concen t ra t ions decreased to approx imate ly 2 0 % of normal values by 2 days post- i r radiat ion and were w ide ly var iable w i t h i n each t rea tmen t g roup unti l t hey stabi l ized by day 14 post- i r radiat ion (Figure 4 .2E) . Blood leukocy te levels of Photofr in®-treated mice were higher than those of the so lvent - in jec ted animals at all sampl ing t imes f r o m day 14 to day 21 post - i r rad ia t ion, a l though these d i f ferences achieved stat is t ical s igni f icance only on day 15 post - i r rad ia t ion. Normal b lood leukocy te concen t ra t ions were achieved by 151 T i m e p o s t - i r r a d i a t i o n ( d a y s ) Figure 4 . 2 In f luence o f Photofr in® on the hematopo ie t ic recovery of sub- lethal ly i rradiated (4 Gy) mice. (A) Relative spleen w e i g h t s , (B) spleen cel lu lar i ty , (C) levels of splenic CFU-GM, (D) BM cel lu lar i ty , (E) levels of BM CFU-GM, and (F) nuc leated blood leukocy te numbers were determined for mice irradiated (4 .0 Gy) on Day 0 and g iven either 5 % dext rose ( • ) or Photofrin® (10 m g / k g , O ) 1 and 4 days later. Day 0 resul ts cor respond to the combined results obta ined w i t h 2 2 un t rea ted , age-and sex-matched DBA/2 mice sacr i f iced at d i f fe rent t imes over the exper imenta l per iod. Results for each data point were obta ined for 5 -10 mice per g roup . T w o exper iments employ ing these cond i t ions were per formed and yielded highly similar results w h i c h w e re comb ined for presentat ion purposes. + , P < 0 . 0 5 ; + + , P < 0 . 0 2 5 ; * , P< 0 . 0 1 ; * * , P < 0 . 0 0 5 . 152 Con A (ug/ml) Figure 4 . 3 Prol i ferat ive response to Con A by spleen cells f r o m irradiated mice . M i togen ic responses were determined for spleen cells obta ined f r o m mice tha t w e re sub- lethal ly (4 Gy) i rradiated (A) 8 or (B) 15 days prev iously and g iven 5 % dext rose ( • ) or Photofrin® (10 m g / k g ; O ) 1 and 4 days after i r radiat ion. Naive mice ( • ) were assayed in parallel on each sampl ing day. Each data point represents the mean result obta ined for 4-5 mice. M T T assay results are presented as absorbance ( 5 9 0 nm) values obta ined in cu l ture wel ls conta in ing mi togen sub t rac ted f r o m the result obta ined in wel ls conta in ing cells alone. Mean absorbance values in cu l tures conta in ing cells alone were 0 . 0 9 5 ± 0 . 0 1 2 , 0 . 0 8 4 ± 0 . 0 0 4 , 0 . 1 4 0 ± 0 . 0 1 2 and 0 . 1 2 2 ± 0 . 0 0 9 , 0 . 1 3 5 ± 0 . 0 1 3 , 0 . 1 0 3 ± 0 . 0 1 6 on day 8 and day 15 post- i r radiat ion for naive, i rradiated cont ro l and irradiated Photofr in®-treated mice, respect ive ly . * + P< 0 . 0 1 ; P < 0 . 0 5 for spleen cells f r o m so lvent - in jec ted , i rradiated mice compared w i t h the result obta ined w i t h spleen cells f r o m Photofr in®-injected, i r radiated mice. 153 mice in bo th t rea tmen t g roups by 21 days post - i r rad ia t ion. Spleen cells obta ined 8 or 15 days post- i r radiat ion f r o m solvent-in jected mice , responded more s t rong ly over a concen t ra t ion range of Con A than cells f r o m mice g iven Photofrin® (Figure 4 . 3 A , B ) . H o w e v e r , the prol i ferat ive responses to Con A by sp lenocytes f r o m mice w i th in bo th t rea tmen t groups at each sampl ing t ime were cons iderably less than those generated by spleen cells prepared f r o m unir radiated mice. 4 . 2 . 2 Hematopo ie t ic recovery by i r radiated mice t rea ted w i t h Photofrin® or H V D (10 mg /kg ) 1, 4 and 7 days post- i r radiat ion In an exper iment per formed to compare the e f fec ts of Photofrin® and HVD on the hematopo ie t ic recovery of sub- lethal ly mice, i rradiated (4 .8 Gy) mice g iven Photofr in® 1,4, and 7 days post- i r radiat ion exhib i ted s ign i f icant ly heavier spleens at days 8, 14 and 17 post- i r radiat ion compared to the irradiated solvent-in jected mice (Figure 4 . 4 A ) and s igni f icant ly greater nucleated spleen cell numbers than the so lvent - in jec ted cont ro l mice on all sampl ing days (Figure 4 .4B) . Levels of splenic (Figure 4 .4C) and BM (Figure 4.4E) CFU-GM were s igni f icant ly elevated 8 and 10 days (spleen) and 8, 10 and 21 days (BM) post- i r radiat ion in mice g iven Photofr in®. BM cel lu lar i ty w a s s igni f icant ly greater in Photofr in®-treated mice 21 days post- i r radiat ion (Figure 4 . 4 D ) . In cont ras t to Photofr in®, HVD did not s ign i f icant ly a f fec t any spleen or BM parameter w i t h i n sub- lethal ly i rradiated mice at any t ime po in t t es ted , excep t at day 17 post- i r radiat ion w h e n relat ive spleen w e i g h t s w e re greater in HVD- in jected animals compared to the so lvent - t reated mice 1 5 4 (Figure 4 . 4 A ) . Blood nucleated cell concent ra t ions were no t determined in this exper iment . 4 . 2 . 3 Hematopo ie t ic recovery b y i r radiated mice t rea ted w i t h FeHp Sub- lethal ly i rradiated (7 Gy) mice g iven FeHp (10 mg/kg) 2 4 hours prior to i r radiat ion and again 4 8 hours or 4 8 and 9 6 hours post - i r rad ia t ion, exhib i ted no s ign i f icant change in relat ive spleen w e i g h t (Figure 4 . 5 A ) bu t did demonst ra te s ign i f icant increases in spleen cel lular i ty (Figure 4.5B) and splenic CFU-GM (Figure 4 .5C) by day 15 post - i r rad ia t ion, compared to the so lvent - in jected cont ro ls . The single post - i r radiat ion dose of FeHp w a s associated w i t h the largest increase in splenic C F U - G M , g iv ing a result approx imate ly 5 0 0 % greater than tha t observed for bo th in tact and i rradiated cont ro l mice. A t day 11 post - i r rad ia t ion, a s igni f icant ly lower number of BM cells were present in mice g iven a to ta l of three inject ions of FeHp compared to animals tha t had received the con t ro l so lvent (Figure 4 . 5 D ) . H o w e v e r , no s ign i f icant d i f ference in BM CFU-GM levels w a s observed in mice t rea ted w i t h FeHp, relat ive to the cont ro l result at any sampl ing t ime (Figure 4 .5E) . 155 B D 0.5 0.4 'g? 0.3 °2 0.2 tn CD "eg CD QC 0.1 0.0 •V /\ \ \ / : \w V C D Q O J X CO C D CJ> CL> C D CO CO "53 O 0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 Time post-irradiation (days) Figure 4 . 4 Inf luence of Photofrin® and HVD on the hematopo ie t ic recovery of sub-lethal ly i r radiated (4 .8 Gy) mice. (A) Relative spleen w e i g h t s , (B) splenic cel lu lar i ty , (C) splenic CFU-GM levels, (D) BM cel lu lar i ty , and (E) BM levels of CFU-GM were determined for mice i rradiated (4 .8 Gy) on day 0 and g iven either 1 0 % DMSO in PBS ( • ), Photofrin® (10 m g / k g , • ) or HVD (10 m g / k g , 0 ) 1 , 4 and 7 days later. A t each sampl ing t ime , 4 or 5 mice f r o m each t rea tmen t g roup were eva luated. , P< 0 . 0 2 5 ; , P< 0 . 0 1 ; , P< 0 . 0 0 1 compared to so lvent - in jected cont ro l mice. 156 Figure 4 . 5 In f luence of FeHp on the hematopo ie t ic recovery of sub-lethal ly-i r radiated (7 Gy) mice. (A) Relative spleen w e i g h t , (B) splenic cel lu lar i ty , (C) splenic CFU-GM levels, (D) BM nuc leated cell numbers , and (E) femora l CFU-GM levels were determined for sub- lethal ly i rradiated (7 Gy) mice g iven the cont ro l so lvent ( • ), FeHp (10 m g / k g , i.p.) 4 8 hours later ( • ), or FeHp (10 m g / k g , i.p.) 4 8 and 9 6 hours (c ross-hatched) . Al l FeHp-treated animals also received FeHp (10 m g / k g , i.v.) 2 4 hours prior to i r radiat ion. Each t rea tmen t g roup cons is ted of 4-5 mice. Day 0 values for each parameter cor respond to the result obta ined for 2 7 in tact male D B A / 2 mice mainta ined under the same cond i t ions as the exper imenta l animals. P < 0 . 0 5 ; P < 0 . 0 2 5 ; P < 0 . 0 0 1 compared to the so lvent - in jected cont ro l mice. 157 4 . 2 . 4 Inf luence of Photofr in® on the myelopoie t ic recovery of mice t rea ted w i t h 5-FU A single dose of 5-FU (1 5 0 mg/kg) drast ical ly reduced relat ive spleen w e i g h t (Figure 4 . 6 A ) , spleen cel lular i ty (Figure 4 .6B) , levels of splenic CFU-GM (Figure 4 . 6 C ) , BM cel lu lar i ty (Figure 4 . 6 D ) , levels of BM CFU-GM (Figure 4 .6E) , and peripheral b lood nucleated cell numbers (Figure 4 .6E) . The magn i tude of the depress ion of these hematopo ie t ic indices w a s similar for mice g iven either the cont ro l so lvent or Photofrin® (10 mg/kg) one and 3 days af ter the 5-FU t rea tment . The f i rs t ind icat ion of hematopo ie t ic recovery w a s observed in the spleen by 6 days pos t -5 -FU. Spleen w e i g h t , cel lular i ty and levels of CFU-GM were greater in the Photofr in®-treated mice at all sampl ing t imes f r o m day 6 to day 9 of the exper iment , a l though mos t of these d i f ferences did not achieve stat ist ical s ign i f icance. By day 9 pos t -5 -FU, levels of splenic CFU-GM in the solvent- in jected cont ro l and Photofr in®-injected mice were approx imate ly 4- fo ld and 9-fold greater, respect ive ly , t han those of unt reated mice. A similar response pat tern was observed in the B M . Recovery in th is t issue w a s f i rst noted by day 7 post -5-FU and bone m a r r o w cel lu lar i ty and levels of CFU-GM were greater in the Photofr in®-treated mice on days 7 and 9. (Figure 4 . 6 D and E), but achieved stat is t ical s igni f icance only on day 7. No s ign i f icant d i f ference in b lood leukocy te concen t ra t ions be tween the t w o t rea tmen t g roups w a s observed at any sampl ing t ime (Figure 4 .6F) . L imited f l o w cy tomet r i c analyses per formed on spleen cells obtained f r o m mice t rea ted w i t h 5-FU 6 days prev ious ly , indicated tha t expression levels (percent posi t ive cells) of the LR-1 ant igen were similar for unt reated cont ro l animals, and mice g iven 5 % dext rose or Photofrin® (Figure 4 . 7 ) . H o w e v e r , tak ing 158 the cel lular i ty of the organ at th is sampl ing t ime into accoun t , it w a s est imated tha t the spleens of Photofr in®-injected mice conta ined a greater number of LR-1 + spleen cells than the spleens of so lvent - in jec ted, 5-FU-treated cont ro ls (Table 4 . 1 ) . In no exper iment , w h i c h tes ted the impact of porphyr in adminis t rat ion on the hematopo ie t ic recovery of sub- lethal ly i rradiated or 5-FU-treated mice, w a s ery thro id progeni tor ac t iv i ty eva luated. 159 Figure 4.6 Hematopo ie t ic response to Photofrin® by mice t rea ted w i t h 5-FU. (A) Relative spleen w e i g h t , (B) splenic cel lu lar i ty, (C) levels of splenic CFU-GM, (D) BM nucleated cell numbers , (E) femora l CFU-GM levels, and (F) peripheral b lood nucleated cell numbers were determined for mice t reated w i t h a myelosuppress ive dosage ( 1 5 0 mg/kg) of 5-FU on day 0 and given either 5 % dext rose ( • ) or Photofrin® (10 m g / k g , O ) 1 and 3 days a f t e r w a r d . The combined results of t w o ident ical ly per formed exper iments are s h o w n and each sample t ime cor responds to 3-9 mice per g roup . Day 0 values represent results obta ined w i t h 1 5 in tac t age- and sex-matched DBA/2 mice sampled w i th in the same t ime period as the exper imenta l animals. + , P < 0 . 0 5 ; * , P < 0 . 0 0 1 . 160 Table 4 . 1 Spleen cell expression of the LR-1 ant igen in normal mice and mice t reated w i t h 5-FU and g iven 5 % dext rose or Photofrin®. Pooled spleen cells (3 animals per g roup) , obta ined f r o m normal or mice t reated w i t h 5-FU 6 days before, were labelled w i t h the LR-1 monoc lona l an t ibody and analyzed by f l o w c y t o m e t r y . Trea tment Group Spleen cel lular i ty (x 10 6 / 2 0 g) L R - 1 + spleen cells (%) L R - 1 + spleen cells (x 10 6 / 2 0 g) Naive 4 1 . 7 3 ± 12 .57 7 0 . 6 2 9 . 5 5-FU + 5 % Dext rose 1 9 . 8 0 ± 4 . 0 7 2 . 2 14 .3 5-FU + Photofr in® 2 8 . 6 4 ± 1.17 7 4 . 3 2 1 . 3 161 Rat IgM LR-1 Naive 5-FU + 5% Dextrose 5-FU + Photofrin® Log Fluorescence Intensity (FITC) Figure 4 . 7 F low cy tomet r i c prof i les of LR-1 ant igen express ion on spleen cells f r o m mice t reated w i t h 5-FU. Mice were t reated w i t h 5-FU ( 1 5 0 mg/kg) on day 0 and given 5 % dext rose or Photofr in® (10 mg/kg) 1 and 3 days later. Spleen cells prepared f r o m three mice per g roup , 6 days after 5-FU w a s admin is tered, were pooled and labelled w i t h the LR-1 monoc lona l an t ibody. Spleen cells obta ined f r o m naive DBA/2 mice were evaluated in paral lel. Cel l-associated rat IgM w a s de tec ted w i t h a FITC-con jugated ant i - rat IgM reagent. The f l o w cy tomet r i c analysis w a s per formed w i t h a Coul ter Epics C cy tomete r . Percentage (%) posi t ive spleen cells (events to the r ight of the ver t ica l line) obta ined w i t h the isotype cont ro l or LR-1 is g iven w i th in each panel . 162 4 . 4 D i s c u s s i o n The photosensi t iz ing propert ies of non-metal l ic porphyr ins have been harnessed for the des t ruc t ion of mal ignancy but their potent ia l as hematos t imu la to ry agents has received litt le a t ten t ion . The body of w o r k reported herein indicates tha t the photosensi t izer Photofrin® can p romote hematopoiet ic recovery w h e n adminis tered to mice myelosuppressed w i t h 5-FU or ^-radiat ion in the absence of ac t iva t ing levels of l ight. Photofrin® w a s relat ively more ef fect ive w h e n g iven to sub- lethal ly i rradiated mice than those t reated w i t h the ant i -metabol i te 5-FU. These observat ions con f i rm the f ind ings of an earlier s tudy by Canti et al. (1989) t ha t c laimed tha t th is photosensi t izer could exer t posi t ive hematopo ie t ic e f fec ts in myelosuppressed mice. However , the exper imenta l readout in the Italian s tudy w a s restr ic ted to a determinat ion of nuc leated cell numbers in peripheral b lood and the hematopo ie t ic t issues, w i t h o u t any evaluat ion of hematopo ie t ic precursor ac t iv i ty . HVD, a monomer ic cons t i tuen t of Photofrin® had litt le ac t ion in ei ther the spleen or BM of irradiated mice suppor t ing the conten t ion tha t the o l igomer ic f rac t ion conta ins the hematos t imu la to ry act ive component (s ) . The tumour -pho tosens i t i z ing c o m p o n e n t also resides in the ol igomeric f rac t ion (Bellnier et al., 1 9 8 9 ) . The explanat ion for the lower e f f icacy of Photofrin® in the 5-FU model of mye losuppress ion , relat ive to the i rradiat ion s y s t e m , is uncer ta in . It is conceivab le tha t an accessory cell w h i c h part ic ipates in the hematos t imu la to ry response to Photofr in® may be vulnerable to the cy to tox i c e f fec ts of 5-FU thereby reduc ing the impac t of the porphyr in in the BM and spleen. It has been s h o w n tha t sub- lethal ly i r radiated mice were more responsive to the hematos t imu la to ry e f fec ts 163 of G-CSF those t reated w i t h 5-FU (Shimamura et al., 1 9 8 7 ) . Enhanced hematopo ie t ic recovery w a s observed in the spleen but not in the BM c o m p a r t m e n t of i rradiated mice g iven FeHp. This observat ion does not necessari ly s igni fy t ha t hematopo ie t ic precursors in these t issues di f fer in their sensi t iv i ty t o FeHp, but may ref lect the fac t t ha t hematopo ie t ic reconst i tu t ion in immunosuppressed mice is associated w i t h the mobi l izat ion of progeni tor cells into the c i rcu lat ion and an expans ion of the spleen as the major site of ext ramedul lary hematopoie is (Moore etal., 1 9 9 0 ) . Thus , in addi t ion to its s t imu la tory e f fec ts upon splenic CFU-GM, FeHp could also increase the mobi l izat ion of progeni tor cells f r o m the bone m a r r o w to the spleen. A var ie ty of agents inc luding rSCF (Fleming et al., 1 993) and rG-CSF (Pojda etal., 1990) p romote the redis t r ibut ion of hematopoiet ic progeni tor ac t iv i ty f r o m the m a r r o w to the spleen. In the exper iments in w h i c h mice were pre- t reated w i t h FeHp prior to i r radiat ion, it is conceivab le tha t the porphyr in migh t have amel iorated some of the d i rect e f fec ts of radiat ion. However , it is unl ikely tha t th is represents authent ic rad iopro tec t ion , such as tha t w h i c h may be promoted by rlL-1 (Neta et al., 1 9 8 6 ) , since FeHp did not prevent the nadir in BM or splenic cel lu lar i ty or CFU-GM tha t occur red by 8 days post - i r rad iat ion. However , it may indicate tha t a larger number of HSC w e re pr imed for ent ry into the cell cyc le by FeHp. It is of interest tha t FeHp exer ted posi t ive hematos t imu la to ry e f fec ts in sub-lethal ly i rradiated mice, since Hp itself exer ted no such e f fec t in normal mice (Chapter 3 ) . The chemica l insert ion of iron into its s t ruc tu re may have altered the molecular con f igura t ion of Hp thereby y ie ld ing a c o m p o u n d w i t h hematos t imu la to ry ac t iv i ty . 1 6 4 The f ind ings repor ted herein indicate tha t the hematopo ie t ic recovery suppor ted by Photofr in®, w h e n given 1 and 4 days fo l l ow ing the del ivery of a sub-lethal radiat ion dose , w a s more p ronounced t h a n w h e n three doses were p rov ided . Mice g iven the addi t ional dose of the drug exhib i ted higher levels of splenic and BM CFU-GM at early sampl ing t imes but fai led to produce the magn i tude of splenic and BM myelopoie t ic ac t iv i ty s t imulated by only t w o doses of the preparat ion. The explanat ion for the fai lure of mul t ip le Photofrin® in ject ions to fur ther augment myelopoies is is not readily apparent . Litt le in format ion is available concern ing the biological e f fec ts of repeated inject ions of Photofrin® since a single dose is character is t ica l ly g iven for PDT purposes. A l t hough the mice in the cur rent s tudy were not d i rect ly exposed to l ight, it is conceivable tha t repeated adminst ra t ion of Photofr in® cou ld lead to a bui ld-up of photosensi t izer in the t issues thereby render ing hematopo ie t ic precursors suscept ib le to p h o t o c y t o t o x i c damage. A t rans ient reduct ion in spleen size occur red in normal mice and g iven Photofrin® and a localized (hind l imb) l ight t rea tment (Gomer et al., 1 9 8 8 ) , indi rect ly suggest ing t h a t mur ine splenic hematopo ie t ic ac t iv i ty m igh t be a f fec ted by PDT. It has been indicated tha t the pho todynamic t rea tment of mur ine per i toneal macrophages causes the release of in f lammatory cy tok ines including TNF-a (Evans et al., 1990) and PGE 2 (Henderson and Donovan , 1 9 8 9 ) , bo th po ten t modu la tors of hematopoies is (Jacobsen etal., 1 9 9 2 ; Pelus, 1989a) . The int raper i toneal admin is t ra t ion of Photofrin® to normal mice w a s associated w i t h an inf lux of cells w i t h an in f lammatory granu locy t ic ( G r - 1 + ) pheno type into the per i toneal cav i ty (see Chapter 5) demons t ra t ing the existence 165 of a localized in f lammatory- l ike response, concur ren t w i t h the hematos t imulary events occur r ing in the BM and spleen. Admins t ra t ion of i ndomethac in , a prostaglandin synthes is inhibi tor , to normal mice g iven a single hematos t imu la to ry dose of Photofr in® fur ther increased splenic hypercel lu lar i ty and CFU-GM fo rmat ion , indicat ing tha t Photofrin® may inf luence the arachidonic acid metabol ic p a t h w a y and PGE 2 f o r m a t i o n , even in the absence of act ivat ing levels of l ight (see Chapter 3 ) . Similar i ly, adminst ra t ion of indomethac in to in tact mice g iven r l L - l a s t rongly augmented splenic CFU-GM fo rmat ion (Pelus, 1 9 8 9 b ) . It w a s s h o w n tha t the hematos t imu la to ry e f fec ts of IL-1 were part ial ly b lunted by the co- induc t ion of PGE 2 f o rmat ion and an inhib i tory act ion of Mac-1 + (CD11b) BM cells (Pelus, 1 9 8 9 b ) . The capac i ty of the hematopo ie t ic sys tem to respond to Photofrin® may be l imited and once a cer ta in level of hematopoie t ic ac t iv i ty is p roduced by the porphyr in preparat ion, negat ive regulatory fac tors may be induced. The pro t rac ted retent ion of Photofrin® c o m p o n e n t s w i th in the organism may pro long the act ion of these puta t ive negat ive hematopo ie t ic regulatory e lements . It has been s h o w n tha t the pro longed admins t ra t ion of cer ta in hematos t imu la to ry agents fo l low ing chemothe rapy may u l t imate ly stall the hematopoie t ic recovery by the concomi tan t induct ion of mye losuppress ive cy tok ines (Gasparet to et al., 1 9 8 9 ) . Sp lenocytes f r o m irradiated mice g iven t w o in ject ions of Photofrin® generated prol i ferat ive responses to the T cell mi togen Con A tha t were s ign i f icant ly less than t h o s e of cells f r o m solvent- in jected irradiated cont ro l mice at sampl ing t imes w h e n spleen cel lular i ty w a s s igni f icant ly greater in the Photofrin®-t reated animals. Canti et al. (1989) repor ted tha t spleen cells f r o m mice g iven a 166 large (75 mg/kg) Photofrin® dose 7 days fo l l ow ing sublethal i r radiat ion and one day prior to sacr i f ice produced greater spleen cell responses to Con A than irradiated cont ro l mice. This result is d i f f icu l t to explain g iven the brief exposure to the drug and in v i e w of the observat ion tha t Photofrin® exerts l itt le e f fec t on splenic T or B l y m p h o c y t e numbers w h e n given to normal mice. It is conce ivab le , however , tha t large doses of Photofrin® could p romote l ymphocy te sequest ra t ion in the spleen. In the present s t u d y , the inferior Con A response by spleen cells prepared f r o m Photofr in®-treated mice may be the consequence of a relat ively greater number of g ranu locy te -macrophage (and presumably erythroid) p rogeny in the spleens of these animals and/or the presence of cy tok ines w h i c h inhibi t l y m p h o c y t e pro l i ferat ion. In sub- lethal ly i rradiated and 5-FU-treated mice, there w a s no indicat ion tha t Photofrin® acts by accelerat ing the ent ry of early HSC into the cell cyc le , since myelopoie t ic recovery in Photofr in®-treated mice w a s not de tec ted unti l the hematopo ie t ic rebound w a s also observable, albeit at a lower level, in the cont ro l an imals. Un fo r tuna te ly , the techn iques to s tudy the impac t of Photofrin® on ery thropoies is in immunosuppressed mice were not in place w h e n these studies were pe r fo rmed . However , the deve lopment of sp lenomegaly in irradiated mice adminis tered Photofr in®, does suggest tha t the preparat ion may have p romoted ery thropo ie t ic ac t iv i ty in the o rgan. In mice pretreated w i t h 5-FU and then given Photofin®, l imited f l o w cy tomet r i c analyses indicated tha t the c o m p o u n d increased the number of spleen cells w h i c h expressed the LR-1 ant igen. For normal mice, it w a s s h o w n in Chapter 3, tha t Photofrin® also increased the number of spleen cells w h i c h expressed the LR-1 marker. This suggests tha t the splenic hematopoiet ic 167 response in mice t reated w i t h 5-FU shares cer ta in features w i t h the splenic response w h i c h occurs in normal mice. Presumably th is w o u l d include a posi t ive e f fec t on splenic ery thropoies is . A l t h o u g h , the relat ive e f fec t iveness of Photofrin® and recombinant hematopo ie t ic g r o w t h fac to rs were not compared exper imenta l ly , it is l ikely tha t recombinan t g r o w t h fac to rs such as GM-CSF and G-CSF are more potent in these sys tems than the porphyr in preparat ion. However , their shor t metabol ic half- l i fe demands tha t these cy tok ines are administered f requent ly (1 -3 t imes daily) in order to p roduce their max imal e f fec t iveness. Porphyr ins of fer the at t ract ive character is t ics of chemical s tabi l i ty , relat ively l ow synthes is cos t , and minimal sys temic tox i c i t y in the absence of d i rected l ight. H o w e v e r , pro longed skin photosens i t i v i t y in subjects t reated w i t h mult ip le doses of Photofr in® w o u l d be a major obstac le to its use as a hematopoie t ic s t imulant . Insert ion of metals into the terapyrro le r ing character is t ica l ly el iminates the photosensi t iz ing act iv i ty of the porphyr in . Thus , in order to consider the use of porphyr ins for hematos t imu la to ry purposes it w i l l be essential to fur ther evaluate the e f fec t iveness of non-photosens i t iz ing porphyr ins on hematopoies is in exper imenta l models of mye losuppress ion . 