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Studies on the mechanism of murine cytomegalovirus induced immunosuppression Whyte, Peter Frederick Muir 1983

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STUDIES ON THE MECHANISM OF MURINE CYTOMEGALOVIRUS INDUCED IMMUNOSUPPRESSION by PETER FREDERICK MUIR WHYTE B.Sc, (Microbiology), University of Bri t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Augus t 1983 (Copyright, Peter F.M. Whyte f 3 9 3 3 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 i t 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 i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Aug. 19, 1983 DE-6 (3/81) i i ABSTRACT The present study was undertaken to elucidate the mechanism of murine cytomegalovirus-induced immunosuppression. Previous studies had established the immunosuppressive effect of murine cytomegalovirus (MCMV) in vivo, and in v i t r o , and had characterized a wide range of immunological function which were impaired during MCMV infection. Canditioned medium, prepared from the tissue culture medium of cultured spleen cells infected with MCMV, contained a soluble immunosuppressive factor which suppressed spleen c e l l response to stimulation by Con A. This virus induced immunosuppressive factor (VISF) was not present i n conditioned media prepared from control cultures or from cultures of spleen c e l l s inoculated with various other viruses or latex beads. VISF was produced within 24 hours after infection of the c e l l s , and was not dependent upon the addition of exogenous fe t a l bovine serum to the culture medium. Attempts to extract VISF by l i p i d extraction techniques wer unsuccessful: no significant difference was observed between the capacities of control and MCMV conditioned media-extracted l i p i d s to suppress Con A stimulation of spleen c e l l s . Also, the production of i i i VISF could not be abrogated by the addition of indomethacin, an inhibitor of prostaglandin synthesis, to the spleen c e l l cultures. From these studies, i t was concluded that VISF was not a prostaglandin and was probably not any other type of l i p i d molecule. It was found that VISF could be concentrated i n the residual volume of an u l t r a f i l t r a t i o n c e l l using an Amicon YM2 membrane f i l t e r (molecular exclusion l i m i t of 1000 daltons), but VISF was not retained when an Amicon YMLO membrane f i l t e r (molecular exclusion limit of 10,000 daltons) was used. This indicated a molecular weight range for VISF of 1000-10,000 daltons. Gel f i l t r a t i o n chromatography of concentrated samples of conditioned media on a Sephadex G-25 matrix revealed a low molecular weight fraction (MW 1400 daltons) of immunosuppressive a c t i v i t y present i n the MCMV infected sample, but not i n the control sample or i n samples from spleen c e l l s infected with other viruses or particles. When MCMV and control samples were digested with the proteolytic enzyme, proteinase K, prior to fractionation, this immunosuppressive acxtivity was significantly diminished, but no significant change occurred in the corresponding control sample fraction. This suggested that VISF was a peptide or was, at least partly, peptide i n nature. To correlate the above findings with immunosuppression in vivo, studies were performed on mice infected with a sublethal dose of MCMV. Virus-free serum collected four days after infection of the mice was found to contain immunosuppressive a c t i v i t y relative to control serum from uninfected mice. Fractionation of the serum by Sephadex G-25 column chromatography demonstrated several fractions i n which the infected mouse serum was more suppressive than the fraction which contained the immunosuppressive a c t i v i t y from the MCMV conditioned medium sample, however the pattern was complicated by the number of other immunosuppressive substances present i n both sera. :> v TABLE OF CONTENTS Chapter Page I . Introduction 1 I . H i s t o r i c a l aspects of cytomegalovirus 1 I I . Nomenclature 2 I I I . P h y s i c a l properties of the v i r u s 3 a.) Morphology 3 b.) DNA 3 c.) Proteins 4 IV. R e p l i c a t i o n 4 