168 CHAPTER FIVE INFLUENCE OF PHOTOFRIN® ON THE HEMATOPOIETIC ACCESSORY ACTIV ITY OF PERITONEAL CELLS 5.1 In t roduc t ion The exper iments descr ibed w i th in th is chapter were per fo rmed in order to determine w h e t h e r the in t roduc t ion of Photofrin® into the per i toneal cav i ty might alter the hematopo ie t ic accessory act iv i ty of cells present w i th in th is region. The per i toneal cav i ty is a unique env i ronment in w h i c h special ized cells of the immune sys tem coord inate ly act to deter in fect ious agents f r o m establ ishing a foo tho ld in th is reg ion, w h i c h in heal thy animals is steri le. In add i t ion , immune cells in the per i toneal cav i ty possess d is t inc t phenotyp ic and func t iona l propert ies in compar ison to the equiva lent cells in other t issues. In a norma l , heal thy mouse approx imate ly 3-5 x 1 0 6 leukocy tes can be harvested f r o m the per i toneal space. Resident per i toneal cells (RPC) are compr ised o f approx imate ly 5 0 % B cel ls, 3 0 -4 0 % macrophages , 1 0 % T cel ls, and < 5 % granu locy tes (Aus tyn and Gordon, 1 9 8 1 ) . The overal l number and relat ive propor t ion of these cells can be great ly mod i f ied by in t raper i toneal admin is t ra t ion o f cer ta in agents . For example , a steri le ar- thioglycol late so lu t ion el icits a substant ia l increase in numbers of per i toneal g ranu locy tes w i t h i n 18 hours and macrophages w i th in 9 6 hours (Aus tyn and Gordon , 1 9 8 1 ) . Immune cells of the per i toneal cav i ty express t issue-speci f ic pat terns of sur face ant igens. A n atypica l B l ymphocy te popula t ion w h i c h exists w i th in the per i toneal and pleural spaces is phenotyp ica l ly d is t inc t f r o m the major i ty of B cells 169 of the sp leen, l ymph nodes and b lood . These l y m p h o c y t e s , des ignated B-1 cel ls, express sur face IgM and secrete Ig as do convent iona l B cel ls, bu t also express the pan-T cell s igna l - t ransduc ing CD5 ant igen and the macrophage-assoc ia ted integrin CD11 b an t igen . B-1 cells possess a remarkable capac i ty for se l f - renewal and appear to mainta in their numbers in the region by repl icat ion of fu l ly mature cells (Hayakawa et al., 1 9 8 6 ) . Dur ing on togeny , B-1 progeni tors can be de tec ted in fetal l iver and o m e n t u m bu t are essential ly absent f r o m adul t BM whereas convent iona l B cell p rogen i to rs are present in the fetal liver and adul t BM bu t absent f r o m fetal o m e n t u m (Kantor and Herzenberger, 1 9 9 3 ) . B-1 cells appear early in deve lopment and are present in the neonatal sp leen. In heavi ly i rradiated mice , syngeneic adult bone m a r r o w cells reconst i tu te convent iona l bone mar row-der i ved B cells but not B-1 cel l popu la t ions , fu r ther ind icat ing t h a t B-1 cells have a d is t inc t or igin (Hayakawa et al., 1 9 8 5 ) . This indicates tha t the deve lopmenta l potent ia l of hematopo ie t ic s tem cells in the generat ion of B cells may change over t ime (Kantor and Herzenberg, 1 9 9 3 ) . However , the B-1 and CD5" B cell d i c h o t o m y may be more comp lex since it has been s h o w n tha t t rea tmen t w i t h an t i - immunog lobu l in ant ibody and IL-6 can induce the expression of CD5 on CD5" splenic B cells (Ying-zi et al., 1991) . Mac rophages , a major resident per i toneal immune cell popu la t ion , express the Mac-1 ant igen C D 1 1 b (Springer et al., 1 9 8 1 ) , the macrophage-rest r ic ted F 4 / 8 0 ant igen (Aus tyn and Gordon , 1981) but l i t t le or no M H C Class II in normal mice . Elicited macrophages express l ow levels of M H C Class II ant igens and exh ib i t a l o w ac t iva t ion state (Ziegler et al., 1 9 8 4 ) . H o w e v e r , f o l l ow ing the 1 7 0 admin is t ra t ion of h ighly immunogen ic mater ials such as live bacter ia , per i toneal macrophages w i t h an ac t iva ted (MHC Class l l + ) pheno type are observed (Ziegler et al., 1 9 8 4 ) . Evidence has suggested tha t per i toneal macrophages in the adult mouse may or ig inate part ly or w h o l l y f r o m w i th in the per i toneal cav i ty as macrophage progen i to rs are demonst rab le w i th in the per i toneal cav i t y (Pelus etal., 1 9 7 9 ; Me tca l f et al., 1 9 9 2 ) . Transgenic mice over-express ing the gene for GM-CSF exhib i ted large increases in per i toneal macrophage numbers , but no change in levels of splenic or BM macrophage progeni tor levels as compared to animals expressing normal levels of t he GM-CSF gene (Metca l f et al., 1 9 9 2 ) . The per i toneal cav i ty may also represent a unique env i ronment for T l y m p h o c y t e s in t ha t it may serve as an ex t ra - thymic site of T cell d i f fe rent ia t ion . Neonata l t h y m e c t o m y p roduces a drast ic reduc t ion in splenic and l y m p h node T cell numbers bu t is associated w i t h an increase in C D 3 + / T h y - 1 . 2 + cells in the peri toneal cav i ty ( K r o e m e r e f al., 1 9 9 3 ) . These observat ions indicate tha t the peri toneal cav i ty may prov ide cond i t ions t h a t favour T cell d i f fe ren t ia t ion . The phys ica l separat ion of the immune sys tem into d is t inc t c o m p a r t m e n t s may serve to enhance the d ivers i ty of hos t p ro tec t ive mechan isms. It has been s h o w n tha t fac tors elaborated by per i toneal macrophages can produce cy tok ines w h i c h suppor t normal bone m a r r o w myelo id co lony fo rmat ion in vitro (Pelus et al., 1 9 7 9 ) . Since Photofrin® w a s adminis tered i.p., it w a s conce ivab le tha t the st imul i for Photofr in®-enhanced hematopoies is in the BM and spleen migh t be a consequence of an in teract ion w i t h cells w i t h i n th is d is t inct c o m p a r t m e n t . To address th is ques t ion , per i toneal cells we re obta ined at var ious 171 t imes af ter the drug w a s adminis tered and assessed for thei r : a) number , b) p h e n o t y p e , c) p roduc t ion of the hematopoie t ic cy tok ine GM-CSF, d) capac i ty to suppor t normal BM CFU-GM g r o w t h in vitro, and e) p roduc t ion of the react ive in termediate nitr ic ox ide (NO) in vitro. The data indicated tha t , a l though Photofrin® el ic i ted a large increase in per i toneal cell numbers and a change in the overal l compos i t i on of th is popu la t ion , no ev idence w a s obta ined tha t these changes were related to the myelopoie t ic responses w h i c h occur in the spleen and B M . 5 .2 Results 5 .2 .1 Inf luence of Photofrin® on the cel lular i ty and leukocy te prof i le w i th in the per i toneal cav i ty Wi th in 6 hours fo l l ow ing a single i.p. in ject ion o f Photofrin®, a s ign i f icant increase in to ta l per i toneal cell numbers w a s ev ident (Figure 5 .1) . Peritoneal cell numbers in Photofr in®-treated mice, a l though d isp lay ing considerable var iabi l i ty among indiv idual animals, were elevated 3-4 fo ld above cont ro l levels at 2 4 and 4 8 hours post - in ject ion and dropped to values tha t were approx imate ly t w i c e tha t of the cont ro ls at bo th the day 3 and day 7 sampl ing t imes . Mon i to r ing the compos i t i on of the per i toneal cell popula t ion f r o m 0 - 4 8 hours post - in jec t ion, indicated tha t a major increase in the number of leukocy tes w h i c h expressed the granu locy te marker Gr-1 (Ly-6G), occur red as early as 6 hours af ter the i.p. admin is t ra t ion of Photofrin® (Figure 5 .2) . Fur thermore, numbers of per i toneal cells w h i c h expressed the myelo id-associated ant igen CD 11b , as recognized by the monoc lona l an t ibody M 1 / 7 0 , were also s t rongly increased by the porphyr in prepara t ion . In con t ras t , there w a s a d iminu t ion in the propor t ion of per i toneal cells 172 w h i c h expressed the B cell i so fo rm of CD45 (B220) . The relat ive expression of the T cel l -associated marker T h y - 1 . 2 w a s litt le changed by the t rea tmen t . Taking the overal l cel lu lar i ty of the region into accoun t , Photofrin® produced a 5-10 fold increase in g ranu locy te numbers at the 2 4 and 4 8 hour sample t imes . F low cy tomet r i c prof i les of per i toneal cells obta ined 6 hours post-in ject ion are s h o w n in Figure 5 .3 . These analyses demons t ra te tha t even at th is early sampl ing t ime the relat ive expression of the Gr-1 and CD11 b sur face ant igens w a s increased in Photofr in®-treated animals, and concomi tan t l y the propor t ion of cells w h i c h expressed the C D 4 5 R - B 2 2 0 , I g M , or M H C Class II ant igens decreased. Expression of T h y - 1 . 2 , the macrophage-assoc ia ted F 4 / 8 0 ant igen and the receptor for t ransfer r in (CD71) among per i toneal cells were relat ively unchanged at th is sampl ing t ime . 5 . 2 . 2 Hematopo ie t ic accessory ac t iv i ty of per i toneal cells W h e n graded numbers of normal per i toneal cells we re mixed w i t h normal BM cells in the semi-sol id agar cul ture s y s t e m , w i t h o u t exogenous g r o w t h fac to rs , s ign i f icant numbers of g ranu locy te -macrophage colonies fo rmed (Figure 5 .4) . A t all sampl ing t imes (6 - 7 2 hours post- in ject ion) examined , the hematopo ie t ic accessory act iv i ty of per i toneal cells obta ined f r o m mice g iven Photofr in® i.p., w a s great ly inferior to t ha t of cells obta ined f r o m cont ro l mice. Litt le or no myelo id progeni tor ac t iv i ty w a s demonst rab le among per i toneal cells obta ined f r o m so lvent or Photofr in®-injected animals. 173 30 c o CD CD O CD CD Q_ 25 20 = 15 10 0 - J I I I I L_ _J I l _ 0 1 2 3 4 Days post-injection F i g u r e 5.1 P e r i t o n e a l ce l l n u m b e r s a f t e r P h o t o f r i n ® a d m i n i s t r a t i o n . Total numbers of viable peritoneal cells were determined from 6 hours to 7 days after the i.p. injection of 5% dextrose (•) or Photofrin® (25 mg/kg, O). At each sampling time 4-8 mice per treatment group were analyzed. Day 0 results correspond to the values obtained with untreated male DBA/2 mice (n = 8). P< 0.01 compared to solvent-injected controls by Student's t-test. 174 C O " C D C D C O o C D C D as C D CJ> as C O " C D O Time post-injection (hours) Figure 5 .2 Expression of d i f ferent leukocy te ant igens by per i toneal cells af ter in t raper i toneal Photofrin® admin is t ra t ion . Peritoneal cells were obta ined f r o m d i f fe rent mice 0 -48 hours after the i.p. in ject ion of 5 % dext rose ( • ) or Photofrin® (25 m g / k g , O ) and labelled w i t h monoc lona l ant ibodies react ive against T h y - 1 . 2 , C D 4 5 R - B 2 2 0 , C D 1 1 b , or Gr-1 sur face ant igens. Equal numbers of per i toneal cells obta ined f r o m 4 mice per t rea tmen t g roup were pooled and analyzed by f l o w c y t o m e t r y . Day 0 values cor respond to the result obta ined w i t h cells f r o m unt reated male DBA/2 mice. 175 5% Dextrose Photofrin J3 T | | m I, ijiiMi 1 O : > riilii'illiftii Anti-Rat IgG-FITC Thy-1.2 CD45R-B220 IgM F4/80 CD11b (M1/70) Gr-1 MHC Class II CD71 Fluorescence Intensity (FITC) Figure 5.3 F low cy tomet r i c prof i les of per i toneal cells obta ined 6 hours after the i.p. in ject ion of Photofrin®. Peritoneal cells obta ined 6 hours fo l l ow ing the i.p. admin is t ra t ion of 5 % dext rose or Photofrin® were evaluated for their expression of var ious leukocyte ant igens using a bat te ry of rat monoc lona l IgG ant ibodies. Cell sur face rat IgG w a s counter- label led w i t h a donkey FITC-conjugated ant i - rat IgG F(ab ' ) 2 reagent. Cell f luorescence in tens i ty w a s determined w i t h an Epics C f l o w cy tomete r . 176 To fur ther examine their hematopo ie t ic accessory ac t iv i t y , peri toneal cells obta ined 2 4 hours or 4 8 after the i.p. admin is t ra t ion of 5 % dext rose or Photofr in®, were cu l tured for a fur ther 2 4 hours . Cell-free supernatants were prepared and analyzed for GM-CSF levels by ELISA. GM-CSF w a s readily detectable in superna tan ts cond i t ioned by cells f r o m the cont ro l mice, exceed ing the levels achieved in cu l tures cond i t ioned by cells f r o m Photofr in®-treated mice by 5-10 fold (Figure 5 .5) . Peri toneal cell supernatants were also added at a f inal concen t ra t ion of 2 0 % to the rout ine BM cell co lony assay. The capaci ty of the med ium cond i t ioned by per i toneal cells f r o m the cont ro l mice to suppor t normal BM cell co lony fo rmat ion in vitro exceeded tha t by per i toneal cells obta ined f r o m Photofr in®-treated animals over 5-fold (Figure 5 .6) . H o w e v e r , the level of co lony fo rmat ion w a s considerably less than tha t observed w h e n an opt imal concent ra t ion of PWM-spleen cell cond i t ioned m e d i u m w a s used as the source of hematopo ie t ic g r o w t h fac to rs . To examine w h e t h e r the fac to rs produced by per i toneal cells f r o m Photofrin®-t reated mice m igh t exer t inhib i tory e f fec ts on normal BM co lony fo rma t ion , per i toneal cells w e re added to the co lony assay sys tem in the presence of opt imal a m o u n t s o f g r o w t h fac to rs as suppl ied by P W M - S C C M . H o w e v e r , l itt le impact on the number of colonies fo rmed in vitro w a s observed w h e t h e r per i toneal cells f r o m cont ro l or Photofr in®-treated mice were present (Figure 5 .7 ) . The capac i ty of Photofrin® to inf luence the fo rmat ion of the react ive in termediate NO w a s assessed. Peritoneal cells obta ined 2 4 hours post- in ject ion we re cu l tu red for a fu r ther 2 4 hours in phenol red-free m e d i u m conta in ing 1 0 % 177 F i g u r e 5.4 H e m a t o p o i e t i c a c c e s s o r y a c t i v i t y o f p e r i t o n e a l c e l l s . Graded numbers of per i toneal cel ls, lavaged f r o m mice g iven either 5 % dext rose ( • ) or Photofrin® (25 m g / k g , O ) i.p., were cu l tured in semi-sol id agar in the presence of 5 x 1 0 4 BM cells prepared f r o m naive male D B A / 2 mice. Mean ± s tandard dev ia t ion number of g ranu locy te -macrophage colonies fo rmed in t r ip l icate cu l tures are s h o w n . Cells obta ined at these sampl ing t imes were tes ted for accessory ac t iv i ty in at least t w o d i f fe rent exper iments , excep t for the 6 hour t ime po in t w h i c h w a s per fo rmed only once. Few colonies (0-1 per plate) fo rmed in vitro w h e n per i toneal cells f r o m either group were plated w i t h or w i t h o u t P W M -SCCM in the absence of BM cells. 178 Figure 5.5 GM-CSF levels in per i toneal ce l l -condi t ioned m e d i u m . Peritoneal cells obta ined either 2 4 or 4 8 hours af ter the i.p. admin is t ra t ion of 5 % dext rose ( • ) or Photofrin® ( • ) were cu l tured for 2 4 hours in I M D M conta in ing 1 0 % FCS. Cell-free supernatants were prepared and analyzed for GM-CSF levels by ELISA. 179 bm cells alone CD ^ bm cells + PWM-SCCM co 2^ bm cells + control pc O bm cells + Photofrin pc -1—j 25 50 75 100 125 150 175 Colonies / 5 x 104 BM cells plated Figure 5 .6 Capac i ty of per i toneal ce l l -condi t ioned med ium t o suppor t the in vitro g r o w t h of normal BM CFU-GM. Peritoneal cells (pc) isolated f r o m mice g iven 5 % dext rose or Photofrin® i.p. 2 4 hours previously were mainta ined in cu l ture for 2 4 hours . Cell-free supernatan ts ( 2 0 % ) prepared f r o m these cul tures we re assessed for their abi l i ty to suppor t the in vitro g r o w t h of normal BM myelo id progeni tors in co lony assays. Each t rea tmen t w a s tes ted in t r ip l icate cu l tures . 1 8 0 200 0 1 5 10 Peritoneal cells added (x 10'4) Figure 5 . 7 In f luence of per i toneal cells on BM CFU-GM deve lopment in the presence or absence of PWM-SCCM. Dif ferent numbers of per i toneal cel ls, obta ined 4 8 hours af ter the i.p. del ivery of 5 % dext rose (closed symbols) or Photofrin® (25 m g / k g ; open symbo ls ) , we re seeded into the semi-sol id agar cul ture sys tem w i t h or w i t h o u t normal BM cel ls, in the presence or absence of an opt imal concen t ra t ion of g r o w t h fac tors suppl ied by P W M - S C C M . Cul tures conta in ing per i toneal cells plus P W M - S C C M are denoted by t r iangular symbo ls , per i toneal cells plus BM cells by circular symbols , and per i toneal cells plus BM cells and P W M - S C C M are denoted by square symbols . 181 FCS. L o w levels of NO were detectable in cul ture med ium cond i t ioned by peri toneal cells f r o m bo th cont ro l and Photofr in®-injected animals (Figure 5 . 8 A ) . However , tak ing into accoun t the cel lular i ty of the peri toneal region at th is sampl ing t ime, overal l NO produc t ion by per i toneal cells f r o m Photofr in®-injected mice great ly exceeded tha t by the cont ro ls (Figure 5.8B) . To evaluate w h e t h e r the fo rmat ion of per i toneal hypercel lu lar i ty w a s rest r ic ted to the i.p. route of Photofrin® admin is t ra t ion , the drug w a s g iven i.v. and per i toneal cell numbers were determined 2 4 hours later. W h e n either 5 % dext rose or Photofr in® (25 mg/kg) w a s g iven i.v., no s igni f icant d i f ference in peri toneal cell numbers compared to unt reated mice w a s apparent (Figure 5 .9) . 182 CO 4 C O o 3 S 2 CJ> o Q _ 1 O 0 B „ 50 o ^ 40 o O 30 CD CJ> To 20 CD CD 10 Q . 0 LI Treatment Group Figure 5 . 8 Peri toneal cell ni tr ic ox ide (NO) fo rma t ion . (A) Peri toneal cel ls, obta ined f r o m mice adminis tered either 5 % dext rose ( • ) or Photofrin® (25 m g / k g , • ) 2 4 hours before , we re cu l tured at 1 x 1 0 6 cel ls /ml for 2 4 hours . Supernatant levels of NO were determined by the Griess reac t ion . Three mice per t rea tment group were analyzed. (B) Tota l peri toneal cell NO p roduc t ion w a s est imated by mul t ip ly ing NO fo rmat ion per 1 0 6 cells by the per i toneal cell y ie ld . Peritoneal cell numbers in th is exper iment cor responded to 2 . 8 7 ± 1.21 x 1 0 6 and 1 2 . 4 3 ± 0 . 7 9 x 1 0 6 for so lvent and Photofr in®-injected mice, respect ive ly . P< 0 . 0 5 compared to the result for so lvent- in jected con t ro l animals. 183 Figure 5.9 Compar ison of the route of Photofr in® admin is t ra t ion on peri toneal cell numbers . Normal male DBA/2 received an in ject ion ( 0 . 2 0 - 0 . 2 5 ml) of either 5 % dext rose or Photofr in® (25 mg/kg) intraper i toneal ly or in t ravenous ly . Mice were sacr i f iced 2 4 hours later and per i toneal cell numbers de te rm ined . Peritoneal cell y ields obta ined f r o m unt reated mice are also s h o w n . The number of mice tes ted w i th in each group is g iven in parentheses. * P < 0 .01 compared to all o ther t rea tment g roups . 1 8 4 5.3 Discussion The response of the cells w h i c h populate the per i toneal cav i ty upon the admin is t ra t ion of Photofrin® w a s of interest since it has been demonst ra ted tha t per i toneal cells e laborate fac to rs w h i c h suppor t the in vitro deve lopment of c o m m i t t e d BM myelo id progeni tors of normal mice (Pelus et al., 1 9 7 9 ) . Since Photofrin® w a s typ ica l ly g iven int raper i toneal ly , it w a s possible tha t the drug might have fu r ther enhanced per i toneal cell hematopoie t ic accessory f u n c t i o n , thereby accoun t ing for at least for some of the greater hematopo ie t ic ac t iv i ty in the spleen and BM of the porphyr in - t reated mice. However , th is w a s no t observed , since as early as six hours and up to at least 7 2 hours pos t - in jec t ion , hematopoiet ic accessory ac t iv i ty by per i toneal cells w a s s t rong ly reduced in mice g iven Photofrin® i.p.. The hematopo ie t ic g r o w t h fac tor GM-CSF w a s readily detectable in supernatan ts cond i t ioned by cells obta ined 2 4 or 4 8 hours post - in ject ion f r o m so lvent - in jec ted , but not Photofr in®-injected mice. Fur thermore, w h e n the 2 4 hour supernatan ts w e re tes ted in the rout ine progeni tor assay, per i toneal ce l l -condi t ioned med ium obta ined f r o m cont ro l bu t not Photofr in®-injected mice , readily suppor ted m a r r o w CFU-GM g r o w t h in vitro. W h e n per i toneal cells were added to cul tures conta in ing normal BM cells and an opt imal concent ra t ion of g r o w t h fac to rs , as suppl ied by P W M - S C C M , the presence of cells f r o m cont ro l or Photofr in®-treated mice did not alter the number of CFU-GM produced in vitro. The above observat ions indicate tha t the lower capac i ty of per i toneal cells f r o m Photofr in®-treated mice to suppor t myelo id g r o w t h is mos t l ikely at t r ibutable to l o w g r o w t h fac to r p roduct ion 185 and is not a consequence of a d i rect physical suppressive in teract ion of peri toneal cells w i t h m a r r o w progeni tors in the cul ture s y s t e m . Mye lo id progeni tor ac t iv i ty w a s essential ly undetectab le w i th in the per i toneal cell popu la t ion of cont ro l or Photofr in®-injected animals at all sampl ing t imes ind icat ing tha t the increase in the number of cells in th is region after Photofrin® admin is t ra t ion w a s not due to greater local progeni tor cell ac t iv i ty . The reduced accessory act iv i ty corre lated w i t h a large increase in to ta l and relat ive numbers of C D 1 1 b + and G r - 1 + per i toneal cells as indicated by monoc lona l an t ibody labell ing and f l o w cy tomet r i c analysis. Gr-1 (Ly-6G) is a g lycosy l -phosphat idy l inos i to l (GPI)-l inked cell sur face g lycopro te in (Fleming et al., 1993) w h o s e express ion is restr ic ted to mature g ranu locy tes (Hestdahl et al., 1 9 9 1 ) . The monoc lona l an t ibody M 1 / 7 0 (Springer et al., 1979) recognizes the integr in CD11 b w h i c h is typ ica l ly expressed by cells of the m o n o c y t e / m a c r o p h a g e l ineage (Springer et al., 1 9 7 9 ) , but may also be expressed by act iva ted neutrophi ls or B cell sub-popu la t ions . A l t hough the d imin ishment in myelopo ie t ic accessory could be largely a "d i l u t i on " of resident per i toneal cells associated w i t h a g ranu locy t i c in f lux , the reduced myelopoie t ic accessory ac t iv i ty w a s greater than the relat ive increase in per i toneal cell numbers , especial ly at the 6 hour and 72 hour sampl ing t imes . This suggests t ha t Photofrin® migh t have reduced hematopoie t ic cy tok ine p roduc t ion by cells w i th in th is region. Converse ly , h o w e v e r , fac tors f o rmed w i t h i n th is cell popula t ion may be more rapidly uti l ized by the peri toneal cells in the cu l ture sys tem if Photofrin® had in some w a y increased receptor-mediated uptake of these cy tok ines . A l t h o u g h granu locy tes are capable of 186 synthes iz ing a w i d e var ie ty of cy tok ines , a l though at m u c h lower levels than mononuc lear cells on a per cell basis (Cassatel la, 1 9 9 5 ) , their con t r ibu t ion to the main tenance of normal hematopoie is is is w ide ly bel ieved to be minor . Format ion of the react ive in termediate NO on a per cell basis was comparab le for per i toneal cells obta ined f r o m mice 2 4 hours af ter the inject ion of the con t ro l so lvent or Photofrin®. Therefore , the inferior in vitro hematopoiet ic accessory cell ac t iv i ty exhib i ted by per i toneal cells obta ined at th is sampl ing t ime f r o m Photofr in®-treated animals canno t be explained by an altered produc t ion of NO. H o w e v e r , w h e n the cel lular i ty of the region at th is sampl ing t ime w a s taken into accoun t , overal l NO fo rmat ion w i th in the per i toneal cav i ty of Photofr in®-treated mice w a s s ign i f icant ly greater. Whe the r , th is w o u l d be suf f ic ient to produce sys temic hematopo ie t ic e f fec ts is uncer ta in . NO has been s h o w n to exert s t rong i m m u n o m o d u l a t o r y (Weinberg et al., 1994) and hematopo ie t ic e f fec ts (Punjabi et al., 1 9 9 2 ) . It has been demons t ra ted tha t in human ery thro leukemic K562 cells (Weiss etal., 1993) and murine macrophages (Drapier et al., 1 9 9 3 ; Weiss etal., 1 9 9 3 ) , NO a f fec ts the s ta tus of the iron regulatory e lement b ind ing prote in (IRE-BP) leading to a stabi l izat ion of th is cy top lasmic fac tor and an increase in the express ion of the t ransfer r in receptor (Oria etal., 1 9 9 5 ; Weiss etal., 1 9 9 5 ) . Since an increase in CD71 expression o c c u r r e d ' ' i n the spleen after Photofrin® admin is t ra t ion and the increase in splenic CD71 expression w a s associated w i t h an increase in ery thropo ie t ic ac t iv i ty , NO may have cons t i tu ted a signal l ing event for ery thro id progeni tor ac t i v i t y . A n evaluat ion of NO sta tus in ery thropo ie t ic t issues after Photofr in® admin is t ra t ion w o u l d help to clar i fy th is issue. 187 It has been indicated tha t nannograbm amoun ts of HpD and Photofrin® increase the phagocy t i c ac t iv i ty of per i toneal macrophages of mice mainta ined under ambien t , bu t not l igh t -p ro tec ted , cond i t ions (Yamamoto etal., 1 9 9 4 ) . Greater doses ( > 1 5 0 / /g /kg) of these c o m p o u n d s were inhib i tory in th is regard (Yamamoto et al., 1 9 9 4 ) . A l t h o u g h these studies did not examine cy tok ine p roduc t ion by these cel ls, these f ind ings indicate tha t the doses of Photofrin® uti l ized in the present s tudy w o u l d l ikely inhibi t per i toneal macrophage func t ion in mice mainta ined under ambient l ight cond i t ions . The in f lammatory- l ike response w h i c h occurs w i t h i n the per i toneal cell popu la t ion appears to represent a bystander c o m p o n e n t of the overall hematos t imu la to ry response to Photofrin®, since splenic hypercel lu lar i ty occurs even w h e n the c o m p o u n d is g iven in t ravenously (Chapter 3 ) . It has been s h o w n tha t Photofr in® clears rapidly (exit ha l f - t ime; TVz = 3 0 minutes) f r o m the peri toneal cav i ty and moves into the c i rcu lat ion (Bellnier et al., 1 9 8 9 ) . This indicates tha t the c o m p o u n d qu ick ly becomes available to other t issues in the body w h e n admin is tered by th is route . However , the dramat ic cellular changes w h i c h take place w i t h i n the per i toneal cav i ty of Photofr in®-treated mice does indicate tha t the c o m p o u n d , never the less, has a s igni f icant physio logical impac t w i t h i n the region. 188 C H A P T E R S I X I N F L U E N C E O F P H O T O F R I N ® O N T H E I M M U N E R E S P O N S E 6.1 I n t roduc t ion In prev ious chapters , it w a s s h o w n tha t Photofr in® s igni f icant ly modi f ied ery thropo ie t ic and myelopoie t ic ac t iv i ty w i th in the m a r r o w and spleen. Since cells w h i c h func t i on in the deve lopment of ant igen speci f ic immun i t y are also present w i t h i n these t issues, it w a s impor tan t to determine w h e t h e r Photofrin® migh t also m o d i f y immune react iv i ty in mice g iven th is mater ia l . 6.1.1 The immune response Ver tebra tes have evo lved complex immunocel lu lar and b iochemical mechan isms to coun te rac t the cons tan t chal lenge by in fect ious agents in their env i ronment . Responses w i th in the immune sys tem are exer ted by t w o d is t inct ye t in t imate ly related par ts : a humoral c o m p o n e n t cons is t ing of soluble immunog lobu l ins (Ig), a p roduc t of B l ymphocy tes and a cellular cons t i tuent represented by C D 8 + c y t o t o x i c and C D 4 + helper T l y m p h o c y t e s . B cell l ymphopo ies is , w h i c h occurs in the fetal l iver and the adul t B M , is cons t i tu ted by the c o m m i t m e n t and matura t ion of hematopoie t ic s tem cells into mature B cells w h i c h co-express IgD and IgM on the cell sur face. T cells also or iginate in the m a r r o w but undergo a s t r ingent select ion and matura t ion process w i th in the t h y m u s . The immune response to a fore ign ant igen is bel ieved to commence 189 at the level of the dendr i t ic cell (DC) (Ste inman, 1 9 9 1 ) . These BM-der ived , ant igen present ing cells (APC), f ound in small numbers in v i r tua l ly all t i ssues, appear to serve a sent inel f unc t i on w i th in the immune s y s t e m . The po ten t immunos t imu la to ry proper t ies of DC are in t imate ly associated w i t h their large sur face area and high cons t i tu t i ve sur face expression of M H C Class I and II gene p roduc ts , cell adhesion and co-s t imu la to ry molecules (Inaba et al, 1 9 9 4 ) . Current ev idence indicates tha t per ipheral DC, such as the Langerhans cells of the sk in , take up fore ign ant igens and process molecules of potent ia l ly in fect ious agents into d iscrete pept ide f ragments intracel lu lar ly. In con t ras t to macrophages , DC possess litt le or no discernable phagocy t i c ac t iv i ty . However , they may obta in external ant igens via a cell sur face receptor (DEC-205) w h i c h possesses a number of lect in-binding domains and is recognized by the DC-speci f ic monoc lona l an t ibody NLDC-145 (J iang etal., 1 9 9 5 ) . An t igens are thereby d i rected to an intracel lular, mul t i -vesicular ant igen process ing c o m p a r t m e n t (Jiang et al., 1 9 9 5 ) . Exogenously or internal ly der ived pept ide ant igens are u l t imate ly expressed at the DC sur face in complex w i t h M H C Class II or Class I molecu les, respect ive ly . DC migrate f r o m peripheral t issues via the b lood to the spleen or t h rough the af ferent l ymph into drain ing l ymph nodes as vei led cel ls. The migra tory phase is associated w i t h a d imin ishment in DC ant igen process ing capac i ty but an enhancement in their immunos t imu la to ry propert ies (Aus tyn et al., 1 9 9 4 ) . A l t h o u g h macrophages and act iva ted B cells can ef f ic ient ly present ant igen to and act ivate memory T cel ls, the capac i ty to act ivate naive T l y m p h o c y t e s appears a lmost comple te ly restr ic ted to DC (Inaba and S te inman, 1 9 8 4 ; S te inman, 1 9 9 1 ) . 190 W i t h i n the l ymph node, in terd ig i ta t ing DC associate w i t h and probe the sur face of T l y m p h o c y t e s . Product ive T cell ac t iva t ion does not occur unless the T cell bears a c lono typ ic cell sur face ant igen receptor w i t h b inding speci f ic i ty for a pept ide presented in the b inding c lef t of the appropr iate M H C molecule. C D 4 + T cells are responsive to APC expressing pept ide in complex w i t h M H C Class II molecules whereas C D 8 + T cells engage w i t h cells expressing speci f ic pept ides in comp lex w i t h M H C Class I molecules. In add i t ion , an appropr iate co-s t imula tory signal such as tha t p roduced by the in teract ion b e t w e e n C D 8 0 on the APC and C D 2 8 on the T cell mus t be de l ivered. In add i t ion , DC:T cell in teract ions are stabi l ized t h r o u g h a number of receptor -co- receptor pairs inc luding I C A M -1: leukocyte func t ion-assoc ia ted ant igen-1 ( L F A - 1 , C D 1 1 a / C D 1 8 ) , LFA-3 ( C D 5 8 ) : C D 2 , and C D 8 6 : C D 2 8 . A l t h o u g h no counter - receptor on the T cell has been ident i f ied, a co-s t imu la to ry role for APC-associated HSA has been advanced (Liu et al., 1 9 9 2 a ; Liu et al., 1 9 9 2 b ) . The early phases of T cell ac t iva t ion are associated w i t h mul t ip le intracel lular protein phosphory la t ion events and an increase in intracel lular ca lc ium (A l tman , 1 9 9 0 ) . These events are f o l l o w e d by de novo gene t ransc r ip t ion , an increase in cell metabo l i sm, the synthes is and release of immunoregu la to ry cy tok ines , the appearance of speci f ic cy tok ine receptors and cell adhesion molecules on the cell sur face, and u l t imate ly the repl icat ion of the ant igen-act iva ted T cel l . The m ic roenv i ronment (ant igen concen t ra t i on , t ype of APC w h i c h presents the an t igen, and the ident i ty and concent ra t ion of cy tok ines present) in w h i c h the T cell encounters ant igen s t rongly inf luences the pat tern of cy tok ines 191 produced by the T cell upon its ac t ivat ion (Fitch et al., 1 9 9 3 ) . In the mouse , at least t w o d is t inc t C D 4 + T helper (H) subsets ex is t : p ro - in f lammatory T H 1 cells w h i c h produce IL-2 and interferon-) / upon ac t iva t ion , and T H 2 cells w h i c h release IL-4, IL-5, IL -10, IL-13 and prov ide help for B cell an t ibody p roduc t ion fo l low ing their ac t i va t ion (Mosmann and C o f f m a n , 1 9 8 9 ) . The T H 1 and T H 2 subsets may arise f r o m a less d i f ferent ia ted T cell (T H 0) w h i c h expresses an over lapping repertoire of T H 1 - and T H 2- l i ke cy tok ines upon act ivat ion (Mosmann and C o f f m a n , 1 9 8 9 ) . The ant igen-speci f ic ac t iva t ion of naive B cells requires the presence of speci f ic cy tok ines and a physical in teract ion w i t h T cel ls. There is no evidence tha t DC can present ant igen to B cel ls. However , it is appeal ing to speculate tha t in order to ini t iate a coord inated T-B cell in teract ion DC may present the ant igen in a processed f o r m to T cells and the ant igen in an unprocessed f o r m to B cells (Banchereau et al., 1 9 9 4 ) . B cel ls, in addi t ion to other APC inc luding DC and macrophages express M H C Class II molecules and can in teract w i t h and st imulate ant igen-speci f ic C D 4 + T cel ls. However , in con t ras t to T cells w h i c h recognize fore ign ant igen only in the c o n t e x t of a MHC-pept ide comp lex , soluble or cell sur face Ig bind epi topes w i t h i n in tac t ta rge t molecules. B cells may internalize ant igens bound by membrane Ig. M a n y of the receptor- l igand pairs w h i c h par t ic ipate in DC-T cell in teract ions, also init iate and stabil ize B cel l-T cell b ind ing. Of major impor tance in B cell-T cell in teract ion is the l inkage of C D 4 0 on the B cell sur face w i t h the C D 4 0 l igand (CD40L, gp39) of the T cell (Armi tage et al., 1 9 9 2 ) . C D 4 0 L appears t rans ient ly on the sur face of recent ly ac t iva ted T cells (Roy et al., 192 1993) and engagement of C D 4 0 st imulates B cell p ro l i fe ra t ion, inhibi ts apoptosis of germinal cent re B cel ls, p romotes immunog lobu l in iso type sw i t ch ing and a matura t ion of an t ibody a f f in i ty . The net consequence of ant igen-speci f ic T cell ac t i va t ion , prol i ferat ion and cy tok ine fo rma t ion and B cell Ig p roduc t ion is t ha t the o rgan ism can moun t a mul t i - t ie red, coord ina ted and speci f ic a t tack against invading pa thogens . 6 . 1 . 2 The role of i ron and heme in the immune response The requi rement for iron in the immune response is underscored by the observat ion tha t ac t iva ted T and B cells character is t ica l ly express high levels of the t ransfer r in receptor (TrfR, CD71) in order to meet the demands of DNA synthes is . In terest ing ly , rest ing mouse splenic B cel ls, bu t not splenic T cells have been s h o w n to express l o w , ye t detectab le levels of CD71 (Weber and F ink leman, 1 9 8 7 ; Futran et al., 1 9 8 9 ) . The major i ty of ant i -CD71 monoc lona l ant ibodies exer t inhib i tory e f fec ts on the pro l i ferat ion of l ymphocy tes cu l tured in the presence of mi togens or a l loant igens. These reagents act by either b lock ing the b inding of Trf to its receptor or caus ing a sur face down- regu la t ion and subsequent degradat ion of the TrfR (Kemp etal., 1 9 8 9 ; Kemp, 1 9 9 3 ) . The in f luence of metal l ic porphyr ins on immune responsiveness has received l imited a t ten t ion . A series of studies indicated tha t hemin , but not PPIX, w a s mi togen ic for human T cells enhanced prol i ferat ive responses to supraopt imal concen t ra t ions of Con A (Stenzel et al., 1 9 8 1 ) . A m o n g s t the other meta l loporphyr ins t es ted , w h i c h included Sn-PPIX, Zn-PPIX and Co-PPIX, only Sn-193 PPIX w a s mi togen ic for human T cells cu l tured w i t h accessory cells (Novogrodsky e r a / , 1 9 8 9 ) . Hemin w a s also s h o w n to enhance natural killer (NK) cell ac t iv i ty of human b lood mononuc lear cells (Novogrodsky et al., 1 9 8 9 ) . Further s tudy revealed tha t a var ie ty of i ron-conta in ing c o m p o u n d s were also mi togenic for human T cel ls, bu t only if these cu l tures w e re supp lemented w i t h r lL-2 (Novogrodsky et al., 1991 ) . Evidence w a s f o r w a r d e d tha t the mi togenic capac i ty of these " fe r ro -m i togens" w a s related to an intracel lular ox idat ive event since scavengers of o x y g e n free radicals and inhib i tors of peroxidase b locked their mi togenic act ion (Novogrodsky et al., 1 9 8 9 ; N o v o g r o d s k y et al., 1 9 9 1 ) . 6 . 1 . 3 Impac t o f porphyr in photosensi t izers on the i m m u n e response Litt le w o r k has been carr ied out on the in f luence of porphyr in photosensi t izers on the normal immune response, especial ly in the absence of ac t iva t ing l ight . H o w e v e r , t rans ient , immunosuppress ive changes have been observed in mice t reated w i t h some of these c o m p o u n d s and l ight. Mice admin is tered ei ther Photofrin® (Lynch et al., 1 9 8 9 ; Musser and Fiel, 1991) or hematoporphy r in der ivat ive (HpD) (Elmets and B o w e n , 1986) and d i rect ly exposed to ac t iva t ing levels of l ight exhib i ted dramat ica l ly reduced c o n t a c t hypersensi t iv i ty (CHS) responses to top ica l ly appl ied haptens. H o w e v e r , the CHS response proceeded normal ly in mice g iven these porphyr ins but not exposed to d i rect l ight. Gomer et al. (1988) noted the deve lopment of sp lenomegaly in mice given Photofr in® (10 or 5 0 mg/kg) but th is w a s not associated w i t h any s igni f icant a l terat ion o f splenic NK cell ac t iv i ty . H o w e v e r , in mice g iven Photofr in® (10 mg/kg) 1 9 4 and exposed to localized l ight there w a s a rapid, t rans ient , d im in ishment in bo th spleen w e i g h t and NK cell ac t iv i ty (Gomer et al., 1 9 8 8 ) . The cause of the t ransient immunosuppress ion produced by PDT is not wel l unders tood but the produc t ion and release of inh ib i tory prostaglandins into the c i rcu lat ion fo l l ow ing the pho todynamic t rea tmen t (Henderson and D o n o v a n , 1992) represents a plausible exp lanat ion . 6 . 1 . 4 Exper imenta l approach Since Photofr in® w a s s h o w n to increase hematopo ie t ic act iv i ty in normal mice , it w a s of interest to determine whe the r th is response migh t also a f fec t the course of an immune react ion in these animals. To examine this l ike l ihood, a number of immunolog ica l parameters were assessed in mice given an hematos t imu la to ry dosage of Photofrin®. In add i t ion , Photofrin® w a s evaluated for intr insic mi togen ic ac t iv i ty and the capac i ty to a f fec t the in vitro prol i ferat ive response of mouse spleen cells cu l tured w i t h the T cell m i togen Con A . In order to assess the d i rect impact of photosensi t izers on immune respons iveness, a exper imenta l series w i t h the fo l l ow ing ob ject ives w a s carr ied ou t : I. Assessment of Photofr in® and BPD for in vitro mi togenic ac t iv i ty . II . Evaluat ion of Photofrin® or BPD admin is t ra t ion on sp lenocyte responses to po lyc lonal m i togens . III. Assessmen t o f the inf luence of Photofrin® on the deve lopment of specif ic immune responses against a prote in ant igen. 195 6.2 Results 6.2.1 Evaluat ion of Photofr in® and BPD for in vitro m i togen ic ac t iv i ty Culture of normal DBA/2 spleen cells w i t h a concen t ra t ion gradient of Photofr in®, under cu l ture cond i t ions w h i c h suppor ted prol i ferat ive responses to the mi togens Con A and LPS, did not generate measurable prol i ferat ive responses, whereas Con A and LPS produced the expected s t rong responses (Figure 6 .1) . BPD also did no t s t imulate a spleen cell prol i ferat ive response in th is cu l ture sys tem (Figure 6 . 2 A ) . To determine whe the r Photofrin® or BPD cou ld act as a co-s t imu la to ry fac to r in l ympho id cell ac t i va t ion , normal spleen cells were cu l tured w i t h an op t ima l (2 .5 / /g /ml) concen t ra t ion o f Con A and va ry ing concen t ra t ions o f each photosensi t izer . Neither BPD (Figure 6 .2A) nor Photofr in® (Figure 6.2B) enhanced the spleen cell prol i ferat ive response to Con A . A t the highest concen t ra t ions t e s t e d , bo th photosensi t izers s t rong ly inhib i ted the prol i ferat ive response to Con A . 6.2.2 In f luence of Photofr in® and BPD admin is t ra t ion on spleen cell mi togenic responses To determine w h e t h e r Photofrin® migh t alter immune responsiveness in vivo, a hematos t imu la to ry dosage (25 mg/kg) w a s admin is tered. Seven days later, cor respond ing to the t ime at w h i c h the Photofr in®-induced splenic hypercel lu lar i ty w a s character is t ica l ly max ima l , the mice were sacr i f iced and spleen cell responses to Con A and LPS assessed. Sp lenocytes f r o m cont ro l and Photofr in®-injected mice responded equal ly over a concen t ra t ion gradient of LPS (F igu re6 .3 ) . H o w e v e r , sp lenocy tes f r o m Photofr in®-treated mice exh ib i ted a d is t inc t 1 9 6 C D £ L O O CD Q _ O CO c= CD C\3 CD ^ CO CD Culture concentration (ug/ml) Figure 6.1 Evaluat ion of Photofr in® for mi togenic ac t iv i ty . Prol i ferat ive responses of normal DBA/2 spleen cel ls, cu l tured w i t h d i f fe rent concen t ra t ions of Con A ( O ), LPS ( • ), or Photofr in® ( • ) for 72 hours at 3 7 ° C, w e re determined by the M T T color imetr ic assay. Mean ± SD absorbance (595 nm) va lues (n = 6 rep l icates/concentrat ion) obta ined for these cells cu l tured in m e d i u m alone ranged b e t w e e n 0 . 1 0 0 ± 0 . 0 0 5 and 0 . 1 1 1 ± 0 . 0 0 6 on the indiv idual micro t i ter p lates. Assay background (cul ture med ium only) absorbance values ranged f r o m 0 . 0 6 8 ± 0 . 0 0 4 to 0 . 0 7 6 ± 0 . 0 0 3 . Results are s h o w n w i t h o u t sub t rac t ion of background M T T ac t iv i ty . 197 o co C D C O S£ O - > C O n C D 0.0001 0.001 0.01 0.1 1 10 BPD ( u g / m l ) B 0.8r 0.01 0.1 10 100 Figure 6 . 2 Evaluat ion o f Photofr in® and BPD for co-mi togen ic ac t i v i t y . (A) Prol i ferat ive responses of normal DBA/2 spleen cells cu l tured w i t h med ium alone ( • ) or an opt imal (2 .5 / /g/ml) concen t ra t ion of Con A ( O ) and increasing a m o u n t s of BPD were eva luated. Mean ± SD absorbance (595 nm) values (n = 4 rep l icates/concent ra t ion) obta ined for these cells cu l tured in med ium alone or w i t h Con A cor responded to absorbance values of 0 . 1 7 0 ± 0 . 0 0 4 and 1.046 ± 0 . 0 4 1 , respect ive ly . Background M T T act iv i ty (medium only) w a s 0 . 0 8 8 ± 0 . 0 1 0 . (B) Prol i ferat ive responses of normal spleen cells cu l tured w i t h med ium alone ( • ) or w i t h Con A (2 .5 /yg/ml, O ) in the presence of d i f ferent concen t ra t ions of Photofrin® w a s also tes ted . Mean ± SD absorbance values (n = 4 rep l ica tes /concent ra t ion) obta ined for these cells cu l tured in med ium alone or w i t h Con A cor responded to absorbance values of 0 . 1 0 2 ± 0 . 0 1 2 and 0 . 6 5 7 ± 0 . 0 1 2 , respect ive ly . In th is exper iment , background M T T ac t iv i ty (medium only) cor responded to an absorbance of 0 . 1 0 2 ± 0 . 0 2 3 . 198 response pa t te rn to Con A (Figure 6 .3 ) . Sp lenocytes obta ined f r o m Photofrin®-t reated mice w e re s igni f icant ly more responsive to Con A at the 1.25 and 2.5 / /g /ml concen t ra t ions than spleen cells f r o m the cont ro ls . In con t ras t , spleen cells prepared f r o m cont ro l mice were s igni f icant ly more responsive to Con A at the 5 / /g /ml concen t ra t i on . In a single exper iment (2 mice per t rea tmen t g roup) , prol i ferat ive responses to Con A (2 .5 / /g/ml) by spleen cells prepared f r o m mice g iven BPD (10 mg/kg) 7 days prev ious ly were v i r tual ly ident ical to those generated by spleen cells f r o m sham- in jec ted cont ro l mice (MTT resul t , absorbance at 5 9 0 n m : cont ro ls = 0 . 9 6 2 ± 0 . 0 5 5 ; BPD-treated = 1.018 ± 0 . 0 1 6 ) . 6 . 2 . 3 Lympho id cell pro l i ferat ive responses to an admin is tered ant igen The admin is t ra t ion of O V A in CFA produced an increase in spleen w e i g h t and cel lu lar i ty , and l ymph node cel lular i ty in bo th so lvent and Photofrin®-t reated animals (Table 6 .1 ) . Prol i ferat ive responses to Con A , but no t to O V A , were observed in cu l tures conta in ing l ympho id cells prepared f r o m naive animals (Table 6 . 1 , Figure 6 .4B) . Prol i ferat ive responses to O V A by l ympho id cells f r o m ant igen-pr imed mice w e re readily de tec ted by means of the M T T co lor imetr ic assay. The in vitro pro l i ferat ive response to the ant igen produced by l ymph node cells f r o m OVA- immu n ize d mice were cons is tent ly s t ronger than tha t generated by spleen cells prepared f r o m the same mice. However , there w a s no s ign i f icant d i f ference b e t w e e n the prol i ferat ive responses against O V A produced by spleen or l ymph node cells prepared f r o m cont ro l mice to tha t of cells obta ined f r o m these organs f r o m 199 CD ° 2 CO 5= CD co cO CD _Q CD O > C O "Il >T n~ " o 0.5 0.4 0.3 0.2 0.1 0.0 1.5 1.2 0.9 0.6 0.3 0.0 ; Con A \ / ^ \ i \ 11111 0.2 0.5 1 2 5 0 Mitogen (ug/ml) Figure 6 . 3 Spleen cell m i togen ic responses f o l l o w i n g Photofr in® admin is t ra t ion . Prol i ferat ive responses to LPS and Con A by spleen cells obta ined f r o m mice in jected w i t h 5 % dext rose ( • ) or Photofrin® (25 m g / k g , O ) 7 days prev ious ly w e re determined by the M T T color imetr ic assay. Data points represent the mean ± SD for three indiv idual mice per t rea tmen t g roup . Mean absorbance values in the con t ro l wel ls (cells plus medium) averaged 0 . 1 6 3 ± 0 . 0 1 8 for the cont ro l mice and 0 . 1 5 0 ± 0 . 0 0 4 for the Photofr in®-injected mice. These values have been sub t rac ted f r o m the result obta ined w i t h each m i togen . Background M T T ac t iv i ty (med ium only) cor responded to an absorbance of 0 . 0 8 7 ± 0 . 0 0 3 . T w o addi t ional exper iments w i t h Con A and one w i t h LPS gave highly similar spleen cell response pat terns for so lvent and Photofr in®-injected mice. P < 0 . 0 5 ; ** P < 0 .01 compared to cells obta ined f r o m solvent - in jected mice. 2 0 0 Photofr in®-injected mice at either sampl ing t ime (Figure 6 . 4 A , B). In addi t ion, admin is t ra t ion of Photofr in®, either 7 days before or at the same t ime as O V A , did not alter the response to Con A by spleen or l ymph node cells compared to cells obta ined f r o m so lvent - in jected animals (Table 6 .1 ) . H o w e v e r , these responses were less than those generated by spleen and l ymph node cells prepared f r o m naive animals. 6 . 2 . 4 . In f luence of Photofr in® on ant igen-speci f ic humora l i m m u n i t y There w a s no s igni f icant d i f ference b e t w e e n serum levels of IgG ant i -O V A ant ibod ies , w h e t h e r the ant igen w a s g iven at the same t ime or 7 days after Photofr in® w a s adminis tered (Figure 6.5 A ,B) . React iv i ty against O V A w a s not demonst rab le for serum samples obta ined f r o m norma l , un immunized mice. Compet i t i on s tudies demons t ra ted tha t the immunoassay w a s speci f ic for O V A , in tha t O V A , but not a separate prote in (HSAlb) , s t rong ly inter fered w i t h the abi l i ty of serum IgG an t i -OVA ant ibodies to bind p la te-bound O V A (Figure 6 .6) . No a t t e m p t w a s made to determine levels of IgM class an t i -OVA ant ibodies in the serum of immunized mice. 201 CD O •D 0 N "E 3 E E < > O •o c cc 0 > 'ra c o CO k-0 4-> cu E CO k_ CO a CD •a o c a E T3 C CO c CD a CA T3 Cl) N "IZ CO E E 3 CO C O . Q CO c LO > © O .E CD E 5 3 cu c w I £ — X CD CD O X I E # o - c CD CD O > E c <D CD > 0 S * i - > O CD **- SZ -D CD >-C <U T 3 CD N > CO c CO CD k-<D CD C J E d CN « < CQ Q J D CO CD k_ CO c E -2 4-> CD > N CD E "° 3 -a CD E E -o c o c CO +-« co _ CD < - M - m +1 E 'co ® 0) -—• +-> r N CD , "a + CD > CO 0 CO CO 1 s E "co T3 > CO c $ ro 0 CO CO > 1_ k-CO O Q . _ CD O "D c Q -C •— Q . CD II T 3 c CO C CO CL CO ^ CO 0 SZ ' 0 < LL. o 0 c H — CO CO CO CO o .E - 0 "O CL 0 x O 0 CO CO CD ro 0 ^ 0 ^ 0 0 1 I TO E to" 0 4-> 0 E CO CO Q. 0 •a o sz a E 0 4-> 0 E CO k-CO Q. c 0 _0 Q . </) 0 (A ^ c — o E s- «= 0 O < LO § < o •z <p JO O J3 *" "5J X u a> w ^ c —• CO o cn < LO §< o ~ o •= CM CO ^ l b 0 X o o 0 t-•5 X — 4-CD SZ oc o> •3 5 .2> « I E o c 0 Q. S5 CO * -CM *~ CM O 0 ci +1 +1 o o CO d +1 CO in o «- o 6 ci +1 +1 in cj) CM Po in *-6 p <J1 CM +1 CN o CM CD +1 o +1 CM CN CN 0 'io ^ IN CD Oo O P 6 ci +1 +1 CD IN ^ »-d p d +1 o CO Po o o d ci +1 +1 00 CO <- CM d p LO I D +1 CD o CM o d +1 CD L O I N CM +1 CO CO CM 0 OT o x — is CD IN Co O P d ci +1 +1 IN o CD d p +1 CO CD CJ) CO St-o p +1 O co LO Lf) r - C\| d p q 1 ^ +1 o [ N o CD d +1 CN CO I N CM +1 o CO CM © O (0 O) IN IN P o d +1 p ci +1 <- CO CD Co LO d p +1 CO CN ^> o d +1 o p +1 00 cj) 00 CO CM * -d p +1 CN CD CT) o d +1 CO CM LO CO +1 I N CO CN 0 CO o X ^ 0 + Q >. N P CO CN CO o d +1 I N LO d CO CN p p +1 p CD CM +1 CO CN LO I N CM o o d ci +1 +1 CM IN CM LO CO d p CO CN CN +1 CO CD o CN d +1 CO CO 00 +1 LO CO CN © _ .E rN J= + I > o ca c T 3 c 0 0 N »+— 'O T 3 0 4—' ita trac are sub one ity CO > J O -4-> 0 "0 CO 0 1- sz 1- +-* N , . T 3 0 0 c C 0 'co CO 4-> Si _co 0 "0 CO 0 4-» '—' D T 3 CO c 0 k-gro 1-1-0 ba SD ted sz CO 4-> +1 0 4—' c J O c CO CO show 0 d anim 0 k_ 0 <4— D) O CO O k-, , 4-> 0 /ml E Si E =s. the nui LO • 1 CN O 11 0 c < nc c 0 0 CO CO 0 0 k- ese 0 Q. ese 4-> 0 sz CO SZ 4-> 0 4-> c CO 0 uods c T 3 0 /— n par 0 •— 1— 'co T 3 0 > +-> X 2 0 4-> O c CO 4-« 0 k_ sul CO 0 sul 0 sul k-0 Q. O k . Q_ he pu T - +-> CO 202 <D 0 5 C O * = o C D C O > _ Q "is ^ o_ or 1 2 . 