a.) Host Range 4 1. Human cytomegalovirus 4 2. Murine cytomeglaovirus 5 b.) Penetration of the c e l l 5 c.) R e p l i c a t i v e c y c l e 5 V. Pathogenesis 7 a.) Human cytomegalovirus 7 b.) Murine cytomegalovirus 8 VI. Immunological Interactions 11 a.) Human cytomegalovirus 11 b.) Murine cyotmegalovirus 12 VII. Purpose of the study 15 v i . I I . Materials and Methods 16 I . Methods 16 1. Mice 16 2. C e l l s 16 3. Viruses 17 4. Tissue c u l t u r e medium 17 5. F e t a l bovine serum 18 6. Solutions 18 7. Chemicals 20 I I . Methods 21 1. Mouse embryo c e l l c u l t u r e s 21 2. BHK-21 c e l l c u l tures 22 3. Spleen c e l l c u l t u r e s 22 4. Propagation of murine cytomegalovirus 23 5. Propagation of Sindbis v i r u s 23 6. Plaque assay of v i r u s 24 7. C o l l e c t i o n of mouse serum 25 8. Preparation of conditioned medium 25 9. Mitogen s t i m u l a t i o n t e s t 26 10. U l t r a f i l t r a t i o n of conditioned media 27 11. E x t r a c t i o n of l i p i d s from conditioned media 28 12. Sephadex G-25 f r a c t i o n a t i o n of conditioned media 29 13. Assay of Sephadex G-25 f r a c t i o n s 29 14. Proteinase K d i g e s t i o n 30 15. Polyacrylamide g e l e l e c t r o p h o r e s i s 30 v i i 16. S t a i n i n g of gels 31 17. L i q u i d s c i n t i l l a t i o n counting of r a d i o a c t i v i t y 32 I I I . Results I: Evidence f o r the presence of a soluble suppressor f a c t o r . 33 I. Immunosuppressive e f f e c t of serum from MCMV i n f e c t e d mice 33 I I . In v i t r o production of a v i r u s induced suppressor f a c t o r 36 I I I . S p e c i f i c i t y of the v i r u s induced suppressor f a c t o r 43 IV. Factors a f f e c t i n g the production of the v i r u s induced suppressor f a c t o r 44 IV. Results I I : Ch a r a c t e r i z a t i o n of the v i r u s induced suppressor f a c t o r . 53 I . E f f e c t of indomethacin on the production of the v i r u s induced suppressor f a c t o r 53 I I . Concentration of the v i r u s induced suppressor f a c t o r by u l t r a f i l t r a t i o n 55 I I I . F r a c t i o n a t i o n of conditioned medium by g e l f i l t r a t i o n chromatography 59 IV. F r a c t i o n a t i o n of mouse serum by g e l f i l t r a t i o n chromatography 64 V. F r a c t i o n a t i o n of Sindbis v i r u s and LB8 conditioned media by g e l f i l t r a t i o n chromatography 69 VI. Proteinase K digestion of the virus induced suppressor factor VII. Electrophoresis of mouse serum and conditioned medium Discussion Bibliography . • i x LIST OF TABLES — Page o I. Effect of conditioned media on the uptake of H-dThd by unstimulated and Con A stimulated spleen c e l l s . 40 II. Effect of the purification of conditioned media on the suppression of Con A stimulation of spleen c e l l s . 42 III. Effect of l i p i d s from conditioned media on the stimulation of spleen c e l l s by Con A. 58 IV. Effect of u l t r a f i l t r a t i o n through a YM2 membrane f i l t e r on the a b i l i t y of conditioned media to suppress Con A stimulation of spleen c e l l s . 61 V. Effect of proteinase K on the fractionation of the virus induced suppressor factor by Sephadex G-25 chromatography. 75 ; .X LIST OF FIGURES Figure Page 1. Effect of normal and infected mouse serum on Con A stimulation of spleen c e l l s . 35 2. Suppression of Con A stimulation of spleen c e l l s by murine cytomegalovirus conditioned medium. 39 3. Effect of various conditioned media on Con A stimulation of spleen c e l l s . 46 4. Kinetics of production of the vir u s induced suppressor f a c t o r . 50 5. Effect of f e t a l bovine serum on the production of the virus induced suppressor f a c t o r . 52 6. Effect of indomethacin on the production of the viru s induced suppressor f a c t o r . 56 7. x i E f f e c t of the concentration of the v i r u s induced suppressor f a c t o r on Con A s t i m u l a t i o n of spleec c e l l s . 63 8. Sephadex G-25 f r a c t i o n a t i o n of c o n t r o l and murine cytomegalovirus conditioned media. 66 9. Sephadex G-25 f r a c t i o n a t i o n of normal and murine cytomegalovirus i n f e c t e d mouse serum. 