5 2 5 5 0 1 0 0 O v a l b u m i n ( u g / m l ) Figure 6 . 4 Spleen and l ymph node cell pro l i ferat ive responses to O V A . Prol i ferat ive responses to O V A were determined for spleen and lymph node cells obta ined f r o m mice adminis tered 5 % dext rose or Photofrin® (25 mg/kg) (A) 7 days before or (B) at the same t ime as the immunizat ion w i t h O V A in CFA. Ten days f o l l o w i n g ant igen sensi t izat ion, four animals in each t rea tmen t group were sacr i f iced. Mean ± SD M T T absorbance values obta ined w i t h l ymph node cells f r o m so lvent - in jec ted ( • ), Photofr in®-injected ( • ), and naive ( A ) mice are s h o w n . Spleen cell pro l i ferat ive responses to O V A generated by the same solvent-in jected ( • ), Photofr in®-injected ( O ), and naive ( v ) animals are also p resented . To determine the magn i tude of speci f ic cell responses to the ant igen, data are s h o w n w i t h the background (cells alone) M T T result sub t rac ted . 2 0 3 O j — CZ CD =3 CCS — -2 o V - Q co ^ O lo O ) CD CD I CO LU 2.5 2.0 1.5 1.0 0.5 0.0 2.5 A : I v V — V — V - V - V - V - ^ - f — f 1 1 1 100 1000 10000 100000 100 1000 10000 100000 S e r u m d i l u t i o n ( 1 / x ) Figure 6.5 In f luence o f Photofr in® on serum an t i -OVA IgG levels. Circulat ing levels of serum IgG ant ibodies against O V A were determined for mice g iven 5 % dex t rose ( • ) or Photofrin® (25 m g / k g , O) either (A) 7 days before or (B) at the same t ime as the immuniz ing dose of O V A . Results obta ined w i t h se rum obta ined f r o m un immunized DBA / 2 mice (V) are also s h o w n . Blood w a s obta ined 21 days fo l l ow ing ant igen admin is t ra t ion and serum ant ibody t i t res we re determined by a speci f ic ELISA. 2 0 4 110 r 90-80-i i i i 1 1 1 — i i ' ' ' ' I I • i i i i i i | 10 100 1000 Competitor added (ug/ml) Figure 6 .6 Spec i f ic i ty o f se rum an t i -OVA IgG ant ibod ies . The spec i f ic i ty of serum IgG ant ibodies generated against O V A by mice g iven 5 % dex t rose (fil led characters) or Photofrin® (open characters) at the same as the immuniza t ion w i t h ant igen w a s determined by compet i t i ve inhibi t ion s tud ies. Pooled serum samples (final d i lu t ion = 1/100) we re incubated overn ight at 4 ° C in PBS-Tween 2 0 , 2 % FCS and increasing amoun ts of ei ther O V A (square symbols) or HSAlb (circular symbo ls ) . Unabsorbed serum IgG an t i -OVA ant ibody levels w e re quant i ta ted w i t h the s tandard ELISA techn ique . Assay results are presented as the percentage of the original (unabsorbed) result minus the assay background ( 0 . 1 0 0 ± 0 .004 ) w h i c h y ielded absorbance (405 nm) values of 1.509 ± 0 . 0 3 3 and 1.238 ± 0 . 0 3 5 for serum f r o m solvent- and Photofr in®-injected mice, respect ive ly . 2 0 5 6 .3 Discuss ion W h e n a single dose of Photofrin® w a s g iven to norma l , un immunized mice there w a s no change in the prol i ferat ive response of spleen cells obtained seven days after in ject ion to the B cell mi togen LPS. In con t ras t , a shi f t in the response to the T cell m i togen Con A w a s observed for Day 7 post - in ject ion spleen cells f r o m Photofr in®-treated mice, in t ha t these cells we re more responsive to lower concen t ra t ions and less responsive to higher concent ra t ions of the mi togen than spleen cells f r o m the cont ro l animals. Bianchi et al. (1992) noted a modest increase in spleen cell responses to Con A , but not LPS, in mice g iven mult iple doses o f Photofr in®. The explanat ion for the modi f ied response to Con A is uncer ta in , s ince Con A-respons ive T cells w o u l d be "d i l u ted " to a degree in the spleens of Photofr in®-treated animals by the increased number of ery thro id and myelo id cells in the o rgan . There fore , a reduced Con A response migh t have been ant ic ipated for spleen cells prepared f r o m Photofr in®-treated animals. However , it is conce ivab le tha t Photofrin® increased numbers of myelo id l ineage APC (macrophages , DC) in the spleen thereby increasing the e f f ic iency of T cell accessory ac t i v i t y . BPD had no discernable e f fec t on spleen responsiveness to Con A w h e n these cells were tes ted 7 days after admin is t ra t ion of th is photosensi t izer . Neither Photofr in® nor BPD had demonst rab le mi togenic or co-mi togen ic act iv i ty in vitro and at higher concen t ra t ions ( > 5 /vg/ml) inhibi ted spleen cell prol i ferat ive responses to Con A . It is not clear whe the r the inh ib i tory e f fec ts at the higher concen t ra t ions are the result of a d i rect c y t o t o x i c e f fec t exer ted by these 2 0 6 c o m p o u n d s or pho todynamic damage produced by the inadver ten t exposure of these cells to small amoun ts of l ight dur ing the cu l ture per iod. Franco etal. (1983) indicated tha t HpD d i rect ly inhibi ted immune cell ac t iv i ty in an number of in vitro assays inc lud ing the b inding of c y t o t o x i c T cells to ta rge t cells and the lyt ic act iv i ty of NK cel ls, w i t h o u t a discernable e f fec t on the v iabi l i ty of these immune ef fector cel ls. Admin is t ra t ion of a Photofrin® dosage (25 m g / k g ) , w h i c h was character is t ica l ly hematos t imu la to ry in normal mice, did not s ign i f icant ly mod i fy the fo rmat ion of a cellular immune response to an admin is tered prote in ant igen, OVA. Sp lenocyte and l ymph node cell prol i ferat ive responses to th is ant igen were similar for so lven t and Photofr in®-injected mice , whe the r the ant igen w a s administered at the same t ime or seven days after the porphyr in preparat ion w a s g i ven . Spleen and l ymph node responses to the T cell m i togen Con A , a l though lower than those of naive l i t te rmates , were comparab le for so lvent and Photofr in®-treated animals. The d imin ished response to Con A in ant igen- immunized mice l ikely ref lects a partial immunosuppress ive e f fec t p roduced by the Mycobacter ia l c o m p o n e n t of the i m m u n o g e n preparat ion. In add i t ion , immunizat ion w i t h O V A in CFA w a s associated w i t h a large increase in spleen w e i g h t and cel lular i ty and an increase in l ymph node cell numbers in bo th cont ro l and Photofr in®-treated mice. Serum ant i -OVA IgG ant ibody levels w e re comparab le in so lvent and Photofr in®-treated mice. A l t hough levels of OVA-spec i f i c IgM ant ibodies were not assessed, a hematos t imu la to ry dosage of Photofr in® did not s igni f icant ly alter the magn i tude of the cellular or humora l immune response generated against a prote in an t igen. These f ind ings are in agreement w i t h an earlier s tudy in the mouse w h i c h did no t ident i fy any major 2 0 7 immuno log ic a l terat ions p roduced by Photofrin®, in the absence of drug pho toac t i va t ion (Bianchi et al., 1 9 9 2 ) . The use of CFA to enhance ant igen pr iming migh t have shrouded d i rect immunolog ic in f luences of Photofr in®, since sp lenomega ly , splenic hypercel lu lar i ty and lymph node hyper t rophy occurred in so lvent and Photofr in®-injected mice g iven O V A w i t h th is ad juvant . The studies descr ibed above demons t ra ted tha t relat ively large, single doses o f Photofr in® or BPD have l i tt le impact on the immuno log ic responsiveness of normal mice. The capac i ty of the immune sys tem to respond normal ly to ant igenic s t imul i in Photofr in®-injected mice fu r ther i l lustrates tha t the hematopo ie t ic response to the c o m p o u n d is restr ic ted to cells of the ery thro id and myelo id l ineages and th is ac t iv i ty does not compromise immune responsiveness. 2 0 8 CHAPTER SEVEN CHARACTERIZATION OF THE LR-1 ANTIGEN 7.1 In t roduc t ion The studies out l ined in th is chapter descr ibe exper iments per formed in order to ident i fy the cell sur face molecule recognized by the monoc lona l ant ibody L R - 1 . The observat ion tha t Photofrin® great ly increased the number of spleen cells t h a t expressed a cell sur face molecule w h i c h labelled w i t h the rat IgM monoc lona l an t ibody LR-1 p rompted a series of s tudies designed to ident i fy th is cell an t igen. The LR-1 an t ibody w a s der ived by immuniz ing a Lou rat w i t h the Friend leukemia cell line 7 0 7 . M 2 and w a s init ial ly descr ibed as being react ive against mouse splenic B cel ls, bu t not splenic T cells (Hutch ings et al., 1 9 8 5 ; Hutch ings et al., 1 9 8 7 ; Serotec technica l in fo rmat ion) . H o w e v e r , Photofr in® produced an e levat ion in the number of spleen cells tha t expressed the LR-1 ant igen w i t h o u t a l ter ing t h e number o f cells t h a t expressed the B l ymphocy te - res t r i c ted IgM or C D 4 5 R - B 2 2 0 ant igens, suggest ing tha t the LR-1 ant igen had a d is t r ibut ion beyond tha t of the B cell l ineage. In the spleens of Photofr in®-injected mice , the increased express ion of the LR-1 ant igen w a s s t rongly associated w i t h cells also expressing ery thro id ant igens. The ant igen w a s also s h o w n to be expressed at high f requenc ies on the m a r r o w cells of cont ro l and Photofr in®-injected mice. No publ ished in fo rmat ion w a s available concern ing the nature of the ant igen recognized by the LR-1 monoc lona l an t ibody . It w a s recognized tha t i ts ident i f icat ion w o u l d 2 0 9 provide a more comple te character izat ion of the hematopo ie t ic response invoked by Photofr in®. 7 .2 Results 7 .2 .1 Tissue d is t r ibu t ion o f the LR-1 ant igen A survey of the expression of the LR-1 ant igen in d i f ferent l ymphohematopo ie t i c organs revealed tha t the ant igen labelled by th is ant ibody w a s s t rong ly expressed on the vas t major i ty ( > 9 5 % ) of normal BM cells and t h y m o c y t e s , approx imate ly 6 0 % of spleen cel ls, and 2 0 % of l ymph node cells (Figure 7 .1 ) . Double label s tudies s h o w e d tha t the LR-1 ant igen w a s s t rongly expressed on B 2 2 0 + , l gM + , CD11 b + ( M 1 / 7 0 ) , G r - 1 + , CD71 + , and T E R - 1 1 9 + BM cells (Figure 7 .2 ) . In the t h y m u s , the LR-1 marker w a s co-expressed on the major i ty of cells d isp lay ing the C D 3 , C D 4 , CD8 , or T h y - 1 . 2 ant igens. In con t ras t , there was l i tt le assoc iat ion of cells bearing T cell ant igens w i t h LR-1 expression in the spleen. In th is t i ssue, LR-1 w a s co-expressed on the vast major i ty o f cells bearing B cel l-rest r ic ted markers ( IgM, B220) . A lso , smal l , bu t d is t inc t , popula t ions of CD71 + / L R -1 + and T E R - 1 1 9 V L R - 1 + cells were de tec ted w i th in the spleen. The LR-1 an t ibody labelled a sizeable p ropor t ion ( 3 1 - 6 8 % ) of spleen cells f r o m the D B A / 1 , PL /J , and Balb/c strains o f mice (Figure 7 .3 ) . The t issue d is t r ibut ion of the LR-1 ant igen w a s similar to tha t of HSA (Kay et al., 1990) and the sta in ing in tens i ty of these spleen cells cor responded wel l t o t ha t exhib i ted by the ant i -HSA ant ibody J 1 1 d (Figure 7 .4 ) . Double label f l o w cy tomet r i c studies of normal D B A / 2 mouse spleen cells w a s per fo rmed w i t h the LR-1 reagent in parallel 2 1 0 w i t h the HSA-spec i f ic an t ibody J 1 1 d . These studies revealed tha t the LR-1 ant igen w a s expressed s t rong ly on splenic IglVT B cel ls, w a s essent ial ly absent f r o m C D 4 + and C D 8 + T cells and w a s present on cells tha t s t rong ly expressed the TER-119 or CD71 cell sur face ant igens. J 1 1 d exhib i ted a s t r ik ingly similar s ta in ing pa t te rn . W h e n doub le label an t ibody staining w a s per formed in parallel on spleen cells f r o m D B A / 1 , D B A / 2 , PL/J , and Balb/c strain mice, LR-1 w a s again s h o w n to be co-expressed on cells t ha t expressed a B cell marker (B220) , but not T cell ant igens (CD4 or CD8) and th is pat tern cor responded closely to t ha t observed w i t h the J 1 1 d an t ibody (Figure 7 .5 ) . In all mouse strains examined , spleen cell sub-populat ions were present w h i c h labelled w i t h LR-1 or J 1 1 d but did not express B 2 2 0 . The LR-1 an t ibody cons is ten t ly labelled a larger propor t ion of th is spleen cell populat ion than did J 1 1 d . 211 Bone Marrow Fluorescence Intensity Figure 7.1 Expression of the LR-1 ant igen on normal D B A / 2 mouse bone m a r r o w cel ls, t h y m o c y t e s , sp lenocytes and l ymph node cel ls. Single cel l suspensions w e r e prepared f r o m t issues obta ined f r o m normal DBA/2 mice and sur face labelled w i t h the LR-1 reagent . Bold lines represent the sta in ing pat tern obta ined w i t h the LR-1 ant ibody whi le the th in lines in each panel represent the result obta ined w i t h the rat IgM isotype cont ro l an t ibody. 212 Bone Marrow Thymus Spleen i i n i l '.1 ICOO .1 .1 IM9 13 n a T T 1 ! S LR-1 Figure 7 .2 Co-express ion of var ious sur face ant igens w i t h the LR-1 marker on normal D B A / 2 mouse BM cel ls, t h y m o c y t e s and sp lenocy tes . Dif ferent cell populat ions were labelled w i t h LR-1 in combina t ion w i t h a monoc lona l ant ibodies react ive against var ious d i f fe rent ia t ion ant igens. Cell sur face LR-1 w a s de tec ted w i t h a PE-conjugated ant i - rat IgM an t ibody , excep t in con junc t ion w i t h ant i -CD71 in w h i c h case a FITC-conjugated ant i - rat IgM ant ibody w a s e m p l o y e d . Evaluat ion of these cell populat ions for LR-1 co-express ion pat terns w a s per fo rmed t w i c e g iv ing essent ial ly identical resul ts. 2 1 3 OVEBUr^SIHCLE PMMHrtER DBA/1 LR-1 (68.1%) Fluorescence Intensity Figure 7 .3 . LR-1 and J l l d labell ing of normal mouse spleen cells. Spleen cells obta ined f r o m normal male D B A / 1 , D B A / 2 , PL/J , and Balb/c mice w e re sta ined w i t h the LR-1 or J 1 1 d rat monoc lona l ant ibodies. Thin l ines in each panel represent the resul t ob ta ined w i t h the rat IgM isotyp ic con t ro l an t ibody . The percentage (%) of LR1 + and J 1 1 d + spleen cells is g iven w i th in each f igure . 2 1 4 Figure 7 . 4 Co-express ion of var ious leukocy te ant igens w i t h the ant igen recognized by LR-1 or the HSA-speci f ic monoc lona l an t ibody J 1 1 d on normal D B A / 2 spleen cells. Spleen cells were prepared f r o m normal DBA/2 mice and double label s tudies w e re per fo rmed w i t h the rat IgM monoc lona l ant ibodies LR-1 or J 1 1 d in comb ina t ion w i t h ant ibodies react ive against a var ie ty of sur face ant igens. Cell sur face-bound J 1 1 d and LR-1 ant ibody w a s counter- label led w i t h an ant i-rat IgM PE-conjugated mouse monoc lona l an t ibody , excep t in con junc t ion w i t h the ant i -CD71-PE an t ibody w h e n an ant i -rat IgM-FITC conjugate w a s e m p l o y e d . „„. 2 1 5 DBA/1 DBA/2 PL/J Balb/c Figure 7 .5 Co-express ion o f T and B cell ant igens w i t h t he LR-1 marker and HSA, as recognized by J 1 1 d , on sp lenocytes prepared f r o m fou r d i f ferent mouse st ra ins. In order to compare the d is t r ibut ion pat tern of the LR-1 ant igen w i t h tha t of HSA, sp lenocy tes obta ined f r o m untreated D B A / 1 , D B A / 2 , PL /J , and Balb/c strain male mice w e re labelled w i t h PE-conjugated (A) an t i -CD4, (B) an t i -CD8, or (C) an t i -CD45R-B220 monoc lona l ant ibodies in combina t ion w i t h the rat IgM ant ibody LR-1 or the rat IgM ant i -HSA ant ibody J 1 1 d . Cell sur face-assoc ia ted rat IgM w a s counter- label led w i t h an ant i - rat IgM FITC-conjugated mouse monoc lona l an t ibody. 2 1 6 7 .2 .2 Expression of the LR-1 ant igen on normal b lood e ry th rocy tes , splenic dendr i t ic cells (DC) and splenic B l ymphocy tes Normal peripheral red blood cel ls, whe the r labelled w i t h LR-1 or the ant i -HSA ant ibodies M 1 / 6 9 or J 1 1 d , exhib i ted highly un i fo rm sta in ing pat terns and expressed equal ly high levels of the ta rge t ant igen (Figure 7 .6 ) . JDUERLAYlSIMQLC PARAMETER •1 Fluorescence Intensity ! j Figure 7 .6 Sur face labell ing of HSA on normal mouse peripheral b lood e ry th rocy tes by M 1 / 6 9 , J 1 1 d and LR-1 monoc lona l ant ibodies. D B A / 2 red blood cells were labelled w i t h three d i f fe rent ant i -HSA monoc lona l ant ibod ies. Each of these ant ibodies p roduced a degree of red cell aggregat ion dur ing the sta in ing procedure and the results s h o w n were obta ined w i t h the f l o w c y t o m e t e r gated on the non-aggregated cell f r a c t i o n . 2 1 7 DC pur i f ied f r o m the spleens of normal DBA/2 mice were s h o w n to s t rong ly s t imulate a MLR at l ow cell numbers w h e n cu l tured w i t h al logeneic DBA/1 strain T cells (Figure 7 .7 ) . This immunos t imu la to ry a t t r ibute suppor ted their ident i ty as DC (Ste inman, 1 9 9 1 ) . Splenic DC exhib i ted a broad sta in ing pat tern w h e n labelled w i t h the LR-1 an t ibody . This labell ing prof i le w a s d is t inc t f r o m tha t associated w i t h the ant i -HSA ant ibody J 1 1 d (Figure 7 .8 ) . The ident i ty of the isolated cells as DC w a s fur ther suppor ted by their h igh sur face expression of MHC Class I and II ant igens and intercel lular adhesion molecule-1 ( I C A M - 1 , CD54) . In add i t ion , these cells expressed the co-s t imula tory molecule C D 8 0 , C D 1 1 b and the DC-rest r ic ted marker DEC-205, but f e w e r than 1 5 % of these cells labelled w i t h the B cel l -speci f ic an t ibody RA3-6B2 react ive w i t h CD45R (B220) . Therefore , the overal l ant igen express ion pat tern w a s cons is tent w i t h t ha t descr ibed for murine splenic DC (Inaba et al., 1 9 9 4 ) . 1 0.4 S 10* 103 10" 106 DC added Figure 7.7 Immunos t imu la to ry ac t iv i ty of pur i f ied splenic DC. The mixed leukocy te react ion (MLR) w a s per fo rmed by co-cu l tur ing t i t ra ted , m i t o m y c i n C-treated DBA/2 splenic DC w i t h 4 x 1 0 5 al logeneic DBA/1 splenic T l y m p h o c y t e s for 3 days . Prol i ferat ive responses were measured by the M T T co lor imet r ic assay. The M T T result obta ined w i t h T cells cu l tured in the absence of al logeneic DC gave an absorbance ( 5 9 0 nm) value of 0 . 0 2 2 ± 0 . 0 0 6 . 2 1 8 'I CD45R-B220 Fluorescence Intensity Figure 7 .8 A n t i g e n express ion pat tern o f splenic DC. Expression levels (bold t racings) of M H C Class I and II ant igens, I C A M -1 (CD54) , B7-1 (CD80) , DEC-205 (NLDC-145) , C D 1 1 b (Mac-1 ) , the LR-1 ant igen and HSA (as recognized by the J 1 1 d monoc lona l ant ibody) we re determined for pur i f ied D B A / 2 splenic DC. Fewer than 1 5 % of these cells labelled w i t h the anti-B cell an t ibody RA3-6B2 w h i c h recognizes the B cel l - restr icted iso form of C D 4 5 . Thin t rac ings cor respond to the result obta ined w i t h the iso typ ica l ly -matched cont ro l an t ibody . 2 1 9 Freshly isolated splenic B l ymphocy tes exhib i ted l o w , ye t detectable a m o u n t s of M H C Class II and C D 8 0 sur face ant igens and modera te levels of the LR-1 ant igen (Figure 7 .9 ) . C D 2 5 w a s absent f r o m these cel ls. A f t e r a 3 day cul ture in the presence of LPS (5 / /g /m l ) , levels of MHC Class I I , C D 8 0 , C D 2 5 , and LR-1 ant igens w e re marked ly increased. 7 . 2 . 3 Evaluat ion of g lycosy l -phosphat idy l inos i to l (GPI)-l inkage Since HSA is k n o w n to be a GPI-anchored cell sur face g lycoprote in and if the ant igen recognized by the LR-1 an t ibody is HSA it should therefore be vu lnerable to removal w i t h PI-PLC enzymolys is . T rea tment of normal sp lenocytes w i t h phosphat idy l inos i to l -spec i f ic phosphol ipase C (PI-PLC) did no t alter the staining pat tern for ei ther C D 4 or I g M . However , th is enzyme t rea tmen t great ly reduced the sta in ing in tens i ty of spleen cells labelled w i t h ant ibodies to the GPI-l inked proteins T h y - 1 . 2 and HSA (as recognized by J11d) (Figure 7 . 1 0 A ) . PI-PLC also substant ia l ly reduced the labell ing of these spleen cells w i t h LR-1 indicat ing tha t the ant igen recognized by LR-1 is also GPI-anchored. Enzymat ic t r ea tmen t of spleen cells prepared f r o m mice injected w i t h cont ro l so lvent (Figure 7.1 OB) or Photofrin® (Figure 7 .10C) indicated tha t the f o r m of the LR-1 ant igen on the sur face of the addi t ional L R - 1 + cells present w i th in the spleens of Photofr in®-treated mice appeared to be relat ively resistant to c leavage w i t h PI-PLC. This feature w a s less apparent for PI-PLC-treated spleen cells f r o m Photofr in®-injected mice since J 1 1 d does not recognize a propor t ion of the cells w h i c h s t rong ly label w i t h L R - 1 . 2 2 0 Day 0 - B lymphocytes Day 3 - LPS Blasts Fluorescence Intensity Figure 7.9 Ef fect of LPS ac t iva t ion on the express ion o f the LR-1 ant igen by splenic B cel ls. Flow cy tomet r i c analyses of the expression of M H C Class II , C D 8 0 , C D 2 5 , and the LR-1 ant igen were per fo rmed on f reshly isolated splenic B cells and the same cell popu la t ion after a 3 day cul ture in the presence of LPS (5 / /g /ml ) . Thin t rac ings cor respond to the result obta ined w i t h the isotype cont ro l wh i le specif ic an t ibody sta in ing is denoted by bold l ines. The exper iment w a s per fo rmed t w i c e and gave h ighly similar resul ts. Mean log channel f luorescence values (in arbi t rary units) for the LR-1 + peak cor responded to 4 7 . 0 ± 8 .2 (n = 2 exper iments) on Day 0 and 1 2 4 . 9 ± 9.7 (n = 2 exper iments) af ter 3 days of cu l tu re . 221 F l u o r e s c e n c e I n t e n s i t y Figure 7 . 1 0 Phosphat idy l inos i to l -speci f ic phosphol ipase C (PI-PLC) enzyme d igest ion s tud ies . (A) Sensitivity of different spleen cell surface antigens to treatment with PI-PLC was tested. Normal DBA/2 spleen cells were treated with or without PI-PLC for one hour at 37 ° C , washed and analyzed for expression of IgM, CD4, Thy-1.2, HSA (J 11 d) and LR-1. Thy-1 and HSA are GPI-linked proteins and therefore are susceptible to PI-PLC digestion. Bold tracings indicate the flow cytometric result obtained with spleen cells not exposed to PI-PLC. (B/C) PI-PLC digestion studies of spleen cells prepared from mice given the control solvent or Photofrin® 7 days previously and labelled with either the (B) J11 d or (C) LR-1 monoclonal antibody. Enzyme digestion studies on spleen cells from solvent and Photofrin®-injected mice were performed three times giving highly similar results. 222 7.2.4 Western blot analysis Probing normal t h y m u s and bone m a r r o w SDS-PAGE-f ract ionated, de te rgen t ex t rac ts w i t h t he LR-1 ant ibody indicated t h a t t he ta rge t ant igen has a large degree of heterogenei ty in molecular mass, exhib i t ing bands ranging f r o m 3 0 -55 kDa (Figure 7 .11 A ) . A n addi t ional intensely stained band w i t h a molecular mass of approx imate ly 21 kDa w a s associated w i t h the BM cell ex t rac ts . Red cell lysates also exh ib i ted considerable heterogenei ty w i t h many indiv idual bands ranging b e t w e e n 3 0 kDa and 55 kDa, a l though the most intense sta in ing occured be tween 3 0 - 4 5 kDa. The sta in ing in tens i ty of spleen cell lysates w a s lower , a l though t w o separate label l ing c lusters ( 3 0 - 4 0 kDa and 4 5 - 6 0 kDa) we re seen. Spleen cell ex t rac ts f r o m Photofr in®-injected mice sta ined m u c h more intensely t h a n ex t rac ts f r o m the con t ro l animals, w i t h a marked enhancement of the 3 0 - 4 0 kDa bands. In add i t ion , a band w i t h an apparent molecular mass of 21 kDa w a s noted w i t h spleen cell lysates f r o m cont ro l and Photofr in®-injected mice, a l though th is band was much more dense in t he lat ter g roup . Thus , t he 21 kDa species w a s associated w i t h spleen and BM cell ex t rac ts , but w a s not present in t h y m i c or red cell ex t rac ts . A st r ik ingly similar LR-1 sta in ing pat tern w a s obta ined w i t h lysates prepared f r o m the spleens o f so lven t and Photofr in®-injected DBA/1 mice (Figure 7 .11B) . No staining w a s observed , w i t h all t issue ex t rac ts examined , w h e n the J 1 1 d ant ibody w a s ut i l ized, despi te tes t ing a w ide range of t ransfer cond i t ions , an t ibody concent ra t ions and incubat ion t imes . 2 2 3 Figure 7 .11 LR-1 Immunob lo t analysis of t issue ex t rac ts . (A) Wes te rn b lot analysis w a s per formed w i t h the LR-1 monoclonal an t ibody against lysates prepared f r o m red blood cells (lane 2) , t h y m o c y t e s (lane 3 ) , bone m a r r o w cells (lane 4) obta ined f r o m normal DBA/2 mice or spleen cells obta ined f r o m 3 d i f fe rent mice injected w i t h 5 % dext rose (lanes 5-7) or 3 d i f ferent mice g iven Photofr in® (25 mg/kg) (lanes 8-10) 7 days prev ious ly . Each sample w a s loaded at 5 / /g prote in/ lane except for the red cell lysate w h i c h w h i c h w a s run at 10 / /g / lane. Molecular w e i g h t s tandards (lane 1) were separated by SDS-PAGE, b lo t ted in parallel w i t h the cell ex t rac ts and stained w i t h amido black. In th is exper iment , f l o w cy tomet r i c analysis demons t ra ted tha t for spleen cells of cont ro l and Photofr in®-treated mice, 5 5 . 0 ± 5 . 3 % and 71 .1 ± 6 . 9 % labelled w i t h the the LR-1 an t ibody , respect ive ly . No bands were observed w h e n the ni t rocel lu lose str ips were probed w i t h the rat IgM isotype cont ro l or the ant i -HSA monoc lona l ant ibody J 1 1 d (data not s h o w n ) . (B) Wes te rn blot analysis w a s per fo rmed on spleen cell lysates prepared f r o m DBA/1 mice injected 7 days before w i t h 5 % dex t rose (lanes 1-3) or Photofr in® (25 mg/kg) (lanes 4 -6 ) . Transferred prote ins (5 / /g/ lane) were probed w i t h the LR-1 an t ibody . W h e n adminis tered to DBA/1 strain mice, Photofrin® s igni f icant ly increased relat ive spleen w e i g h t , numbers of nuc leated cel ls, ery thro id p rogen i to rs , and the propor t ion of spleen cells w h i c h expressed the LR-1 ant igen (contro ls = 5 4 . 2 ± 2 . 3 % L R - 1 + ; Photofr in®-injected = 7 0 . 6 ± 3 . 0 % L R - 1 + ) (see Figures 3 . 2 6 and 3 . 2 7 ) . 2 2 4 225 7 . 2 . 5 LR-1 b ind ing spec i f ic i ty for HSA W h e n normal spleen cells w e r e t rea ted w i t h t h e LR-1 an t ibody mixed w i t h the HSA-spec i f ic reagents J 1 1 d or M1 / 6 9 (Figure 7 .12A) overal l labell ing w a s indist inguishable f r o m tha t observed w i t h either an t ibody alone. These f ind ings indicate t h a t the ant igen recognized by LR-1 is HSA or a separate ant igen pos i t ioned in c lose p rox im i ty t o HSA on the cell membrane . To fu r ther evaluate the f ine spec i f ic i ty of these ant ibodies, spleen cells were pre- incubated in buf fer alone or w i t h unlabel led rat I g M , J 1 1 d , or LR-1 after w h i c h FITC-conjugated M 1 / 6 9 w a s added. Purif ied rat IgM did not alter the binding of M1 /69 -F ITC (Figure 7 .12B) . H o w e v e r , in th ree exper iments LR-1 reduced M 1 / 6 9 b ind ing an average of 8 2 . 9 ± 2 . 3 % whi le J 1 1 d had a m u c h lower e f fec t in th is s y s t e m , reduc ing M 1 / 6 9 staining in tens i ty by only 18.1 ± 9 . 0 % . In con t ras t , none of these IgM ant ibodies prevented labell ing w i t h an an t i -MHC Class I reagent s h o w i n g tha t the b lock ing e f fec t w a s spec i f ic for HSA. W h e n the reverse exper iment w a s p e r f o r m e d , M 1 / 6 9 s t rong ly b locked the b inding of LR-1 to spleen cells (data not s h o w n ) . 7 . 2 . 6 . In f luence of Photofrin® and Hp on spleen cell express ion of HSA as recognized by four d i f ferent ant i -HSA reagents Labell ing of spleen cells obta ined 7 days post - in jec t ion w i t h 4 d i f ferent ant i -HSA monoc lona l ant ibodies (LR-1 , M 1 / 6 9 , J 1 1 d and M 1 / 7 5 ) revealed tha t Photofr in®, bu t no t Hp, s ign i f icant ly increased the number of cells w h i c h bound each of these reagents (Figure 7 . 1 3 ) . The observa t ion t h a t Photofr in® increased the number 2 2 6 Unblocked rrj 0.1 1000 0.1 1000 0.1 1000 0.1 1000 Rat IgM LR-1 0.1 1000 0.1 1000 0.1 1000 0.1 1000 J11d 0.1 1000 0.1 1000 Fluorescence Intensity Figure 7 . 1 2 HSA b ind ing speci f ic i t ies o f L R - 1 , J 1 1 d and M 1 / 6 9 ant ibod ies. . To compare the binding character is t ics of the L R - 1 , J 1 1 d , and M 1 / 6 9 monoc lona l ant ibodies (A) normal DBA/2 mouse spleen cells we re labelled w i t h the HSA-react ive monoc lona l ant ibodies J 1 1 d or M1 /69-F ITC, in the absence or presence of the LR-1 reagent . FITC-l inked ant i -rat IgM w a s subsequent ly added to ident i fy cel l-associated LR-1 or J 1 1 d . (B) Levels of M H C Class I and HSA (as recognized by M1/69-F ITC) w e re determined for normal sp lenocytes pre- incubated w i t h either rat IgM or the rat IgM monoc lona l ant ibodies LR-1 or J 1 1 d . 2 2 7 o c o >< C O C D O C D C D CO C D CO O <c CO " o 60i 50 = 40 ^ 30h 20 10 0 1 7,. I X* Treatment: I Solvent A • Photofrin H Solvent B B Hp 1 r LR-1 M1/69 J11d M1/75 Anti-HSA monoclonal antibody Figure 7 . 1 3 Numbers of spleen cells w h i c h express HSA as determined by 4 d i f ferent an t i -HSA monoc lona l ant ibodies. Spleen cells (3 mice per group) were analyzed for HSA expression 7 days after the admin is t ra t ion of Photofrin® (25 m g / k g ) , Hp (25 m g / k g ) , or the appropr iate ly matched so lvent as indicated w i th in the f igure. + P< 0 . 0 5 ; * * P< 0 . 0 0 0 1 compared to the result obta ined for mice g iven 5 % dex t rose . 2 2 8 of spleen cells w h i c h labelled w i t h the ery thro id HSA-spec i f ic an t ibody M 1 / 7 5 (matched so lvent - in jec ted cont ro ls = 3 .2 ± 1.4% M 1 / 7 5 + ; Photofr in®-injected = 14.1 ± 7 . 0 % M 1 / 7 5 + ) fu r ther suppor ts the con ten t ion tha t the c o m p o u n d had s t imula ted ery thropoies is in the o rgan . FACS stain ing prof i les of spleen cells obta ined w i t h the four ant i -HSA monoc lona l ant ibodies are presented in Figure 7 . 1 4 . 7 .2 .7 Spleen HSA co-express ion studies - in f luence of Photofrin® Analys is of the co-express ion of HSA, as recognized by the LR-1 reagent , w i t h a var ie ty of leukocy te ant igens on spleen cells prepared 7 days f o l l o w i n g Photofr in® t rea tmen t revealed a s t rong associat ion of HSA w i t h I g M , CD71 and TER-119 ant igens, l itt le associat ion w i t h the T cel l - restr ic ted CD3, CD4, and CD8 ant igens and presence on approx imate ly 5 0 % of the cells w h i c h expressed the g ranu locy te marker G r - 1 , in bo th cont ro l and Photofr in®-injected mice (Figure 7.1 5 A ) . H o w e v e r , cells d isplaying a CD71 + / H S A + , T E R - 1 1 9 + / H S A + , TER-1 1 9 + / C D 7 1 + or T E R - 1 1 9 V C D 7 1 " phenotype were found in s ign i f icant ly greater numbers in the spleens of Photofr in®-treated animals (Table 7 .1 ) . Approx imate ly 5 0 % of T E R - 1 1 9 + spleen cells co-expressed C D 7 1 . The spleen cells w h i c h most s t rong ly expressed CD71 co-expressed the TER-119 ant igen (Figure 7.1 5B). Low, but detec tab le a m o u n t s of CD71 are present on splenic B cells (Futran et a l . , 1989) we re gated w i t h i n the C D 7 1 " popu la t ion . A d is t inc t popula t ion of H S A + / C D 4 5 " / l 0 cells w a s ev ident in the spleens of bo th cont ro l and Photofr in®-injected mice but p ropor t iona l ly , the HSA + /CD45" / | 0 popula t ion w a s approx imate ly t w i c e as large in the Photofr in®-treated animals. 2 2 9 M1/75 LR-1 J11d M1/69 Fluorescence Intensity Figure 7 . 1 4 Spleen cell expression of HSA as labelled w i t h 4 d i f ferent ant i -HSA ant ibodies - FACS prof i les. Representat ive f l o w cy tomet r i c prof i les of spleen cells labelled w i t h the ery thro id HSA-spec i f ic an t ibody M 1 / 7 5 , or the HSA-react ive ant ibodies L R - 1 , J 1 1 d , and M 1 / 6 9 (bold t rac ings) are s h o w n . Results obta ined w i t h the iso type-matched con t ro l ant ibodies are s h o w n as th in t rac ings . Spleen cells were prepared f r o m DBA/2 mice 7 days after the in ject ion of 5 % dext rose (Solvent A ) , Photofrin® (25 mg /kg ) , Solvent B, or Hp (25 m g / k g ) . 2 3 0 In an exper iment per fo rmed to compare the co-express ion of CD71 w i t h HSA on spleen cells obta ined 4 days after the admin is t ra t ion of Photofrin®, similar resul ts w e re obta ined whe the r LR-1 or J 1 1 d w a s used to sur face label HSA. H o w e v e r , s o m e w h a t f e w e r H S A + / C D 7 1 + cells were ident i f ied w i t h J 1 1 d (Figure 7 . 1 6 ) . V i r tua l ly all C D 7 1 b r i g h t spleen cells also expressed HSA. These studies s h o w e d t h a t Photofr in® increased to ta l numbers of C D 7 1 + / H S A + spleen cells approx imate ly f ive- fo ld above cont ro l levels by four days pos t - in jec t ion , whe the r the LR-1 or J 1 1 d an t ibody w a s uti l ized for HSA labell ing (Table 7 .2 . ) . 231 Table 7.1 Summar ized results of spleen cell double label FACS analyses. Co-express ion of d i f ferent leukocyte ant igens w i t h HSA, as recognized by the monoc lona l an t ibody L R - 1 , and co-express ion o f t he ery thro id- res t r ic ted ant igen TER-119 w i t h CD71 ( t ransferr in receptor) w a s evaluated for spleen cells obta ined f r o m mice in jected w i t h 5 % dext rose or Photofrin® 7 days previously. Results w e re obta ined w i t h 3-8 animals per g roup . % of spleen cells Tota l cells per sp leen 1 Ant igen co-express ion pat te rn 5 % dext rose Photofrin® 5 % dex t rose Photofrin® L R - 1 + / C D 4 5 + 5 0 . 3 ± 2 .4 4 7 . 9 ± 2.1 2 2 . 9 ± 5.5 3 3 . 2 ± 1.3* L R - 1 + / C D 4 5 / l 0 4 . 2 ± 1.70 8 .4 ± 1 .3* 1.9 ± 1.2 6 .0 ± 0 . 7 * * L R - 1 + / l g M + 3 4 . 0 ± 3 .8 2 9 . 5 ± 1.4 13 .3 ± 1.4 15 .8 ± 1.6 L R - 1 + / G r - 1 + 3 .4 ± 1.0 3 .7 ± 0 .5 1.4 ± 0 . 6 2 .2 ± 0 .4 L R - 1 + / C D 3 + 2.7 ± 0 .8 1.8 ± 0 . 1 * 1.1 ± 0 .3 0 .8 ± 0 .7 L R - 1 + / C D 4 + 3.1 ± 0 . 4 2.5 ± 0 . 4 1.2 ± 0 .3 1.5 ± 0.1 L R - 1 + / C D 8 + 1.3 ± 0 .7 1.5 ± 0 . 4 0 .5 ± 0 .3 0 .9 ± 0 . 4 L R - 1 + / C D 7 1 + 3.6 ± 1.4 8 .4 ± 1 .3* 1.6 ± 0 .9 6 .0 ± 0 . 7 * * C D 7 1 + / L R - 1 0 0 - -L R - 1 + / T E R - 1 1 9 + 3.3 ± 0 .3 * * 7.4 ± 1.1 1.7 ± 0 .3 * * 5.1 ± 1.1 TER-119 + / C D 7 1 + 1.6 ± 0 .5 * * 3.7 ± 0 .3 0 .7 ± 0 .3 2 .6 ± 0 . 3 * * TER-119 + / C D 7 1 1.7 ± 0 . 4 2 .8 ± 0 . 3 * 0 .8 ± 0 .3 1.9 ± 0 . 3 * 1 g iven as the number of spleen cells (x 10~6) normal ized to a b o d y w e i g h t of 2 0 g rams. * P < 0 . 0 5 ; * * P < 0 .01 compared to the result obta ined w i t h cells f r o m cont ro l mice. 2 3 2 Table 7 .2 Compar ison of the in f luence of Photofrin® on spleen cell co-express ion of C D 7 1 and HSA, as labelled by LR-1 and J 1 1 d monoc lona l ant ibod ies . Spleen cells obta ined f r o m DBA/2 mice in jected 4 days previously w i t h 5 % dex t rose or Photofr in® (25 mg/kg) were labelled w i t h the LR-1 or J 1 1 d monoc lona l an t ibody . Sur face CD71 w a s ident i f ied w i t h t he an t i -CD71 ant ibody C2F2 . Spleen w e i g h t and cel lular i ty data for these mice have been included and exhib i t the character is t ic increases in these parameters p roduced by Photofrin®. Trea tment Spleen parameter 5 % dext rose (n = 4) Photofr in® (n = 4) Relative w e i g h t (x 100) 0 . 3 6 6 ± 0 . 0 2 0 0 . 6 3 4 ± 0 . 0 7 4 * * Nucleated cell numbers (x 1 0 6 / 2 0 g) 3 3 . 3 1 ± 3 .43 5 9 . 9 1 ± 6 . 3 0 * * C D 7 1 + / L R - 1 + (% of cells) 2 . 4 0 ± 0 . 1 4 7 . 6 8 ± 2 . 1 1 * C D 7 1 + / J 1 1 d + (% of cells) 1.68 ± 0 .41 4 . 4 0 ± 0 . 6 3 * * Tota l C D 7 1 + / L R - 1 + cells (x 1 0 5 / 2 0 g) 7 .98 ± 0 .81 4 6 . 7 7 ± 1 7 . 5 2 * Tota l C D 7 1 + / J 1 1 d + cells (x 1 0 5 / 2 0 g) 5.7 ± 1.92 2 6 . 6 0 ± 6 . 5 0 * * * P< 0 . 0 5 ; * * P< 0 . 0 0 1 compared to the so lvent - in jected cont ro l mice by S tuden t ' s t - tes t . 2 3 3 F i g u r e 7 . 1 5 I n f l u e n c e o f P h o t o f r i n ® o n s p l e e n ce l l c o - e x p r e s s i o n o f H S A w i t h v a r i o u s l e u k o c y t e a n t i g e n s . ( A ) Spleen cells obta ined 7 days fo l l ow ing the in ject ion of 5 % dext rose or Photofr in® (25 mg/kg) w e re analyzed for HSA expression using the LR-1 ant ibody and a bat te ry of reagents to counter label other cell sur face ant igens. Cell sur face bound LR-1 w a s revealed using PE-conjugated ant i - rat IgM an t ibody , excep t in con junc t ion w i t h ant i -CD71-PE w h e n a FITC-conjugated an t ibody w a s ut i l ized. For cells labelled w i t h an t i -CD45 and L R - 1 , the CD45~ / I 7HSA + spleen cell populat ion is demarca ted by a rectangle. (B) Co-expression of TER-119 and CD71 ant igens by spleen cells 7 days post - in jec t ion is s h o w n . Summar ized f l o w cy tomet r i c f ind ings and est imates of to ta l double posi t ive spleen cell numbers are presented in Table 7 . 1 . 2 3 4 Figure 7 . 1 5 235 Figure 7 . 1 6 Spleen cell co-express ion o f C D 7 1 w i t h HSA. Co-express ion of CD71 w i t h HSA (as recognized by LR-1 and J 1 1 d monoc lona l ant ibodies) w a s determined for spleen cells obta ined 4 days after the in ject ion of 5 % dext rose or Photofrin®. Summar ized f ind ings are presented in Table 7 .2 . 2 3 6 7 . 2 . 8 Inf luence of Photofrin® on BM cell HSA and C D 7 1 expression W h e n BM cells were evaluated 2 or 4 days pos t - in jec t ion , HSA, as recognized by the LR-1 an t ibody , w a s expressed on the vas t major i ty ( — 9 5 % ) of these cells obta ined f r o m mice g iven 5 % dext rose or Photofr in® at bo th sampl ing t imes (Figure 7 . 1 7 A ) . H o w e v e r by day 4 post - in jec t ion, there w a s a s igni f icant shi f t in the overal l in tens i ty of LR-1 labell ing for BM cells f r o m mice g iven Photofrin®. BM cells f r o m the cont ro ls exhib i ted a b imodal HSA expression prof i le whereas BM cells f r o m Photofr in®-treated mice displayed a large enhancement of the lower intensi ty c o m p o n e n t o f the H S A + peak. The mean channel f luorescence for HSA labell ing o f BM cells obta ined f r o m Photofr in®-injected mice on bo th days w a s s igni f icant ly less than tha t of BM cells f r o m the solvent- in jected mice. These values fell to a lmost one-hal f o f t ha t o f the cont ro ls by day 4 (Table 7 .3 ) . A t the day 2 sampl ing t ime , there w a s a greater p ropor t ion of H S A V C D 7 1 + cells in the m a r r o w of Photofrin®-t reated mice than the so lvent - in jected cont ro ls , a l though th is d i f ference w a s not s tat is t ica l ly s ign i f icant . However , by day 4 post - in jec t ion , there we re propor t ional ly f e w e r cells w h i c h expressed bo th CD71 and HSA w i th in the m a r r o w of Photofrin®-t reated mice , w h i c h represented a stat is t ical ly s ign i f icant d i f ference (Table 7 .3 , Figure 7 . 1 7 C ) . The f l o w cy tomet r i c labell ing pat terns of normal BM cells for the ant i -CD71 and ant i -HSA ant ibodies and the appropr iate isotype cont ro l ant ibodies are s h o w n in Figure 7 . 1 7 B . 2 3 7 Figure 7 . 1 7 In f luence of Photofr in® on BM cell HSA express ion and numbers o f BM cells w h i c h co-express HSA and C D 7 1 . (A) Expression of HSA w a s determined for BM cells obta ined f r o m D B A / 2 mice g iven either 5 % dext rose or Photofrin® (25 mg/kg) 2 (upper panels) or 4 ( lower panels) days prev ious ly . Cell sur face HSA w a s labelled w i t h the monoc lona l an t ibody LR-1 (bold t rac ings) . The result obta ined w i t h the rat IgM isotype con t ro l is s h o w n by th in t rac ings . (B) Normal DBA/2 mouse BM cells were analyzed for their co-express ion of HSA and CD71 ant igens. HSA w a s labelled w i t h the LR-1 reagent wh i le CD71 w a s recognized w i t h the PE-conjugated rat l g G 2 a an t ibody C2F2. Flow cy tomet r i c resul ts (left to r ight) obta ined w i t h the isotype con t ro ls , ant i -HSA alone, ant i -CD71 alone, or the ant i -HSA and ant i -CD71 ant ibody pair are s h o w n to demons t ra te the va l id i ty of the colour compensa t ion set t ings . (C) BM cells obta ined f r o m mice g iven 5 % dext rose or Photofrin® 2 days (upper panels) or 4 days ( lower panels) prev iously were analyzed for co-express ion o f HSA and CD71 sur face ant igens. Summar ized results of these studies are prov ided in Table 7 .3 . 2 3 8 Figure 7 . 1 7 239 Table 7 .3 Inf luence of Photofrin® on bone m a r r o w HSA expression and H S A / C D 7 1 co-express ion levels. Expression levels of HSA and co-express ion of HSA w i t h CD71 were de termined for BM cells obta ined 2 and 4 days after the admin is t ra t ion of cont ro l so lvent or Photofr in® (25 m g / k g ) . n = number of mice per g roup analyzed. Time post-in ject ion HSA mean channel f luorescence (FITC) H S A + / C D 7 1 + BM cells (%) 2 days H S A + BM cells (%) 5 % dex t rose 9 6 . 3 ± 0 .2 (n = 3) 1 2 7 . 3 ± 5.9 (n = 3) 14 .3 ± 1.9 (n = 3) Photofr in® 9 7 . 3 ± 0 . 4 (n = 3) 1 0 9 . 4 ± 4 . 7 * (n = 3) 1 9 . 0 ± 2 .6 (n = 3) 4 days 5 % dex t rose 9 4 . 3 ± 0 . 4 (n = 7) 1 1 0 . 4 ± 1 2 . 0 ( n . = 7) 1 6 . 8 ± 2 .3 (n = 3) Photofr in® 9 3 . 8 ± 1.0 6 5 . 5 ± 2 . 4 * * 8 .2 ± 1 . 6 * * (n = 7). (n = 7). (n = 3) * P< 0 . 0 2 5 ; * * P< 0 . 0 0 1 by S tuden t ' s t - tes t compared to the result obta ined w i t h BM cells f r o m solvent - in jected cont ro l mice. 7.3 Discuss ion In th is series of exper iments , it w a s demons t ra ted tha t the rat monoc lona l an t ibody LR-1 recognizes mouse HSA. F low cy tomet r i c studies s h o w e d tha t LR-1 labelled mos t splenic B cells but f e w splenic T cells in all mouse strains tes ted . This feature is cons is ten t w i t h the an t i - l ymphocy te react iv i ty pat tern exhib i ted by other HSA-speci f ic monoc lona l ant ibodies (Bruce et al., 1 9 8 1 ; Kay et 2 4 0 al., 1 9 9 1 ; Liu e r a / . , 1 9 9 2 ) . In add i t ion , LR-1 s t rong ly labelled t h y m o c y t e s , BM cells, and per ipheral b lood e ry th rocy tes . This part icular d is t r ibut ion amongs t these t issues w a s suggest ive of tha t of heat-stable ant igen (HSA, Nectadr in) , init ially ident i f ied by Springer et al. (1978) using the rat monoc lona l an t ibody M 1 / 6 9 and by Bruce et al. ( 1981 ) by its labell ing w i t h the rat monoc lona l an t ibody J 1 1 d . Both of these reagents mark mur ine neut rophi ls , e ry th rocy tes , m o n o c y t e s but not t issue macrophages , mos t t h y m o c y t e s and B cel ls, and a minor p ropor t ion of peripheral T cel ls. HSA levels are part icular i ly h igh on immature T (Crispe and Bevan, 1987) and B l y m p h o c y t e s (Hardy et al., 1 9 9 1 ) . In terest ingly, HSA has been s h o w n to be t rans ient ly expressed on T cells t ha t have been recent ly ac t iva ted in vivo or in vitro (Hubbe and A l t e v o g t , 1 9 9 4 ) . Of part icular impor tance is the observat ion tha t mur ine m e m o r y B cells do no t express HSA (Bruce etal., 1 9 8 1 ; Pillai etal, 1 9 8 6 ; L inton et al., 1 9 8 9 ) , the only such ant igen expression pat tern tha t unequivocal ly ident i f ies th is l y m p h o c y t e subset . Fur thermore, HSA is present on splenic DC (Crowley et al., 1 9 8 9 ; Inaba et al., 1994) and levels of HSA are increased on mouse splenic B l ymphocy tes upon their ac t iva t ion w i t h LPS (Kennedy et al., 1 9 9 4 ) , features observed w h e n these cell t ypes were labelled w i t h the LR-1 reagent . Evidence indicates tha t mur ine hematopo ie t ic s tem cells are HSA", whereas d i f fe rent ia t ing ery thro id and g ranu locy te -macrophage precursors are H S A + (Bruce et al., 1 9 8 1 ) . A m o n g non-hematopo ie t ic t issues, HSA mRNA t ranscr ip ts are detec tab le in normal mouse brain t issues (Wenger et al., 1 9 9 1 ) . The heat-stable des ignat ion refers to the resistance of the ant igenic de te rminants to heat ( 1 2 0 ° C ) 2 4 1 denatura t ion fo l l ow ing f i xa t ion (Springer et al., 1 9 7 8 ) . The react iv i ty of the rat monoc lona l ant ibodies B 2 A 2 (Scollay et al., 1 9 8 4 ) , 7 9 (Hubbe and A l tevog t , 1 9 9 4 ) , 30F1 (Ledbetter and Herzenberg, 1979) and the hamster monoclonal an t ibody 2 0 C 9 (Liu et al., 1991) against mouse HSA has been del ineated. Fur thermore, Mil ler et al. (1985) demons t ra ted tha t the rat monoc lona l ant ibody M 1 / 7 5 (Springer et al., 1978) labelled a var iant of HSA present only on mur ine red b lood cells and ery th rob las ts . HSA belongs to a large, heterogeneous group of cell sur face molecules in w h i c h the pro te in is anchored in the cel l membrane t h r o u g h cova len t l inkage to a g lycosy l -phosphat idy l inos i to l (GPI) moie ty (Lisanti etal., 1 9 9 1 ; Robinson, 1991 ) . The major i ty of GPI-l inked molecules are sensi t ive to release f r o m the cell membrane by bacter ial phosphat idy l inos i to l -spec i f ic phosphol ipase C (PI-PLC, Robinson, 1 9 9 1 ) . Studies by Pierres e f al. (1987) indicated tha t mur ine HSA w a s a t tached t o t h e cell membrane t h r o u g h GPI l inkage. Other GPI-l inked molecules include Thy-1 ( C D 9 0 , L o w and Kincade, 1 9 8 5 ) , C D 1 4 (Haziot et a l . , 1988) and p roduc ts of the Ly-6 gene locus (Su et al., 1 9 9 1 ) . Enzyme digest s tudies conf i rmed tha t HSA on normal spleen cells as recognized by LR-1 is GPI- l inked. For Photofrin®-t reated mice , HSA on spleen cells w h i c h mos t s t rong ly expressed th is marker, as recognized by L R - 1 , appeared s o m e w h a t resistant to the act ion of PI-PLC. The s igni f icance of th is f ind ing is unclear. Certain cell sur face molecules such as LFA-3 exist as t ransmembrane or GPI-anchored fo rms as the result of a l ternat ive RNA spl ic ing (Barclay et al., 1 9 9 3 ) . It is u n k n o w n whe the r HSA migh t exist in a t ransmembrane f o r m , a l though th is possibi l i ty has been suggested (Kay et al., 2 4 2 1 9 9 1 ; Wenger e f a/ . , 1 9 9 1 ) . St ructura l var ia t ions w i th in the GPI-anchor itself may impar t resistance to PI-PLC cleavage f r o m the cell sur face (Robinson, 1 9 9 1 ) . B iochemical analysis and molecular c lon ing s tud ies have s h o w n tha t the mature f o r m of mouse HSA is an unusual ly small prote in conta in ing only 27 amino acids w i t h an est imated molecular w e i g h t of 3 kDa (Kay et al., 1 9 9 0 ; W e n g e r et al., 1 9 9 1 , Figure 7 . 