68 10. Sephadex G-25 f r a c t i o n a t i o n of Sindbis and LB8 conditioned media. 1 72 11. Sephadex G-25 f r a c t i o n a t i o n of proteinase K digested c o n t r o l and murine cytomegalovirus conditioned media. 74 ABBREVIATIONS N,N'-bis-methylene acrylamide concanavalin A deoxythymidine fetal bovine serum gravitational force tritium Hanks' balanced salt solution human cytomegalovirus immunoglobulin infected mouse serum modified Eagle's medium murine cytomegalovirus mul t i p l i c i t y of infection normal mouse serum phosphate buffered saline sodium dodecyl sulfate virus induced suppressor factor x i i i ACKNOWLEDGEMENTS I would like to extend my most sincere thanks to my supervisor, Dr. J.B. Hudson, for allowing me this opportunity to study and carry out this project in his laboratory, and for his guidance throughout the course of this study. My gratitude is also extended to the members of my supervisory committee, Dr. C.E. Slonecker, Dr. W.H. Chase, Dr. S. Sacks and Dr. R.H. Pearce, for their guidance and helpful suggestions. I would also lik e to thank Ms. Cathy Moore and Ms. Elizabeth Graham for their technical assistance, and Mr. Douglas Walker for his helpful discussions. 1 CHAPTER I  INTRODUCTION I. HISTORICAL ASPECTS OF CYTOMEGALOVIRUS In 1926, the discovery of a f i l t e r a b l e , heat l a b i l e f a c t o r capable of tra n s m i t t i n g 'cytomegalia' i n guinea pigs brought an end to a twenty-two year long debate concerning the nature and e t i o l o g y of the cytomegalic c e l l s observed i n various t i s s u e s of i n f a n t s (22,74). The e a r l i e r theory of a protozoan nature of the large c e l l u l a r bodies had been gradually g i v i n g way to the theory of a v i r a l e t i o l o g y , r e s u l t i n g i n swollen host c e l l s . The cytomegalic l e s i o n s observed i n the s a l i v a r y glands of se v e r a l animals were included i n the o r i g i n a l Type A nuclear i n c l u s i o n s described by Cowdry i n 1934 (23). Today, the majority of Cowdry's Type A i n c l u s i o n s are recognized as being caused by members of the herpes f a m i l y . McCordock and Smith discovered a s i m i l a r transmissible f a c t o r present i n the s a l i v a r y glands of mice i n 1936, however i t was not u n t i l 1954 that the s a l i v a r y gland v i r u s of mice was i s o l a t e d and propagated i n ti s s u e c u l t u r e (122). S h o r t l y a f t e r , i s o l a t e s of the hunam s a l i v a r y gland v i r u s were made (112,123,135). By 1960, the s a l i v a r y gland v i r u s had been demonstrated to produce i t s c h a r a c t e r i s t i c l e s i o n s i n tis s u e s other than the s a l i v a r y 2 glands, and therefore was renamed cytomegalovirus, a f t e r the microscopic morphology of the l e s i o n s i t caused (135) . I I . Nomenclature The development of the negative s t a i n i n g procedure f o r e l e c t r o n microscopy i n 1959 made possible the examination of the s i z e and morphology of vir u s e s , allowing grouping of vir u s e s by these c r i t e r i a (12). S h o r t l y a f t e r t h i s development, C.H. Andrewes proposed a r e v i s e d scheme f or the taxonomic grouping of v i r u s e s based upon four c r i t e r i a , one of which was morphology ( 1 ) . In h i s proposed c l a s s i f i c a t i o n , the cytomegaloviruses formed a group w i t h i n the NITA (nuclear i n c l u s i o n Type A) v i r u s c l a s s . Later that year the proposed c r i t e r i a were adopted by the Virus Subcommittee at the 1962 I n t e r n a t i o n a l Nomenclature Committee meeting, however the NITA group was renamed herpes. Present taxonomic groupings designate the human cytomegalovirus (HCMV) and the murine cytomegalovirus (MCMV) as separate genus of the Herpesviridae family (89). 