1 8 ) . H o w e v e r , in po lyacry lamide gels HSA migrates w i t h an apparent molecular mass ranging b e t w e e n 2 8 - 7 0 kDa , dependent upon the cell ex t rac t examined . Wes te rn blot analysis indicated tha t HSA had a molecular mass o f 3 5 - 4 5 kDa o n e ry th rocy tes , 5 0 - 6 0 kDa on t h y m o c y t e s and 4 5 - 5 5 kD on sp lenocy tes (Barclay etal., 1 9 9 3 ) . This suggests t ha t the HSA prote in core is most l ikely ex tens ive ly , bu t var iab ly , g lycosy la ted among d i f fe rent cell l ineages. The pred ic ted amino acid sequence o f mur ine HSA indicates t he presence of three potent ia l s i tes for N-linked g lycosy la t ion and a number of possible sites of O-linked g lycosy la t ion (Kay et al., 1 9 9 0 ) . Cellular processing of the molecule includes the remova l o f a s ignal sequence and rep lacement of t he C-terminal reg ion w i t h the GPl g roup (Kay etal., 1 9 9 0 ) . Human C D 2 4 may be a homologue of mur ine HSA in tha t it is also a smal l , heavi ly g lycosy la ted GPI-l inked protein (Kay etal. 1 9 9 1 ) . A l t hough there is s t rong sequence ident i ty b e t w e e n the leader pept ide and GPl anchor signal sequences of human C D 2 4 and mouse HSA, the mature prote ins exhib i t only 3 0 % sequence ident i ty (Kay et al., 1 9 9 1 ) . In th is w o r k , i m m u n o b l o t analysis o f de te rgen t lysates ind icated tha t the LR-1 reagent recognized molecules ranging b e t w e e n 3 0 and 55 kDa in t h y m o c y t e s and BM cel ls, b e t w e e n 3 0 - 4 0 kDa and 4 5 - 5 5 kDa in sp lenocy tes , and 2 4 3 3 0 - 4 5 kDa in e ry th rocy tes . These molecular w e i g h t est imates are in reasonable agreement w i t h those demons t ra ted by others for HSA in these t issues (Al terman et al., 1 9 9 0 ; Kay et al., 1 9 9 0 ) . Fur thermore, a d is t inc t band w i t h an est imated molecular mass of 21 kDa w a s demonst rab le in bone m a r r o w and spleen, but not in t h y m o c y t e or e r y t h r o c y t e de tergent lysates probed w i t h t he LR-1 ant ibody. Expression of th is molecular species w a s great ly increased in the spleens of Photofr in®-injected mice. A l te rman et al. (1990) repor ted the presence of t w o low ly g lycosy la ted HSA prote in cores w i t h apparent molecular masses of 2 0 and 17 kDa, ex is t ing in a rat io of approx imate ly 5 : 1 , w h e n HSA-express ing cell l ines were g r o w n in the presence of t u n a c a m y c i n , an ant ib iot ic w h i c h prevents the addi t ion of re-l inked carbohydra te side chains. In our exper iments , the 21 kDa prote in recognized by the LR-1 an t ibody may represent a less g lycosy la ted precursor of ery thro id HSA since it w a s de tec ted only in ery thropo ie t ic t issues and w a s not associated w i t h the mature red cel l . The var ious monoc lona l ant ibodies w h i c h react w i t h HSA may recognize d i f ferent ia l ly g lycosy la ted fo rms or are sensi t ive to speci f ic surface env i ronments on these cells (Miller et al., 1 9 8 5 ; Hardy etal., 1 9 9 1 ) . The potent ia l f ine speci f ic i t ies o f these ant ibodies for d is t inc t HSA ep i topes have no t been r igorously pu rsued . H o w e v e r it has been indicated tha t for t h y m o c y t e HSA by f l o w cy tomet r i c analys is, all ant i -HSA monoc lona l ant ibodies react w i t h the same ant igenic de te rminan t on the prote in core of the molecule and th is b inding is not in f luenced by ca rbohydra te side chains (A l terman et al., 1 9 9 0 ) . 2 4 4 Amino acid codes: Ala = alanine Arg = arginine Asn = asparagine Gin = glutamine Gly = glycine He = isoleucine Phe = phenylalanine Pro = proline Ser = serine Thr = threonine Val = valine mannose mannose mannose cell membrane PI-PLC cleavage site fatty acid Figure 7 . 1 8 Molecular organizat ion and amino acid sequence of mur ine heat-stable ant igen (HSA) . Graphical presentat ion of the GPI-l inkage has been adapted f r o m Robinson ( 1 9 9 1 ) . A m i n o acid sequence w a s determined by Kay et al. (1990) and Wenger et al. ( 1 9 9 1 ) . The C-terminal of n e w l y synthes ized HSA is c leaved immedia te ly af ter synthes is in the endoplasmic re t icu lum and replaced w i t h a p re fo rmed GPI-anchor ing domain by means of a t ransamidase enzyme (Doering etal., 1 9 9 0 ) . A 2 6 amino acid leader pept ide is also c leaved f r o m the precursor protein (Kay et al., 1 9 9 0 ) . Cross-hatched areas indicate potent ia l si tes of N-l inked g lycosy la t ion . The a r r o w pos i t ion denotes the c leavage site for phosphat idy lphospho inos i to l -spec i f i c phosphol ipase C (PI-PLC). Relative sizes of the molecular s t ruc tures are not d r a w n to scale. P = phospha te . 2 4 5 In the present s tudy , the M 1 / 6 9 and LR-1 ant ibodies cons is tent ly labelled a higher p ropor t ion of spleen cells than J 1 1 d . In add i t ion , M 1 / 6 9 labell ing of spleen cells w a s s t rong ly b locked by the ant i -HSA an t ibody L R - 1 , but only w e a k l y by J 1 1 d . This f ind ing indicates tha t M 1 / 6 9 and J 1 1 d label over lapp ing, but not ident ical splenic HSA epi topes or tha t J 1 1 d is an an t ibody of l ow af f in i ty . LR-1 and M 1 / 6 9 appear to recognize all spleen HSA var iants , wh i le J 1 1 d fails to label a small p ropor t ion of H S A + spleen cel ls, typ ica l ly those w i t h the h ighest levels of the g l ycopro te in . These studies demons t ra te tha t LR-1 and M 1 / 6 9 share similar, if no t ident ical ep i tope speci f ic i t ies in the spleen and tha t J 1 1 d recognizes either a d is t inc t site on HSA or possesses displaceable, lower b inding af f in i ty for HSA than either LR-1 or M 1 / 6 9 . The abi l i ty of LR-1 but not J 1 1 d to label t issue ex t rac ts m igh t be related to a d i f ference in the b inding af f in i ty of these rat IgM ant ibodies. A l t h o u g h HSA is w ide ly expressed w i th in the immunohematopo ie t i c sys tem and on deve lop ing neural cells (Nedelec e r a / . , 1 9 9 2 ) , the cellular func t ion of HSA is no t we l l unders tood . The apparent size of the molecule var ies w i th in and among d i f fe rent t issues. Given its shor t po lypept ide cha in , the bulk o f the molecule is ca rbohydra te and , there fo re , var ia t ions in the nature and number of sugar moiet ies may confer d is t inc t func t iona l propert ies and/or l igand speci f ic i t ies. The ex is tence of coun te r l igands for HSA has been proposed and it has been indicated tha t mur ine HSA cou ld faci l i tate the b inding of g ranu locy tes and m o n o c y t e s to platelets and endothel ia l cells t h r o u g h an in teract ion w i t h P-selectin (CD62P, Sammar e r a / . , 1 9 9 4 ) . In con t ras t to the major i ty of GPI-l inked membrane proteins such as members of the Ly-6 fami ly ( M a l e k e f al., 1 9 8 6 ; Fleming and Malek, 1 9 9 4 ) , 2 4 6 Thy-1 (Thomas and Samelson, 1992) and C D 1 4 (Mac in ty re et al., 1 9 8 7 ) , cross-l inking of mur ine HSA does not induce cellular ac t iva t ion or convey a t ransmembrane signal (Pierres et al, 1 9 8 7 ) . Incubat ion of human B chronic lymphoblas t ic leukemia cell line w i t h ant i -human C D 2 4 monoc lona l an t ibody in comb ina t ion w i t h a cross- l ink ing ant i -mouse Ig an t ibody s t imula ted the tyros ine phosphory la t ion of several cellular prote ins (Stefanaova et al., 1991) suggest ing a possible s ignal ing role for human C D 2 4 . A role for HSA in early l ymphocy te deve lopmen t w a s suggested by studies in w h i c h it w a s s h o w n tha t d isrupt ion of HSA express ion in mur ine pre-B cells in f luences the abi l i ty of very late ant igen-4 ( V L A 4 , CD49d) to bind to f ib ronect in and the vascular cell adhesion molecule-1 ( V C A M - 1 ) , t w o of its natural l igands (Hahne et al., 1 9 9 4 ) . H o w HSA migh t a f fect these molecular in teract ions is not unders tood . A role for HSA as a co-s t imula tory molecule invo lved in T cell ac t iva t ion has been advanced . For the act ivat ion of rest ing T l ymphocy tes , a m i n i m u m of t w o d is t inc t intracel lular signals mus t be received (June et al., 1 9 9 4 ) . The f i rs t is del ivered t h r o u g h the T cell recep tor /CD3 comp lex and current k n o w l e d g e indicates tha t signal l ing via C D 2 8 suppl ies an appropr ia te cos t imu la tory message for T cell ac t iva t ion (Linsley and Ledbet ter , 1 9 9 3 ) . Liu et al., (1991) demons t ra ted t h a t the hamster ant i -HSA monoc lona l an t ibody 2 0 C 9 inhibi ted the cos t imu la to ry ac t iv i ty of ac t iva ted mouse splenic B cells par t ic ipat ing in the prol i ferat ive response of ant i -CD3 monoc lona l an t ibody-cu l tu red T l ymphocy tes , w i t h o u t in ter fer ing w i t h B cell - T cell c luster f o r m a t i o n . In add i t ion , f ibroblasts co-t rans fec ted w i t h Fc receptor II and HSA cDNA cons t ruc ts also s t imulated T cell 2 4 7 prol i ferat ion in the presence of the mi togenic ant i -CD3 an t ibody (Liu et al., 1991 ) . Cur ious ly , the rat an t i -HSA monoc lona l ant ibodies J 1 1 d and M 1 / 6 9 had no inhib i tory e f fec t on T cell prol i ferat ion w h e n assayed in th is sys tem (Liu et al., 1 9 9 1 ) . In con t ras t to the ant igen-present ing cell (APC)-restr ic ted sur face molecules B7 ( C D 8 0 , Linsley and Ledbet ter , 1993) and B7-2 (Powers et al., 1 9 9 4 ) , the func t i on of HSA as a B cell co-s t imula tory molecule in the ac t iva t ion of T cells is more d i f f i cu l t to envisage. HSA is present at h igh levels on bo th unst imula ted and act iva ted B cel ls, a l though only ac t iva ted B cells per fo rm ef f ic ient ly as APC (Inaba and S te inman, 1 9 8 4 ; Met lay et al., 1 9 8 9 ) . Levels of cell sur face HSA on murine splenic B cel ls are fu r ther upregula ted upon ac t iva t ion w i t h LPS, bu t no t fo l l ow ing l igat ion w i t h C D 4 0 l igand (Kennedy et al., 1 9 9 4 ) . Enk and Katz (1994) more recent ly demons t ra ted tha t HSA plays a co-s t imula tory role in suppor t ing the ac t iva t ion of T cells by ant i -CD3 and the s t imula t ion of al logeneic T cells by epidermal Langerhans ' cells and th is ac t iv i ty w a s part ial ly b locked by the 2 0 C 9 monoc lona l an t ibody . The presence of h ighly g lycosy la ted , negat ive ly -charged molecules such as HSA on the cell sur face may coun te rac t the T cel l -act ivat ing capac i ty of APC, an a t t r ibu te w h i c h may be reversed by enzymat ic removal of sialic acid residues w i t h neuraminidase (Boog et al., 1 9 8 9 ; Neefjes et al., 1 9 9 0 ) . It is conceivab le tha t cellular ac t iva t ion could alter the g lycosy la t ion pat tern of HSA on B l y m p h o c y t e s thereby fac i l i ta t ing their APC f u n c t i o n . Liu et al. ( 1991 ) postu la ted tha t rest ing B cells may express an HSA inhibi tor tha t is repressed or lost upon cell ac t i va t ion . It may also be possible t ha t an ant i -HSA monoc lona l an t ibody could 2 4 8 ster ical ly hinder the func t i on of accessory cell molecules adjacent to HSA in the cell membrane , the reby inhibi t ing authent ic co-s t imu la tory ac t iv i t y . However , its p romiscuous d is t r ibu t ion among a w ide var ie ty of leukocy tes (Bruce et al., 1 9 8 1 ) , he terogeneous g lycosy la t ion pat terns , and l ow (Crowley et al., 1989) to moderate express ion (Inaba et al., 1 9 9 4 ; Liu et al., 1992) by mur ine splenic DC, po tent ant igen present ing cells of the immune s y s t e m , w o u l d be incons is tent w i t h the no t ion tha t an overal l f unc t ion of HSA is tha t of a co-s t imu la tory molecule in the ac t iva t ion o f T cel ls. The HSA pept ide appears t o represent a versat i le scaf fo ld on to w h i c h var ious carbohydra te residues can be a t tached , the reby generat ing l igands w i t h d is t inc t b ind ing proper t ies. Erythropoiet ic ac t iv i ty in hematopo ie t ic t issues is typ ica l ly evaluated and quant i f ied w i t h in vitro progeni tor assays. In con t ras t , the mon i to r ing of e ry thropo ie t ic changes using f l o w cy tomet r i c analysis is compara t i ve ly used less f requent ly . Few markers exist ' w h i c h unequivocal ly ident i fy mur ine ery thro id precursor cel ls. The screening of a bat tery of ant i -mouse leukocy te ant ibodies ident i f ied a reagent ( M 1 / 7 5 ) w h i c h ident i f ied an e ry th rocy te - res t r i c ted var iant of HSA (Springer et al., 1 9 7 8 ) . The M 1 / 7 5 ant igen is f i rs t detectab le at the ery throb las t s tage of deve lopment (Miller et al., 1 9 8 5 ) . Ikuta et al. (1991) deve loped the monoc lona l an t ibody TER-119 w h i c h labelled mature red cells and cer ta in e ry thro id precursors . No in format ion exists concern ing the nature of th is an t igen . The major i ty of T E R - 1 1 9 + cells in adul t mouse bone m a r r o w were ery th rob las ts morpho log ica l ly , and ery thro id progeni tor assays indicated tha t cells w i t h BFU-E or CFU-E ac t iv i ty were TER-119" ( Ikuta et al., 1 9 9 0 ) . The receptor for 2 4 9 t ransfer r in (CD71) , is expressed to a degree by mos t cells and at high levels on pro l i ferat ing cel ls. This ant igen is also s t rongly expressed on deve lop ing ery thro id cells in order to sat is fy the demands of hemoglob in synthes is for i ron. In ery thro id t issues, express ion of CD71 is largely associated w i t h cells w i t h CFU-E, but not BFU-E ac t i v i t y , and is absent f r o m the mature red cell (Lesley et al., 1 9 8 4 ) . For ery thro id t issues, in con t ras t t o non-ery thro id regions, high express ion of CD71 is associated w i t h cell d i f fe rent ia t ion rather than cell pro l i ferat ion (Ponka, 1994 ) . T h u s , the ant ibodies w h i c h speci f ical ly ident i fy deve lop ing mur ine ery thro id cells label cells at the more advanced stages of ery thro id d i f fe ren t ia t ion . An t igens w h i c h are down- regu la ted and u l t imate ly el iminated dur ing ery thro id deve lopment include C D 4 5 and M H C Class I. C D 45 is present on the major i ty of bone mar row-der ived cells but absent f r o m ery throb las ts and e ry th rocy tes of the mouse (Schied and Tr ig l ia, 1979) and human (Shah et al., 1 9 8 8 ) . The progressive loss of MHC Class I ant igens in deve lop ing ery thro id cells appears to closely parallel the d imin ishment in C D 4 5 express ion (B rown etal., 1 9 8 1 ; Robinson et al., 1 9 8 1 ; Sieff etal., 1 9 8 2 ) . Admin is t ra t ion of Photofrin®, but not its monomer ic equivalent Hp, e levated numbers o f splenic ery thro id progeni tors and produced phenotyp ic changes in th is organ w h i c h paralleled the increased ery thropo ie t ic ac t iv i ty inc luding an increase in t he number o f cel ls w h i c h expressed the TER-119 , C D 7 1 , and HSA prote ins and those tha t expressed l o w or undetectab le amoun ts of CD45 or MHC Class I molecu les . Double label FACS analysis fu r ther suppor ted the observat ion tha t the major hematopo ie t ic e f fec t of Photofrin® in the spleen w a s w i th in the ery thro id d i f fe rent ia t ion p a t h w a y , in tha t the number of nuc leated cells w i t h an 2 5 0 erythro id- l ike C D 4 5 7 H S A \ CD71 + / H S A + , T E R - 1 1 9 + / H S A + , or T E R - 1 1 9 V C D 7 1 + co-express ion pat tern w a s s igni f icant ly increased fo l l ow ing admin is t ra t ion of the po rphy r in . The s tudy of HSA expression w i th in the major leukocy te l ineages, as recognized by the LR-1 an t ibody , accoun ted for mos t bu t not all H S A + spleen cells o f Photofr in®-injected mice . Presumably, t he remainder o f splenic H S A + cells are deve lop ing immature ery th ro id /mye lo id cells w h o s e fo rmat ion w a s also promoted by Photofr in®. Perhaps it is no t supr is ing tha t it w a s the LR-1 reagent w h i c h init ially ind icated t h a t Photofr in® enhanced erythropoies is in the sp leen, since th is ant ibody w a s der ived f r o m a Lou rat immunized w i t h mur ine Friend v i rus- t rans formed ery th ro leukemia cells (Hutch ings et al., 1985) and cells of th is t ype express high levels of HSA (Chapter 8 ) . The func t iona l s igni f icance of HSA on the mature red cell is u n k n o w n a l though a p ro tec t i ve role against homologous comp lemen t a t tack has been proposed (H i tsumoto et al., 1 9 9 3 ) . It has been s h o w n tha t o ther GPI-anchored pro te ins , inc luding decay accelerat ing fac tor (CD55) and homo logous restr ic t ion fac to r (CD59) , regulate facets of comp lemen t act iv i ty (Lisanti etal., 1 9 9 0 . A recent s tudy has indicated tha t C D 5 5 and C D 5 9 expressed on the sur face of mur ine red b lood cells can be t ransfer red as fu l ly func t iona l complement - inh ib i t ing prote ins to endothel ia l cells in vivo (Kooyman et al., 1 9 9 5 ) . A l t h o u g h the bio logical re levance of th is observa t ion is uncer ta in it does suggest t ha t c i rcu la t ing red cells may represent a reservoir of comp lemen t regulat ing prote ins w h i c h cou ld rapidly replace endothel ia l prote ins depleted by comp lemen t ac t ivat ion events (Kooyman et al., 1 9 9 5 ) . The h igh lateral membrane mobi l i ty of GPI-l inked prote ins permi t these 2 5 1 ant igens to be readily removed and re- inserted into cell membranes funct ional ly in tac t (Kooyman et al., 1 9 9 5 ) . Whe ther e ry th rocy te HSA can t ransfer to hetero logous cell membranes such as those of the endothe l ium remains to be tes ted . A l t h o u g h HSA expression w a s increased in the spleens of Photofrin®-t reated mice , th is response w a s not associated w i t h any s ign i f icant modi f ica t ion of immune responsiveness (Chapter Six) . HSA-t ransgenic mice (the HSA t ransgene conta ined the T cell receptor /? gene promoter and the Ig heavy chain /v-enhancer) w h i c h over -express HSA at the t h y m i c T cell double posi t ive ( C D 4 + CD8 + ) and single posi t ive ( C D 4 + or CD8 + ) s tages but not at the double negat ive (CD4" CD8") stage exh ib i ted a severe reduct ion in levels of peripheral o/?T cell receptor- l ineage cells (Hough et al., 1 9 9 4 ) . These studies indicated tha t HSA expression in the t h y m u s plays some role in the thymic select ion process (Hough et al., 1 9 9 4 ) . Recent ev idence suggests t ha t HSA may be t rans ient ly expressed by act ivated mur ine T cells and tha t the emergence of HSA on the cell sur face coincides w i t h the appearance of the IL-2R (Hube and A l t evog t , 1 9 9 4 ) . A l t h o u g h HSA w a s present on a small m inor i t y of splenic T cells in cont ro l and porphyr in - t reated mice, there w a s no ind icat ion tha t Photofrin® induced HSA expression by splenic T cells or indeed had p roduced T cell ac t i va t ion . The latter con ten t ion is suppor ted by the l ow level o f express ion of the l y m p h o c y t e act ivat ion ant igen C D 2 5 and unaltered respons iveness to r lL-2 by spleen cells f r o m Photofr in®-treated mice (Chapter 3 ) . A l t h o u g h the level of HSA expression on B l ymphocy tes may be increased by speci f ic fac to rs such as LPS (Kennedy et al., 1 9 9 4 ) , Photofr in® did not alter HSA 2 5 2 labell ing pat tern for splenic B cel ls. In the present s t u d y , splenic DC were also s h o w n to express HSA, as descr ibed by others (Inaba et al., 1 9 9 4 ) , but whe the r Photofr in® m igh t modu la te DC HSA levels remains un tes ted . Nielsen et al. (1993) s h o w e d tha t in HSA-t ransgenic mice produced w i t h an HSA t ransgene conta in ing an H-2K gene promoter and the Ig heavy chain / / -enhancer , T cell deve lopment proceeded normal ly and tha t peripheral B and T cell numbers or immunolog ica l responses were unchanged . H o w e v e r , fo l l ow ing immuniza t ion w i t h O V A , HSA-t ransgenic mice produced a normal pr imary ant ibody response bu t p roduced a great ly exaggerated secondary IgG! an t ibody response (Nielsen et al., 1993) ind icat ing tha t expression of HSA by mature T cells may enhance i m m u n e responsiveness in exper imenta l animals. The fo rma t ion of secondary an t ibody responses after Photofrin® t rea tmen t w a s not assessed. H o w e v e r , it seems unl ikely tha t Photofrin® w o u l d s ign i f icant ly alter secondary an t ibody responses since the observed increases in splenic HSA expression p roduced by Photofr in® appear restr ic ted to the ery thro id and perhaps myelo id l ineages. The above studies demonst ra te tha t LR-1 recognizes mur ine HSA on a diverse assor tment of cell t ypes w i t h f l o w cy tomet r i c techn iques . In add i t ion , LR-1 also ident i f ies HSA in cell ex t rac ts by means of immunob lo t analysis. It should be a useful reagent w i t h w h i c h to invest igate fur ther the role of HSA in hematopoies is and as a molecular c o m p o n e n t of the immune response. 2 5 3 CHAPTER EIGHT IN VITRO STUDIES 8.1 In t roduc t ion This thesis has demons t ra ted tha t Photofrin® produces s igni f icant a l terat ions in the hematopo ie t ic s ta tus w i th in the mur ine spleen and B M . However , a cellular mechan ism w h i c h migh t accoun t for these changes awa i ts ident i f icat ion. In order to invest igate potent ia l p a t h w a y s by w h i c h Photofrin® migh t inf luence hematopo ie t ic ac t i v i t y , a series of in vitro s tudies were carr ied ou t In the f i rs t , the photosensi t izer w a s evaluated for its capac i ty to in f luence the deve lopmen t of splenic CFU-GM, in the presence or absence of a mix ture of hematopo ie t ic g r o w t h fac to rs . Secondly , Photofrin® w a s tes ted for its abi l i ty to in f luence the prol i ferat ive response of normal mouse spleen cells to indiv idual or combina t ions of recombinant hematopo ie t ic g r o w t h fac to rs . Finally, the c o m p o u n d w a s tes ted for its capac i ty to inf luence the g r o w t h , d i f ferent ia t ion and cell sur face ant igen expression pat tern of the DBA/2 mur ine ery thro leukemia cell (MEL) line D1B (Chesebro etal., 1 9 7 6 ) . The s tudy of MEL and human ery thro leukemia (HEL) cell lines has suppl ied a large a m o u n t of in fo rmat ion concern ing the cellular regulat ion of iron and heme metabo l i sm and the cont ro l of ery thro id d i f fe rent ia t ion (Ben-David and Bernste in, 1 9 9 1 ) . Induced MEL and HEL cells provide useful models for genet ic and b iochemica l s tud ies , w h i c h are not possible w i t h techn iques cur rent ly available for the analysis of e ry thro id deve lopment in normal cells (Sassa, 1 9 8 9 ) . MEL can be 2 5 4 produced by the po lycy themic var iant of the Friend spleen focus - fo rm ing v i rus and is character ized by the polyc lonal prol i ferat ion of normal ery thro id progeni tor cells (BFU-E, CFU-E) and marked sp lenomegaly in in fected mice (Ben-Davis and Bernste in, 1 9 9 1 ) . These progeni tor cells retain the capac i ty for terminal d i f fe rent ia t ion to mature e ry th rocy tes . H o w e v e r , w i th in 4 -6 w e e k s mal ignant c lones emerge w h i c h are b locked at a stage prior to c o m m i t m e n t to terminal d i f fe ren t ia t ion . Erythroid d i f ferent ia t ion of MEL and HEL cells can be produced by hemin wh i le MEL cells can be induced t o w a r d s ery thro id d i f fe rent ia t ion in vitro upon t rea tmen t w i t h a var ie ty of chemical agents inc luding D M S O , hexamethy lene-6/s-acetamide, pur ines, ac t inomyc in D, and shor t chain fa t t y acids (Chang and Sassa, 1 9 8 2 ; Sassa, 1 9 8 8 ) . This event is character ized by a sequent ia l increase in the ac t iv i ty of speci f ic enzymes of the heme b iosynthet ic p a t h w a y , the accumula t ion of hemog lob in prote in and mRNA, expression of red cell membrane prote ins, and a l imited capac i ty for cell d iv is ion (Chang and Sassa, 1 9 8 2 ) . 8.2 Results 8.2.1 In f luence of Photofr in® on splenic g ranu locy te -macrophage co lony f o r m a t i o n In co lony assays per fo rmed w i t h normal spleen cells in the presence of an opt imal concen t ra t ion of P W M - S C C M as a source of g r o w t h fac tors and d i f fe rent a m o u n t s of Photofr in®, high levels (25 or 5 0 / /g /ml) of the porphyr in preparat ion w e re inh ib i tory for co lony fo rmat ion in all exper iments (Figure 8 .1 ) . Inhibi t ion of CFU-GM g r o w t h w a s also noted at the 5 or 10 / /g /ml concent ra t ions 2 5 5 in 2 of these exper iments . H o w e v e r , a 3 0 - 7 5 % increase in CFU-GM numbers w a s observed at some (10 - 2 5 0 ng/ml) concent ra t ions of the c o m p o u n d , a l though a degree of in ter -exper iment var iabi l i ty in these response w a s associated w i t h these tes ts . W h e n spleen cells w e re cu l tured w i t h these concent ra t ions of Photofrin®, in the absence of P W M - S C C M , no co lony fo rmat ion w a s observed . 8.2.2 Splenic responses to Photofr in® in comb ina t ion w i t h d i f ferent recombinan t cy tok ines Cul ture of normal spleen cells w i t h d i f fe rent indiv idual or combinat ions of recombinan t cy tok ines in serum-r ich med ium produced an increase in MTT ac t iv i ty above background values (cells plus med ium alone) w i t h some of these fac to rs (Figure 8 .2 ) . W h e n cy tok ines were tes ted indiv idual ly , on ly r lL-3 and rSCF s ign i f icant ly increased M T T ac t iv i t y . Neither rlL-1jff, r lL -2 , r lL -6 , r lL -7 , rG-CSF, r G M -CSF, or rEpo p roduced a measurable increase in spleen cell ac t iv i ty at the cy tok ine concen t ra t ions e m p l o y e d . In cu l tures conta in ing r lL -7 , rG-CSF, or rEpo, M T T ac t iv i ty w a s s o m e w h a t lower than tha t w i th in cu l tures conta in ing cells alone. The addi t ion of rG-CSF, rM-CSF, rGM-CSF, or rSCF to cu l tures conta in ing r lL-3 fur ther increased spleen cell responses. H o w e v e r , the addi t ion of r lL-6 to cul tures conta in ing r lL-3 and rG-CSF had no addi t ional e f fec t on the spleen cell response. Add i t i on of rG-CSF or rGM-CSF to cu l tures conta in ing rSCF also enhanced the spleen cell response beyond tha t obta ined w i t h rSCF alone. Interest ingly, the response of spleen cells to rSCF in the presence of rEpo w a s less than tha t p roduced w i t h rSCF alone. A modes t spleen cell response occur red in the presence of rll_-10 w i t h IL-6, and rGM-CSF. 2 5 6 • o CD C O C D O O O C O C D O O 0.001 0.01 0.1 1 10 100 P h o t o f r i n ( u g / m l ) Figure 8.1 In f luence of Photofr in® on spleen cell g ranu locy te -macrophage co lony (CFU-GM) f o r m a t i o n . Normal DBA/2 spleen cells were cu l tured for 7 days in semi-sol id agarose gels in the presence of an opt imal concen t ra t ion of PWM-SCCM and d i f fe rent a m o u n t s o f Photofrin®. The results of 3 consecu t ive exper iments are s h o w n . Each assay plate conta ined 5 x 10 5 spleen cells excep t in Exper iment 2 in w h i c h 6.7 x 10 5 cel ls/plate were emp loyed . Each concen t ra t ion w a s tes ted in t r ip l icate excep t for Exper iment 2 in w h i c h dupl icate plates we re used. Responses are s h o w n as the mean ± SD percentage (%) of the result (100%) obta ined w i t h spleen cells cu l tu red in the absence of Photofrin® and cor responded to 39.0 ± 6 . 6 colonies per plate (100 ± 16.8%, Exper iment 1), 69.0 ± 2.8 colonies per plate (100 ± 4 . 1 % , Exper iment 2), and 27.0 ± 3.5 colonies per plate (100 ± 12.8%, Exper iment 3). 257 W h e n spleen cells were cu l tured w i t h d i f fe rent cy tok ines in the presence of a concen t ra t ion gradient of Photofrin® some modi f i ca t ion of the overal l M T T result w a s noted (Figure 8 .3 ) . Photofrin® produced spleen cell responses tha t were greater t han those produced w i t h the indiv idual cy tok ines w h e n Photofrin® w a s added to cu l tures conta in ing r lL-3, rGSF, rGM-CSF or rEpo (Figure 8 . 3 ; panels 4 , 7, 9 , 10 respect ive ly ) . H o w e v e r , no s igni f icant change in spleen cell M T T ac t iv i ty w a s p roduced by Photofrin® in the presence of med ium alone, rlL-1/?, r lL-2, r lL -6 , r lL -7 , rSCF (Figure 8 . 3 ; panels 1 , 2 , 3, 5, 6, and 11 respect ively) or combina t ions of t w o (Figure 8 . 3 ; panels 12-21) or three (Figure 8 . 3 ; panels 22-23) recombinan t g r o w t h fac to rs . Photofrin® at a concen t ra t ion of 2 5 / /g /ml s t rongly inhibi ted cell ac t iv i ty w i t h all fac tor combina t ions . 8 . 2 . 3 Response of MEL cells cu l tured w i t h hemin , D M S O , or Photofrin® W h e n MEL cells were cu l tured for 7 2 hours in the presence of a w ide concen t ra t ion range of Photofr in®, there w a s no s ign i f icant al terat ion in their g r o w t h response, excep t at the h ighest concent ra t ions of the c o m p o u n d at w h i c h cell g r o w t h w a s s t rong ly inhibi ted (Figure 8 .4 ) . Uti l izing the Griess react ion, NO w a s undetec tab le in the supernatants of MEL cells cu l tured for ei ther 2 4 or 4 8 hours in m e d i u m alone or in the presence of a w ide range (20 ng - 2 5 / /g/ml) of Photofrin® concen t ra t ions . 2 5 8 CD " O CO CO p CJ> co Medium IL-1 IL-2 IL-3 IL-3/IL-6 IL-3/G-CSF IL-3/M-CSF IL-3/GM-CSF IL-3/Epo IL-6 IL-7 G-CSF M-CSF GM-CSF Epo SCF SCF/IL-1 SCF/IL-3 SCF/G-CSF H SCF/GM-CSF SCF/Epo IL-3/IL-6/G-CSF IL-1/IL-6/GM-CSF 0.0 0.1 0.2 0.3 0.4 0.5 0.6 MTT activity (absorbance 590 nm) 0.7 Figure 8 . 2 M T T dye reduct ion ac t iv i ty in spleen cell cu l tures con ta in ing d i f ferent recombinan t g r o w t h fac to rs . Normal DBA/2 spleen cells were cu l tured for 9 6 hours in the presence of m e d i u m alone or d i f fe rent indiv idual or combina t ions of recombinan t cy tok ines . Each concen t ra t ion w a s tes ted in quadrupl icate wel ls and cellular responses were evaluated by the M T T assay. Data represents baseline spleen cell responses to these fac to rs in the absence of Photofrin®. Factor concent ra t ions cor responded to 5 0 ng/ml (rll_-1£, r lL -3 , r lL-6, rll_-7, rSCF, rEpo and GM-CSF), 2 0 ng/ml (rG-CSF and rM-CSF) , or 1 0 0 U/ml of r lL-2. Al l cy tok ine combina t ions were tes ted in parallel w i t h the same spleen cell preparat ion and these responses represent levels of baseline M T T dye reduct ion for the evaluat ion of Photofrin® e f fec ts . 2 5 9 U O CL> C_> co o C O co C O 0.4 0.3 0.2 0.1 0.0 0.4 0.3 0.2 0.1 0.0 0.4 0.3 0.2 0.1 0.0 0.4 0.3 0.2 0.1 0.0 0.8 0.6 0.4 0.2 0.0 1. Medium J I I L_L 6.IL-7 11. SCF 16. IL-3 + Epo i i i i i 19. SCF + IL-3 J I I L 2. IL-1B i I I i i 7. G-CSF 12. SCF + Epo -Ms 17. IL-3 + IL-6 J L_J I L 3. IL-2 8. M-CSF j i i i i 13. SCF + IL-1B 18. IL-3 + G-CSF Ar, J I I I L 20. IL-3 + GM-CSF J i i i i t 21. IL-3 + M-CSF J I I I L 4. IL-3 J I I I L 5. IL-6 i i i i«i»» 9. GM-CSF J I I I u 10. Epo j i i i_ 14. SCF + G-CSF 1S.SCFtGIKSI _l I I I L_ 22. IL-1B + IL-6 + GM-CSF -J I I I T 23. IL-3 + IL-6 + G-CSF J I I I L 0 .01 .1 1 10 100 0 .01 .1 1 10 100 0.01 .1 1 10100 0 .01 .1 1 10100 0 .01 .1 1 10100 Figure 8 . 3 M T T dye reduct ion ac t iv i ty in spleen cell cu l tures conta in ing d i f ferent recombinan t g r o w t h fac tors and Photofrin®. Normal DBA/2 spleen cells were cu l tured for 9 6 hours in the presence of m e d i u m alone or d i f fe rent indiv idual or combina t ions of recombinant g r o w t h fac to rs w i t h or w i t h o u t Photofrin®. Cytok ine concent ra t ions cor responded to 5 0 ng/ml (rll_-10, r lL -3 , r lL-6, rll_-7, rSCF, rEpo and GM-CSF), 2 0 ng /ml (rG-CSF and rM-CSF) , or 1 0 0 U/ml of r lL -2 . Photofrin® concent ra t ions cor responded to 0, 0 . 0 2 , 0 . 0 5 , 0 . 1 , 0 . 2 5 , 0 . 5 , 1 , 2 . 5 , 5, 10 , or 25 / / g / m l . Each combina t ion w a s tes ted in quadrup l ica te wel ls and cellular responses were evaluated by the M T T assay. All f ac to rs w e re evaluated w i t h Photofrin® in a single exper iment excep t w i t h rEpo, w h i c h w a s assayed in three indiv idual exper iments g iv ing similar resul ts. 2 6 0 CO CD O CJ> CTJ 0.01 0.1 1.0 10 100 Photofrin (ug/ml) F i g u r e 8 . 4 P r o l i f e r a t i v e r e s p o n s e o f M E L c e l l s t o Photofr in®. MEL cells w e re cu l tured w i t h d i f ferent concen t ra t ions of Photofrin® for 4 8 hours . Cellular ac t iv i ty w a s assessed by the M T T co lor imetr ic assay. Each concen t ra t ion w a s tes ted in repl icates of six. The mean result for the cont ro l cu l tures (cells alone) cor responded to an absorbance of 0 . 9 4 8 ± 0 . 0 9 3 . The mean M T T result (absorbance = 0 . 0 8 5 ± 0 .004 ) for wel ls conta in ing cu l ture med ium alone w a s sub t rac ted f r o m each tes t resul t . Cell responses are s h o w n as the percentage (%) of the result p roduced by cells cu l tured in m e d i u m alone. 2 6 1 W i t h i n a l iquid cu l ture s y s t e m , MEL cells mainta ined high ( > 9 0 % ) v iabi l i ty over a 3 day per iod w h e t h e r these cells were cu l tu red in med ium alone, or w i t h 0.1 m M hemin , or Photofrin® at either the 2 or 10 / /g /ml concent ra t ion (Figure 8 . 5 A ) . Cell v iabi l i ty w a s approx imate ly 7 0 % after 3 days in the presence of 1.5% D M S O , wh i le f e w cells surv ived after 3 days w i t h Photofrin® at the 25 / /g /ml concen t ra t i on . MEL cells g r o w n in med ium alone expanded their numbers over 25 -fo ld w i t h i n the 3 day logar i thmic g r o w t h period (Figure 8 . 5 A ) . For MEL cells cu l tured w i t h 1 .5% D M S O , 0.1 m M hemin , or Photofrin® (2 or 10 / /g/ml) the change in cell numbers cor responded to 4 - fo ld , 15 - fo ld , 12 - fo ld , and 9-fold increases, respect ive ly , over the same per iod. Add i t ion of Photofrin® (2 , 10 , or 25 / /g/ml) t o MEL cell cu l tures did not produce any ev idence of e ry th ro id d i f ferent ia t ion as cells cu l tu red w i t h Photofrin® were un i fo rmly negat ive for hemoglob in w h e n t reated w i t h acidic benzidine up to 7 2 hours after the ini t iat ion of cu l ture (Figure 8 .5B) . H o w e v e r , addi t ion of 1.5% DMSO or 0.1 m M hemin produced direct ev idence (acidic benzidine staining) t ha t ery thro id d i f fe rent ia t ion had occur red in approx imate ly 2 0 % of these cells by the 7 2 hour sampl ing t ime (Figure 8.5C) Hemin w a s a more e f fec t ive agent for el ic i t ing ery thro id d i f ferent ia t ion as it p roduced approx imate ly 3-fold more benzidine" 1" cells t han D M S O . 2 6 2 Time of culture (hours) Figure 8.5 In f luence of D M S O , h e m i n , and Photofr in® on the g r o w t h and ery th ro id d i f fe rent ia t ion o f MEL cel ls. MEL cells were cu l tured at 1 x 1 0 5 / m l in RPMI 1 6 4 0 med ium w i t h 1 0 % FCS alone ( • ), or in the presence of 1.5% DMSO ( • ), 0.1 m M hemin ( • ), or Photofr in® at 2 ( O ), 10 ( A ), or 25 ( O ) / /g /ml for 7 2 hours . (A) Percentage viable cel ls, (B) to ta l v iable cell numbers and (C) the propor t ion of cells s ta in ing w i t h acidic benzidine were determined every 2 4 hours . 2 6 3 Flow cy tomet r i c analysis indicated tha t normal ly passaged MEL cells expressed high levels of HSA and C D 7 1 , moderate amoun ts of H-2D, but did not express the leukocy te c o m m o n ant igen CD45 (Figure 8 .6 ) . W h e n these cells were cu l tured for 3 days in the presence of 1.5% D M S O , 0.1 m M hemin or Photofrin® (2 , or 10 / / g /m l ) , pheno typ ic changes were observed (Figure 8 .7 ) . MEL cells cu l tured w i t h DMSO or hemin exhib i ted a marked reduct ion in sur face expression (mean channel f luorescence in tens i ty = MCFI) of C D 7 1 . H o w e v e r , cells cu l tured w i t h Photof r in at 10 / /g /ml but not at 2 / /g /ml had higher levels of CD71 cells than cells cu l tu red in med ium alone. HSA levels were s t rong ly expressed by MEL cells under all cu l ture cond i t ions whi le CD45 w a s un i fo rmly absent f r o m these cells regardless o f the cu l ture cond i t ions . W h e n CD71 express ion w a s mon i to red over the 3 day cu l ture per iod, MCFI values for th is receptor we re reduced on cells cu l tured w i t h DMSO or hemin w i th in 2 4 hours , whereas CD71 expression was s t rong ly e levated by th is t ime on cells cu l tured w i t h Photof r in at the 10 / /g /ml concen t ra t ion (Figure 8 .8 ) . Litt le or no change in background MCFI levels were observed w i t h the d i f fe rent t rea tmen ts . MEL cells cu l tured in DMSO or Photofrin® (10 / /g /ml) w e re s l ight ly smaller than cells cu l tured in med ium alone by d i rect mic roscop ic examina t ion and on the basis of their f o r w a r d scat ter prof i le in the f l o w cy tomete r . W h e n the MCFI values obta ined for MEL cells cu l tured w i t h the d i f ferent fac to rs w e re compared to the MCFI values obta ined for cells cu l tured in med ium alone, it w a s ev ident t ha t Photofrin® at 10 / /g /ml caused a 6 0 - 8 5 % increase in CD71 MCFI (Figure 8 .9 ) . DMSO and hemin reduced the relat ive express ion of CD71 2 6 4 as m u c h as 8 0 % for DMSO on day 1 and by 7 0 % for hemin on day 3. Photofrin® at 2 / /g /ml had no discernable e f fec t on CD71 express ion. W h e n the relat ive express ion of HSA w a s evaluated in th is manner , only small ( < 2 0 % ) changes in relat ive levels of th is marker were ev ident for cells cu l tured under the d i f ferent cond i t ions . Log Fluorescence Intensity Figure 8 .6 Cell sur face ant igen expression by the MEL cell line D1B. MEL cel ls, mainta ined in RPMI 1 6 4 0 med ium and 1 0 % FCS, were subjected to sur face labell ing w i t h rat monoc lona l ant ibodies against C D 4 5 , HSA, C D 7 1 , or H-2D ant igens (bold lines) and f luorescence in tens i ty analyzed w i t h the Coulter XL f l o w cy tomete r . Results obta ined w i t h iso typ ica l ly -matched cont ro l ant ibodies are s h o w n as th in t rac ings . 2 6 5 CD45 HSA C D 7 1 CD E 3 CD o CD > _ra cu CC Medium DMSO Hemin Photofrin® (2 //g/ml) Photofrin® (10//g/ml) Log Fluorescence Intensity Figure 8 .7 Expression o f C D 4 5 , HSA, and C D 7 1 by MEL cells cu l tured w i t h D M S O , hemin or Photofrin®. MEL cells w e re cu l tured for 3 days w i t h main tenance med ium alone, or w i t h 1 .5% D M S O , 0.1 m M hemin or one of t w o d i f fe rent concent ra t ions of Photofr in®. Cell sur face C D 4 5 , HSA, and CD71 were labelled w i t h the rat monoc lona l ant ibodies (bold lines) 3 0 F 1 1 . 1 , L R - 1 , or C2F2 , respect ive ly . The result obta ined w i t h the iso typ ica l ly -matched cont ro l ant ibodies are s h o w n as l ight t rac ings . Mean channel f luorescence in tens i ty (MCFI) va lues obta ined w i t h the ant i -HSA and ant i -CD71 reagents are g iven w i t h i n the indiv idual f igure boxes . 2 6 6 Day 1 Day 2 Day 3 F l u o r e s c e n c e I n t e n s i t y (PE) Figure 8 .8 F low cy tomet r i c prof i les of C D 7 1 express ion by MEL cells cu l tured w i t h D M S O , hemin or Photofrin®. MEL cells w e re cu l tured at 1 x 1 0 5 / m l in RPMI 1 6 4 0 med ium w i t h 1 0 % FCS alone, or w i t h the inclusion of DMSO ( 1 . 5 % ) , hemin (0.1 mM) or t w o d i f fe rent concen t ra t ions of Photofr in®. Individual petr i dishes were harvested daily and sur face express ion of CD71 w a s determined by f l o w cy tomet r i c techn iques using the PE-conjugated rat monoc lona l an t ibody C2F2 (bold t rac ings) . The result ob ta ined w i t h a PE-conjugated rat l g G 2 a cont ro l an t ibody is s h o w n by th in l ines. The MCFI va lues obta ined w i t h the isotype cont ro l an t ibody and for an t i -CD71 are given adjacent to the respect ive f l o w cy tomet r i c peaks. 2 6 7 + 100 +50 o C D C D CO O o h -50 h -100 +100 +50 h 0 -50 h •100 Time of culture (days) Figure 8 . 9 Relative express ion levels of HSA and C D 7 1 by MEL cells cu l tured w i t h D M S O , hemin or Photofrin®. HSA and CD71 mean channel f luorescence in tens i ty (MCFI) levels (in arb i t rary units) de termined for MEL cells cu l tured w i t h DMSO ( • ), hemin ( • ), or Photofr in® at 2 ( • ) or 10 ( O ) / /g /ml were compared to the levels determined for cells main ta ined in cu l ture med ium alone. Changes in HSA and CD71 levels we re expressed as the % change in MCFI in tensi ty (Liu et al., 1993) as fo l l ows : % change in MCFI = MCFI of tes t cells - MCFI of con t ro l cells MCFI of cont ro l cells x 1 0 0 . F low c y t o m e t r i c prof i les obta ined w i t h these cells are s h o w n in Figure 8 .8 . 2 6 8 To fur ther establ ish the abi l i ty of Photofrin® to in f luence cell sur face CD71 levels, MEL cells w e re cu l tured in the presence of t w o (5 or 10 / /g/ml) d i f fe rent concen t ra t ions of Photofrin®. Both concent ra t ions of Photofrin® s lowed MEL cell g r o w t h in vitro, w i t h the 10 / /g /ml concen t ra t ion produc ing the larger inh ib i tory e f fec t (Figure 8 . 1 0 ) . F low cy tomet r i c studies indicated tha t surface express ion of H-2D d M H C Class I molecules w a s essential ly unchanged for cells cu l tured w i t h or w i t h o u t Photofrin® for ei ther 2 4 (Figure 8 .11 A) or 4 8 hours (Figure 8 .11B) . H o w e v e r , sur face levels of CD71 were increased above con t ro l levels for MEL cells t rea ted w i t h ei ther a m o u n t of Photofrin® at the 2 4 hour (Figure 8 .11 A) and the 4 8 hour (Figure 8 .11B) sampl ing t imes . The higher concent ra t ion of Photofr in® p roduced the greatest increase in the level of C D 7 1 . These exper iments we re per fo rmed a number of t imes g iv ing highly similar resul ts (Table 8 .1) . A n increase in background f luorescence in tens i ty w a s ev ident for cells cu l tured w i t h Photofr in® and th is w a s s o m e w h a t greater w i t h the higher concen t ra t ion of the mater ia l . 8 . 2 . 4 In f luence of Photofr in® on t ransfer r in b ind ing To determine whe the r the increase in cell sur face express ion of CD71 p roduced by Photofr in® corre lated w i t h a change in the b inding of the natural l igand for C D 7 1 , cu l tured MEL cells were incubated w i t h a f luoresceinated human holo-t ransfer r in (Trf-F) preparat ion (Figure 8 . 1 2 ) . These studies indicated tha t cells cu l tured for 4 8 hours w i t h Photofrin® at concent ra t ions of 5 or 10 / /g /ml bound greater a m o u n t s of Trf-F than cells cu l tured in med ium alone. 2 6 9 20 0 5 10 Figure 8 . 1 0 Inf luence of Photofrin® on MEL cell g r o w t h . MEL cells ( 1 0 6 per 10 ml) were cu l tured for 4 8 hours in RPMI 1 6 4 0 m e d i u m conta in ing 1 0 % FCS in the presence of t w o d i f fe rent concent ra t ions of Photofr in®. Cells were harvested and to ta l viable cell numbers were determined by T rypan blue dye exc lus ion . Six exper iments were per fo rmed w i t h Photofrin® at 0 and 10 /vg/ml and four t imes at the 5 ji/g/ml concen t ra t ion . 2 7 0 H-2D C C D 7 1 1.3/70.6 Medium Photofrin (5 ug/ml) Photofrin (10 ug/ml) Medium Photofrin (5 ug/ml) Photofrin (10 ug/ml) Fluorescence Intensity Figure 8 . 1 1 Inf luence of t w o d i f ferent concent ra t ions of Photofr in® on MEL cell express ion of H-2D d and C D 7 1 ant igens. MEL cells w e re cu l tured for ei ther (A) 2 4 or (B) 4 8 hours in med ium alone or in m e d i u m conta in ing Photofrin® at 5 or 10 / / g / m l . Expression levels of H-2 D d and CD71 markers w a s determined by f l o w cy tomet r i c analysis. MCFI values obta ined for the isotyp ic cont ro ls and each cell sur face ant igen are g iven w i th in each f igure . 2 7 1 OVERLAY:SINGLE PARAMETER J3 E 3 0) O CD > JS v CC OVERLAY:SINGLE PARAMETER 1.2 /2 .9 0 . 9 / 1 . 4 jijy \ I M e d i u m 1 r * ! i I r m 1 1 10 t i n i i • i • i r i r r tea 1800 OVERLAY:SINGLE PARAMETER 1.0 /2 .5 j . ' I " <l I TTTII — P h o t o f r i n ® ( 5 u g / m l ) i i 1 1 1 1 1 I I i i 1 1 1 1 1 1 P h o t o f r i n ® ( 1 0 u g / m l ) Fluorescence Intensity (FITC) Figure 8 . 1 2 In f luence of Photofr in® on MEL cell b ind ing o f t rans fer r in . MEL cells were cu l tured w i t h o u t or in the presence of t w o d i f ferent concen t ra t ions o f Photofrin® for 4 8 hours . Cells were harves ted , w a s h e d t w i c e w i t h serum-f ree RPMI 1 6 4 0 med ium and placed in serum-f ree med ium at 3 7 ° for an addi t ional hour . To evaluate non-speci f ic b inding of the Trf-F probe, 1 0 6 cells were incubated for 10 minutes on ice w i t h or w i t h o u t a 200 - fo ld ( 2 0 0 jjg) excess of unlabel led human holo-Tr f , prior to the addi t ion of 1 / /g of Tr f -F . A f te r one hour on ice, cells w e re w a s h e d t w i c e , and cell f luorescence analyzed by f l o w c y t o m e t r y . Results obta ined w i t h Trf-F w i t h (thin t racings) or w i t h o u t (bold t rac ings) the 2 0 0 -fo ld excess of Tr f are s h o w n . MCFI values determined for cells incubated under these cond i t ions are g iven w i th in each f igure. 2 7 2 Table 8 .1 In f luence of Photofr in® on MEL cell express ion o f C D 7 1 . MEL cel ls, cu l tured for 4 8 hours w i t h or w i t h o u t Photofr in®, were assessed for their expression of the receptor for t ransfer r in (CD71) by f l o w cy tomet r i c analys is. Mean channel f luorescence in tens i ty va lues obta ined w i t h the ant i -CD71 an t ibody and the l g G 2 a isotype cont ro l an t ibody are g iven . Results of 5 consecu t i ve exper iments are s h o w n . Mean Channel F luorescence Intensi ty (MCFI) - PE Exper iment Photofr in® Increase in (//g/ml) " g G 2 a C D 7 1 MCFI (%) 1 0 1.4 1 7 4 . 5 _ 2 1.2 1 8 1 . 0 3.7 10 2 .0 2 7 1 . 7 5 5 . 7 II 0 1.4 1 3 6 . 8 _ 10 2 .0 169 .1 2 3 . 6 III 0 1.0 6 7 . 5 _ 5 1.1 9 3 . 9 39 .1 10 1.5 1 2 9 . 2 9 1 . 4 IV 0 1.5 6 8 . 7 _ 5 1.9 1 1 1 . 3 4 2 . 6 10 2 .0 1 3 0 . 4 8 9 . 8 V 0 0 .7 4 2 . 0 _ 5 0 .8 9 2 . 4 1 2 0 . 0 10 1.6 1 2 6 . 8 2 0 1 . 9 8 . 2 . 5 Inf luence of BPD on MEL cell express ion o f C D 7 1 and Tr f b ind ing In t w o separate exper iments , MEL cells cu l tured for 4 8 hours in the presence of d i f fe rent amoun ts of benzoporphyr in der ivat ive (BPD), d isplayed no d i f fe rence in their express ion of CD71 or the b inding of Tr f -F compared to cells mainta ined in m e d i u m alone (Figure 8 . 1 3 ) . Higher concen t ra t ions (5 or 10 / /g/ml) of BPD proved tox ic to these cells w h e n tes ted in th is sys tem and no cells surv ived the cu l ture per iod. 2 7 3 GD71 T r f - F cu X I E 3 Z "55 O co > J O CU cc B CD X I E 3 z "53 O CD > J O CD CC Medium BPD (0.1 ug/ml) BPD (1 ug/ml) Photofrin® (5 ug/ml) Medium BPD (1 ug/ml) BPD (2.5 ug/ml) F l u o r e s c e n c e Intensity Figure 8 . 1 3 In f luence of BPD on MEL cell C D 7 1 express ion and Trf-F b ind ing. In t w o d i f fe rent exper iments , MEL cells were cu l tured for 4 8 hours in the presence of d i f fe rent concent ra t ions of a l iposomal fo rmu la t ion of BPD. Levels of cell sur face of CD71 and binding of Trf-F were determined by f l o w cy tomet r i c analys is. In the f i rs t exper iment (A) , the response of these cells to Photofr in® w a s mon i to red in paral lel. The second exper iment (B), w h i c h tes ted the impac t o f higher a m o u n t s of BPD on t ransfer r in receptor express ion, w a s per formed in a manner ident ical to the f i rs t exper iment , excep t t ha t cells we re placed in serum-f ree m e d i u m for 2 hours before cell sur face Trf-F b inding w a s assessed. 2 7 4 8 . 2 . 6 In f luence of Photofr in® on P 8 1 5 cells Since MEL cells increased their expression of CD71 w h e n cul tured w i t h Photofr in®, it w a s impor tan t to determine w h e t h e r a non-ery thro id cell line m igh t respond in a similar fash ion . The DBA/2 m a s t o c y t o m a cell line P815 has a C D 4 5 + , HSA", C D 7 1 + , F 4 / 8 0 + /" pheno type (Figure 8 . 1 4 ) . The presence of the mur ine macrophage F 4 / 8 0 ant igen on P815 cells has been descr ibed previously (Aus tyn and G o r d o n , 1 9 8 1 ) . P815 cells cu l tured w i t h Photofrin® (10 / /g /ml) exhib i ted a s lower g r o w t h rate than cells mainta ined in med ium (Figure 8 . 1 5 ) . H o w e v e r , upon cul ture in the presence of Photofr in®, P815 cells increased their express ion of CD71 w i th in 2 4 hours . A t all tes t t imes up to 7 2 hours , MCFI values for CD71 were approx imate ly t w i c e as great for P81 5 cells cu l tured in the presence of Photofrin® than on the sur face of cells cu l tured in med ium alone (Figure 8 . 1 6 ) . In cont ras t , express ion levels of C D 4 5 remained cons tan t at all tes t t imes for cells in bo th t rea tmen t g roups (Figure 8 . 1 6 ) . 2 7 5 Log Fluorescence Intensity Figure 8 . 1 4 Di f ferent cell sur face ant igens of mur ine P 8 1 5 m a s t o c y t o m a cells. P81 5 cells were mainta ined under cul ture cond i t ions ident ical to those emp loyed w i t h MEL cel ls. Normal ly passaged cells we re labelled w i t h rat monoc lona l ant ibod ies , as g iven in parentheses, react ive against leukocy te c o m m o n ant igen C D 4 5 ( 3 0 F 1 1 . 1 ) , HSA (LR-1), CD71 (C2F2) , or F 4 / 8 0 ant igen (F4/80) and analyzed w i t h the Coul ter XL f l o w cy tomete r . Results obta ined w i t h the isotype-matched cont ro ls are s h o w n by th in line t rac ings . 2 7 6 1 I 1 I I I I 0 24 48 72 Time of culture (hours) Figure 8 . 1 5 In f luence of Photofr in® on the g r o w t h o f P 8 1 5 cel ls. Mur ine P815 m a s t o c y t o m a cells were cu l tured for 72 hours w i t h o u t ( • ) or w i t h Photofr in® (10 u g / m l , O). Viable cell numbers were determined by T rypan blue dye exc lus ion on each tes t day . 277 0 Hours 24 Hours 48 Hours 72 Hours Figure 8 . 1 6 Inf luence of Photofrin® on P 8 1 5 cell express ion of C D 4 5 and C D 7 1 markers . P81 5 cells were cu l tured for 7 2 hours w i t h o u t or w i t h Photofrin® (10 /yg /ml ) . Cell sur face levels of C D 4 5 and CD71 were assessed dai ly by f l o w c y t o m e t r y . MCFI va lues obta ined w i t h the isotype cont ro l reagents (thin t rac ings) and ant igen-speci f ic monoc lona l ant ibodies (bold t rac ings) are g iven w i t h i n each f igure. 2 7 8 8 . 3 Discussion In an e f fo r t to accoun t for hematopo ie t ic e f fec ts of Photofrin®, normal spleen cells w e re co-cu l tu red w i t h the c o m p o u n d in the rout ine co lony assay s y s t e m . A l t h o u g h Photofrin® exhib i ted no intr insic co lony-s t imu la t ing ac t iv i ty , it did enhance numbers of g ranu locy te -macrophage colonies f o rmed by spleen cells plated in the presence of a cond i t ioned-med ium as a source of g r o w t h fac to rs . For th is cu l ture s y s t e m it is d i f f icu l t to ascribe the cellular level at w h i c h Photofrin® migh t act . It cou ld have d i rect ly p romoted the ent ry of hematopo ie t ic progeni tor cells into the cell cyc le and/or s t imula ted the release of hematopo ie t ic cy tok ines by accessory cel ls. Fur thermore , w h e n normal spleen cells were co-cu l tu red w i t h d i f ferent cy tok ines in the presence of Photofrin® in co lor imetr ic pro l i ferat ion assays, an enhancemen t o f cell ac t iv i ty w a s noted at some concen t ra t ions of the photosensi t izer w h e n it w a s added w i t h G-CSF, M-CSF, GM-CSF, IL-3, and Epo. In the presence of IL-3 or SCF and a var ie ty of g r o w t h fac to rs , no augmenta t ion of the spleen cell response w a s produced by Photofrin®. This assay sys tem has a major l imi tat ion in t ha t it is impossible to ident i fy w h i c h cells are responsible for changes in overal l spleen cell ac t iv i ty . Receptors for many of these fac tors are present on a var ie ty of cell t y p e s , o ther than hematopo ie t ic progeni tor cells (Metca l f , 1 9 8 9 ; Miya j ima et al., 1 9 9 3 ) . A l t hough increased cell metabol ic ac t iv i ty typ ica l ly accompan ies cell prol i ferat ion the M T T assay canno t d i f ferent ia te be tween these t w o processes. To examine the possibi l i ty t ha t Photofrin® migh t d i rect ly inf luence HSC it w o u l d be necessary to isolate these cells and tes t their g r o w t h and phenotyp ic 2 7 9 responses to Photofr in® in combina t ion w i t h ear ly-act ing g r o w t h fac tors such as SCF and IL-3. Techn iques to isolate mur ine cell popula t ions enr iched for ery thro id progeni tors (BFU-E and CFU-E) by cent r i fugal e lutr iat ion have been descr ibed (Lesley et al., 1 9 8 4 ; Landschulz et al., 1 9 8 9 ) . These cells could be used to d i rect ly evaluate the in f luence of Photofrin® on the g r o w t h of ery thro id precursors in vitro. Since Photofrin® s t rong ly p romoted splenic ery thropo ies is , it w a s impor tan t to determine w h e t h e r Photofrin® migh t be a fac to r w h i c h could induce ery thro id d i f fe ren t ia t ion . H o w e v e r , in con t ras t to the e f fec ts of the we l l -d o c u m e n t e d ery thro id d i f fe rent ia t ion fac tors hemin and DMSO (Chang and Sassa, 1 9 8 2 ; Sassa, 1 9 8 9 ) , Photofrin® did not produce any ev idence of hemoglob in synthes is in MEL cel ls. MEL cell g r o w t h s lowed in the presence of Photofrin® whi le express ion of the receptor for t ransferr in and Trf b ind ing w a s increased by the t rea tmen t . In con t ras t , BPD had no comparab le in f luence on these cells. In the presence of hemin or D M S O , expression of the TrfR w a s lower than tha t upon un induced MEL cel ls. The abi l i ty of hemin to down- regu la te sur face TrfR expression has been wel l d o c u m e n t e d (Thiel, 1 9 9 0 ) . A l t h o u g h DMSO s lowed MEL cell p ro l i fe ra t ion, as descr ibed by others (Chan et al., 1 9 9 3 ) , it has been observed tha t the induc t ion of e ry thro id d i f ferent ia t ion in MEL cells by DMSO is accompanied by a large increase in TrfR mRNA levels (Chan et al., 1993) and an increase in the b inding of Tr f to these cells (Hu et al., 1 9 7 7 ; Wi lczynska et al., 1 9 8 4 ; Coccia et al., 1 9 9 5 ) . For the present s tudy , no explanat ion for the d im in ishment in CD71 express ion by d i f fe rent ia t ing MEL cells cu l tured w i t h DMSO is apparent , unless th is t r ea tmen t had altered the ant igenic character of the Tr fR. Cotner et al. (1989) 2 8 0 descr ibed the ex is tence of t w o ant igenical ly d is t inc t f o r m s of the human TrfR on normal human ery thro id progen i to rs , but it is u n k n o w n w h e t h e r al ternate fo rms of the mur ine TrfR exis t . In con t ras t to C D 7 1 , sur face levels of HSA remained at cont ro l levels w h e t h e r these cells were cu l tured in the presence of hemin , DMSO, or Photofr in®. Fur thermore, Photofrin® did not s igni f icant ly a f fec t MEL cell sur face express ion of M H C Class I H-2D d an t igen. W h e n Photofrin® w a s tes ted against the non-ery thro id P815 DBA/2 mouse m a s t o c y t o m a cell l ine, expression of CD71 w a s elevated above cont ro l levels at all t imes over a three day cul ture per iod, a l though the g r o w t h of these cells w a s also s lowed by the t rea tment . In con t ras t , express ion of CD45 for cells cu l tu red in the presence of Photofrin® remained at cont ro l levels t h r o u g h o u t the test per iod . These f ind ings suggest tha t Photofrin® may have in some w a y altered the iron s ta tus w i t h i n bo th MEL and P815 cel ls. For the measurement of cell sur face TrfR by FACS analysis, a PE-con jugated rat monoc lona l an t ibody C2F2 (Kemp e r a / . , 1987) w a s emp loyed . It w a s of some concern tha t Photofrin® w h i c h had accumula ted w i t h i n cu l tured cells migh t in f luence the f luorescence profi le of these cells in the f l o w cy tomete r . H o w e v e r , the intr insic f luorescence character is t ics of Photofr in® are unl ikely to inter fere w i t h th is analysis since the wave leng th ( 4 8 8 nm) of l ight used to exci te the cells as they pass t h r o u g h the f l o w cy tomete r has prev iously been s h o w n to produce relat ively l o w exc i ta t ion of Photofrin® (Prof io, 1 9 9 0 ) . In add i t ion , f luorescence emiss ions in the PE (FL2) channel are gathered at 5 7 5 n m , a w a v e l e n g t h of l ight at w h i c h Photofrin® emits l itt le signal s t rength (Prof io, 1 9 9 0 ) . 281 A small increase in isotype cont ro l (background) mean channel f luorescence values w a s no ted for cells cu l tured in the presence of Photofr in®, bu t these changes were minor in compar ison to the m u c h larger increase in labell ing w i t h the PE-conjugated ant i -Tr fR an t ibody . Fur thermore, the increased b inding of a TrF-F probe to MEL cells cu l tured w i t h Photofr in® suppor ts the con ten t ion tha t the photosensi t izer did elevate the express ion of func t iona l receptors for th is iron t ranspor t pro te in . In order to help unders tand h o w Photofrin® migh t mod i fy TrfR express ion , it is impor tan t to rev iew h o w cell sur face TrfR levels are regula ted. The level of i ron w i t h i n cells is precisely moni to red and the avai labi l i ty of iron in pro l i ferat ing non-ery th ro id cells modula tes the b iosynthet ic rate of the TrfR and ferr i t in prote ins (Casey et al., 1 9 8 8 ) . A l t h o u g h there is l i t t le ev idence for t ranscr ip t iona l con t ro l of TrfR expression in non-ery thro id cells in response to al tered g r o w t h rates or iron depr iva t ion , large increases in the t ranscr ip t ion of TrfR mRNA have been observed after the induct ion of e ry thro id d i f ferent ia t ion in MEL cells (Ponka and Schu lman , 1993) and in d i f ferent ia t ing ch ick embryo ery thoblasts (Chan and Gerhar t , 1 9 9 2 ) . In non-pro l i fe ra t ing, non-ery thro id cel ls, the demand for iron may be largely met by intracel lular recycl ing since heme synthes is occurs on a relat ively smal l scale. However , deve lop ing ery thro id cells uti l ize the ef f ic ient i ron-gather ing Tr f -Tr fR s y s t e m in order to glean the high a m o u n t s of iron required to meet the high demand for hemoglob in synthes is w i th in th is t issue. In cu l tured mammal ian cel ls, Tr fR and ferr i t in levels have been s h o w n to be cont ro l led p redominant ly at the post - t ranscr ip t ional level t h rough iron-responsive e lements (IRE). A single IRE is posi t ioned w i t h i n the 5 ' -unt rans la ted 2 8 2 region (UTR1) of ferr i t in mRNA and at mult ip le sites w i t h i n the 3 ' -UTR of TrfR mRNA. More recent ly , it w a s s h o w n tha t the 5 ' -UTR of ery thro id A L A S also conta ins an IRE (Dandekar et al., 1 9 9 1 ) . Specif ic b inding prote ins (IRE-BP) a t tach to these s tem- loop s t ruc tures thereby either modu la t ing the stabi l i ty or the t rans la t ion of these mRNA species (Thei l , 1 9 9 0 ) . A soluble 9 0 kDa prote in tha t exists in ei ther a l o w or high af f in i ty s tate and binds to the IRE of ferr i t in mRNA w a s isolated (Walden et al., 1 9 8 9 ) . The IRE-BP, also te rmed the ferr i t in repressor protein (FRP, Wa lden et al., 1 9 8 9 ) , i ron-regula tory fac to r (IRF, Mul lner et al., 1 9 9 2 ) , or iron regulatory prote in 1 (Guo etal., 1995) is present in all mur ine t issues (Mullner et al., 1 9 9 2 ) . IRF ac t iv i ty w a s s h o w n to be greatest in k idney, l iver and intest ine and lowes t in musc le , l ung , spleen, B M , and b lood (Mullner et al., 1 9 9 2 ) . W h e n intracel lular iron is in excess of immediate requ i rements , a greater p ropor t ion of the cel l 's IRE-BP is conver ted to its l ow af f in i ty s tate and cells uti l ize previously masked ferr i t in mRNA to synthesize addi t ional ferr i t in for iron s torage. Ferrit in prote in levels can be increased w i t h o u t al ter ing ferr i t in m R N A levels as s tored mRNA moves f r o m the r ibonucleoprote in f rac t ion to the po lysomes to p romote greater t rans la t ion of ferr i t in sub-uni t po lypept ide chains (Rogers and M u n r o , 1 9 8 7 ) . In the presence of the l ow af f in i ty con f igura t ion of the IRE-BP, the stabi l i ty of TrfR mRNA is reduced and its intracel lular degradat ion increased. TrfR synthes is is thereby decreased and consequent l y the uptake of iron is reduced. In con t ras t , at l o w iron levels the IRE-BP is conver ted to its high af f in i ty s tate and ferr i t in mRNA is masked and TrfR mRNA is stabi l ized thereby leading to an increase in TrfR synthes is , a decrease in 2 8 3 ferritin synthesis, an increase in iron uptake and a decline in iron storage (Thiel, 1990). The post-translational switch between low and high RNA-binding activity by the IRE-BP does not require de novo protein synthesis (Haile et al., 1992). Binding of IRE-BP to the IRE in the 5' untranslated region of eALAS mRNA stops translation of this RNA species, decreasing A L A S production and diminishing the utilization of iron within the heme biosynthetic pathway (Bhasker et al., 1993; Melefors et al., 1993). Characterization of the IRE-BP has been the focus of intense research. The IRE-BP was found to possess sequence homology (31%) with mitochondrial aconitase, the enzyme of the Krebs cycle which catalyzes the stereospecific conversion of citrate to isocitrate (Roualt et al., 1991). Mitochondrial aconitase contains an iron-sulphur cluster and requires this cubane 4Fe-4S cluster for full enzymatic activity (Klausner etal., 1993). The cytoplasmic variant of aconitase was characterized and was shown to possess structural characteristics and physicochemical properties identical to that of the IRE-BP (Kennedy et al., 1992). A cytoplasmic aconitase was enzymatically active (4Fe-4S state) when cells were replete with iron and inactive (3Fe-4S state) under low iron conditions suggesting that aconitase/IRE-BP Fe-S cluster acts as a regulatory switch (Haile et al., 1992a). However, the 4Fe -»3Fe interconversion was not accompanied with an activation of mRNA binding, which may indicate that cells utilize additional, yet undefined factors to interconvert the different forms of the IRE-BP (Haile et al., 1992b). A second iron regulatory protein (IRP2) has been isolated and characterized and although it has 61 % amino acid sequence homology with IRP1, it does not possess 284 aconi tase ac t iv i ty (Guo et al., 1 9 9 5 ) . In con t ras t to IRP1 It is bel ieved tha t intracel lular i ron levels regulate the degradat ion rate of IRP2 t h r o u g h the act ion of an inducib le, labile prote in (Guo e r a / . , 1 9 9 5 ) . The addi t ion of hemin (ferric chlor ide PPIX), bu t not inorganic i ron, to a cel l- free w h e a t germ ex t rac t w a s s h o w n to inhibi t the in teract ion of the IRE-BP w i t h the IRE thereby permi t t ing an increase in the t rans la t ion of ferr i t in mRNA (Lin et al., 1 9 9 0 a , 1 9 9 0 b ) . Exper imental manipu la t ion of the heme b iosynthet ic p a t h w a y p roduced ev idence tha t the level of intracel lular heme migh t d i rect ly a f fec t the ac t iv i ty of the IRE-BP (Ward et al., 1 9 8 4 ; Lin e r a / . , 1 9 9 0 b ) . However , the spec i f ic i ty of th is ac t ion of hemin w a s quest ioned in a s tudy by Haile et al. (1990) w h i c h s h o w e d t h a t hemin and other porphyr ins could inhibi t the b inding of a w ide var ie ty of RNA- and DNA-regu la to ry prote ins in vitro. Current ev idence indicates tha t the level of chelatable intracel lular iron d i rect ly determines IRE-BP binding ac t iv i ty (Klausner et al., 1 9 9 3 ) . This v i e w is suppor ted by studies in w h i c h iron chela tors (desfer r ioxamine, Desferal) , and fac to rs w h i c h s t imulate heme synthesis such as A L A or PPIX were s h o w n to lower intracel lular i ron , decrease ferr i t in synthes is (Eisenstein et al., 1 9 9 1 ; Mul lner e r a / . , 1992) and p romote an increase in TrfR express ion (Louache et al., 1 9 8 4 ) . In the presence of Photofr in®, MEL and P815 cells increased their sur face expression of the Tr fR. The present studies were not des igned to ident i fy the b iochemical changes w h i c h accoun t for these observat ions bu t the observed increase in TrfR expression is of a similar magni tude to the response of var ious cell lines cu l tured in the presence of PPIX, A L A , or iron che la tors . 2 8 5 Nitr ic ox ide (NO) w a s s h o w n to augment the IRE-binding o f the IRE-BP in the human J 7 7 4 macrophage and K 5 6 2 ery thro leukemic cell l ines (Weiss e r a / . , 1 9 9 3 ) , normal mur ine macrophages and the mur ine macrophage cell line RAW 2 6 4 . 7 (Drapier et al., 1 9 9 3 ) . It w a s proposed tha t NO reacts w i t h the 4Fe-4S cluster of the IRE-BP fo rm ing i ron-ni t rosyl complexes w h i c h produce an al losteric change of the prote in the reby p romot ing IRE binding ac t iv i ty (Weiss et al., 1 9 9 3 ) . NO has been s h o w n to d i rect ly in teract w i t h and inhibi t the ac t iv i ty of enzymes w h i c h conta in i ron-sulphur centres (Lancaster and Hibbs, 1 9 9 0 ) . Exposure of K 5 6 2 cells to NO increased TrfR mRNA and protein expression but had l i tt le e f fec t on ferr i t in levels (Oria et al, 1 9 9 5 ) . These observat ions may be of s igni f icance in cons ider ing the act ion of porphyr ins since it has been s h o w n tha t certain non-metal l ic porphyr ins mimic the act ion of NO on heme prote ins inc luding guanylate cyc lase (Ignarro et al. 1 9 8 9 ) . However , Photofrin® did not cause the release of detec tab le a m o u n t s of NO into cul ture supernatants even at photosensi t izer concen t ra t ions w h i c h increased the expression of TrfR on MEL cel ls. These f indings indicate t h a t the increase in TrfR expression produced by Photofr in® w a s not associated w i t h an in t roduc t ion of NO into the cell env i ronment . Few studies have been per formed in w h i c h intracel lular b iochemical responses to Photofr in® have been eva luated, especial ly in the absence of act ivat ing levels of l ight . Gomer et al. (1991) s h o w e d tha t levels of heme oxygenase mRNA and prote in w e r e e levated in Chinese hamster f ibroblasts incubated w i t h Photofrin®. Exposure of these cells to l ight fu r ther increased levels of th is degradat ive enzyme of heme (Gomer et al., 1 9 9 1 ) . A l t h o u g h heme oxygenase induc t ion can occur in 2 8 6 cells exposed to a var ie ty of s t imul i other than porphyr ins , the above studies do s igni fy t ha t Photofr in® does mod i fy e lements of the heme p a t h w a y . Further studies indicated tha t Photofrin® also induced the t ranscr ip t ion and express ion of the early response genes c- fos , c - jun, and erg-1 in l ight -protected cells (Gomer et al., 1 9 9 3 ; personal commun ica t ion ) indicat ing tha t the c o m p o u n d does produce responses at the nuclear level . The increase in TrfR expression produced in P815 and MEL cells by Photofr in® may have been caused by any of a number of intracel lular events inc lud ing: 1) chelat ion of iron thereby increasing the demand for i ron; 2) stabi l izat ion of the IRE-BP faci l i ta t ing TrfR mRNA t rans la t ion ; 3) increased t ranscr ip t ion of the TrfR gene; or 4) an al terat ion in the recyc l ing rate of the Tr fR. PPIX at concen t ra t ions of 1-10 / /M w a s s h o w n to increase express ion of the TrfR (Louache et al., 1984) and the b inding act iv i ty of the IRE-BP (Mullner et al., 1992) in human K 5 6 2 ery thro leukemia cel ls. It is not apparent w h e t h e r the amounts of PPIX ( ~ 5%) present in the Photofr in® fo rmu la t ion are suf f ic ient to alter MEL and P815 cell Tr fR expression since the levels of PPIX in the e f fec t ive (5 -10 / /g/ml) concen t ra t ions of Photofrin® w o u l d equate to approx imate ly 0 .5-1 / / M . It w o u l d be highly speculat ive to assert t ha t a change in the s ta tus of the Tr f /Tr fR sys tem represents a pr imary s t imulus for the hematopo ie t ic ac t iva t ion w h i c h occurs in mice g iven Photofr in®. Certa in ly, the number of spleen cells w h i c h s t rong ly express the TrfR is increased after Photofrin® admin is t ra t ion . H o w e v e r , th is s tate is character is t ical ly associated w i t h the later (CFU-E, ery throb last ) s tages of e ry thro id d i f ferent ia t ion (Lesley etal., 1 9 8 4 ) . PPIX 2 8 7 can increase TrfR expression by hematopo ie t ic cells (Louache et al., 1 9 9 4 ; Eisenstein et al., 1 9 9 1 ; Mi l l iner et al., 1 9 9 2 ) , a l though the present s tudy s h o w e d tha t PPIX has no hematos t imu la to ry e f fec t in vivo and fu r thermore PPIX is not k n o w n to cause ery thro id cell d i f ferent ia t ion (Sassa, 1 9 8 8 ) . Even t h o u g h Photofrin® did no t increase the g r o w t h rate of MEL or P815 cel ls, the c o m p o u n d might faci l i tate the del ivery of iron to d i f ferent ia t ing ery thro id cells by fu r ther increasing their express ion of the Tr fR. It w o u l d be of interest to determine w h e t h e r Photofrin® migh t fac i l i ta te the ery thro id d i f ferent ia t ion of MEL cells cu l tured in the presence of DMSO or hemin . To more comple te ly del ineate the impact of Photofrin® on regulatory aspects of the iron metabol ic p a t h w a y , it w o u l d be in fo rmat ive to per fo rm gel e lec t rophoret ic mobi l i ty sh i f t assays. In these techn ique , cy toso l ic ex t rac ts prepared f r o m Photofr in®-treated cells cou ld be incubated w i t h radio- label led IRE probes (Mil l iner et al., 1992) and f rac t ionated on po lyacry lamide gels. Relative levels of these IRE-BP/IRE-RNA complexes could be compared by dens i tomet r ic analysis of the subsequent au torad iographs. 2 8 8 CHAPTER NINE GENERAL DISCUSSION A N D OVERVIEW In the exper imenta l appraisal of a series of photosens i t iz ing porphyr ins , only Photofr in® produced any measurable s t imu la tory e f fec t on murine hematopoies is (Chapter 3 ) . Unfor tuna te ly , any def in i t ive analysis of the biological e f fec ts of Photofr in® is con founded by the heterogeneous compos i t i on of th is mater ia l . A l t h o u g h Photofrin® has received approval in a number of countr ies for use in h u m a n s , the var ie ty of porphyr in s t ruc tures conta ined w i t h i n Photofrin® c o n f o u n d s regula tory appraisal and compl icates manu fac tu r ing qual i ty con t ro l . H o w e v e r , a large body of ev idence has indicated tha t the s t rong ly photosensi t iz ing e lements reside in the o l igomer ic , but not in the monomer ic f rac t ion of Photofrin® (Dougher ty and M a n g , 1 9 9 2 ) . A l t h o u g h the major i ty of the s t ruc tures w i t h i n Photofrin® do not occur in nature , the immediate precursor to heme, PPIX, is present in the fo rmula t ion but never theless by i tself had litt le hematopo ie t ic impact in vivo. The other monomer ic s t ruc tures also k n o w n to be present in Photofrin®, HVD and Hp, indiv idual ly had no discernable hematos t imu la to ry ac t ion . A l t hough extens ive dos ing studies for the monomer ic c o m p o n e n t s mater ials were not pe r fo rmed, it seems apparent tha t the hematopo ie t ica l ly -ac t ive c o m p o n e n t of Photofrin® resides in the Hp ol igomeric f rac t ion . The exp lanat ion to as w h y the ol igomeric porphyr ins in Photofr in®, but not monomer ic Hp, are hematos t imu la to ry is unclear especial ly since many porphyr ins have a natural t e n d e n c y to aggregate in so lu t ion . Perhaps, the chemical o l igomer izat ion of monomer ic porphyr ins w h i c h occurs dur ing the synthes is of 2 8 9 Photofrin® generates a unique molecular s t ruc ture tha t is capable o f t r igger ing the hematopo ie t ic appara tus . The ol igomer ic f rac t ion ( > 8 0 % ) cons is ts of d imers and t r imers of Hp and HVD jo ined mainly by ether bonds , a l though in ter -porphyr in ester bonds may also be present (Dougher ty , 1 9 8 7 ) . Whe the r Photofr in® init iates its hematopo ie t ic e f fec ts d i rect ly at the genet ic level or upon an in teract ion w i t h intracel lular regula tory or sur face signal l ing prote ins awa i ts c lar i f ica t ion. However , the abi l i ty of Photofr in® to increase expression of the receptor for t ransfer r in in MEL and P815 cells (Chapter 8) suggests tha t a c o m p o n e n t of the response to Photofr in® may include an al terat ion in i ron-heme metabo l ism w i th in certain cells. The ident i f icat ion of the ant igen recognized by the monoclonal an t ibody LR-1 w a s a p ivota l step in def in ing the hematopo ie t ic response to Photofr in® (Chapter 7 ) . Initial s tudies indicated tha t mos t of the addi t ional cells present in the spleens of mice g iven Photofrin® expressed the LR-1 marker. A l t h o u g h the LR-1 reagent w a s or iginal ly descr ibed as being react ive w i t h mouse splenic B cells (Hutch ings e f al., 1 9 8 5 ) , Photofrin® did not increase the number of spleen cells w h i c h expressed B lymphocy te- res t r i c ted markers ( I gM, CD45R-B220) . The increase in the number of cells w h i c h expressed high levels of the LR-1 marker in the spleens of Photofr in®-treated mice w a s likely related to the increased myelopoie t ic and ery thropo ie t ic ac t iv i ty de tec ted w i th in the o rgan . This indicated tha t the LR-1 marker may be present on deve lop ing cells of the ery thro id and/or myelo id l ineages. Using a var ie ty of techn iques , it w a s s h o w n tha t the LR-1 monoc lona l an t ibody recognized heat stable ant igen (HSA) , w i t h an epi tope binding spec i f ic i ty ident ical t o t ha t of a previously character ized ant i -HSA an t ibody , M1 / 6 9 2 9 0 (Springer et al., 1 9 7 8 ; Mil ler et al., 1 9 8 5 ) . HSA is present on immature cells of all mur ine leukocy te l ineages (Bruce et al., 1 9 8 1 ; Mil ler e r a / . , 1985) and is found at par t icular ly high levels on immature (Hardy e r a / . , 1993) and LPS-act ivated B l ymphocy tes (Kennedy et al., 1994) and ery thro id cells (Bruce et al., 1 9 8 1 ) . Wi th in the m a r r o w of Photofrin®-t reated animals there w a s a dramat ic shi f t in the overal l HSA expression pat te rn . A l t h o u g h v i r tua l ly all mur ine BM cells are H S A + , cells express ing in termediate levels of HSA predomina ted in the BM of Photofr in®-treated mice , whereas in cont ro l mice there w a s equal d is t r ibut ion in the propor t ion of BM cells w h i c h expressed in termediate or high levels of HSA. The d imin ishment in the average dens i ty of HSA on BM cells of Photofr in®-treated animals occur red along w i t h a decrease in the propor t ion o f cells w h i c h expressed the B cel l - restr icted i so fo rm of CD45 and the ery thro id- re la ted marker CD71 and a concomi tan t increase in cells bearing the myelo id Gr-1 or C D 1 1 b ant igens. It is unl ikely tha t Photofr in® modula tes HSA express ion on indiv idual BM or spleen cel ls. The changes in HSA expression levels in these t issues are mos t l ikely due to the c o m p o u n d ' s inf luence on the fo rmat ion of l eukocy tes , w h i c h express l ineage-specif ic amounts of HSA. Several