3 I I I . P h y s i c a l Properties of the Virus a. ) Morphology Comparison of herpes v i r u s e s by e l e c t r o n microscopy revealed i n d i s t i n g u i s h a b l e v i r i o n p a r t i c l e s f o r many of the members of the herpes v i r u s family i n c l u d i n g HCMV and MCMV (87,104,121,138,140). Features of the v i r i o n include an inner nucleoprotein core c o n s i s t i n g of 162 hexagonal and pentagonal elongated capsomers i n a 5:3:2 symmetrical arrangement (54). Each elongated capsomer i s 9.5 x 12.0-13.5 nm. The capsid i t s e l f i s approximately 100 nm i n diameter and i s surrounded by an envelope, derived from the nuclear membrane of the i n f e c t e d c e l l , measuring 4.0-10.0 nm i n thickness and bearing projections (87,138,140). A d i s t i n g u i s h i n g feature of MCMV i s the presence of multi c a p s i d v i r i o n s which are often observed i n a d d i t i o n to s i n g l e capsid v i r i o n s (60). b. ) DNA The cytomegaloviruses contain large double stranded, l i n e a r , DNA genomes. HCMV has a genome of 150 x 10^ daltons with a buoyant 4 3 density of 1.718 g/cm and a G + C content of 58% (27,30,58). MCMV contains a genome of 132 x 10^ daltons with a buoyant density of 1.718 g/cm3 and a G + C content of 59% (92). c.) Proteins Studies on HCMV reve a l at l e a s t 35 s t r u c t u r a l p r o t e i n s , i n c l u d i n g a minimum of 7 glycoproteins (125) . MCMV appears to be quite s i m i l a r ; 33 s t r u c t u r a l p r o t e i n s , i n c l u d i n g at l e a s t 6 gly c o p r o t e i n s , have been reported (19,71). IV REPLICATION a.) Host Range 1. Human Cytomegalovirus HCMV can be i s o l a t e d from many t i s s u e s and body f l u i d s i n c l u d i n g u r i n e , m i l k , blood, semen, s a l i v a r y glands, kidneys, c e r v i x , l i v e r and s t o o l s (24,31,65,77,137). The v i r u s appears to be capable of r e p l i c a t i o n and persistence i n v i r t u a l l y a l l t i s s u e s as we l l as i n severa l tumours (16,43,59,103). Propagation i n v i t r o i s much more d i f f i c u l t , productive i n f e c t i o n occurs i n few c e l l s other than human f i b r o b l a s t s and f i b r o b l a s t c e l l l i n e s (61,73,105,118). 5 2. Murine Cytomegalovirus Many murine t i s s u e s are su s c e p t i b l e to i n f e c t i o n by MCMV i n v i v o , although i n many cases the c e l l types have not been determined, nor has the extent of r e p l i c a t i o n i n those c e l l s (11,48,61,141). In v i t r o i n f e c t i o n of embryonic f i b r o b l a s t c e l l l i n e s , spleen c e l l s or various primary c u l t u r e s a l l y i e l d i n f e c t i o u s v i r u s (7,51,70,81). b. ) Penetration of the C e l l V i r a l entry i n t o the c e l l i s f a c i l i t a t e d by a temperature independent attachment mechanism i n v o l v i n g f u s i o n of the v i r a l envelope to the c e l l membrane. The nucleocapsid i s then i n t e r n a l i z e d i n t o the cytoplasm i n a temperature dependent event and then migrates toward the nucleus (119,131). c. ) R e p l i c a t i v e Cycle The r e p l i c a t i v e c y c l e f o r cytomegaloviruses i s s i m i l a r to other herpes v i r u s e s . Approximately four days are required f o r the release of the f i r s t progeny v i r u s (118). The cycles of HCMV and MCMV r e p l i c a t i o n are not well c h a r a c t e r i z e d but appear to be s i m i l a r . Several d i f f e r e n c e s have been reported and are discussed below. 6 Af t e r penetration i n t o the c e l l the nucleocapsid migrates q u i c k l y toward the nucleus where the capsid i s thought to be degraded and the v i r a l DNA enters the nucleus (87). Expression of the v i r a l genome i s regulated by a cascade system s i m i l a r to that described by Honess and Roizman f or the r e p l i c a t i o n of herpes simplex v i r u s (53). The genome i s divided i n t o immediate e a r l y , e a r l y , and l a t e gene c l a s s e s . Only the immediate e a r l y genes of HCMV have been mapped, and no genes have been mapped i n MCMV (126) . A DNA polymerase has been i d e n t i f i e d and i s probably a member of the e a r l y gene c l a s s (63,96,99). Although e f f o r t s to f i n d thymidine kinase genes i n the v i r a l genome have f a i l e d , increases i n c e l l u l a r thymidine kinase a c t i v i t y during HCMV i n f e c t i o n s have been reported (35,142). This i s i n contrast to r e s u l t s obtained using MCMV, which have demonstrated a decrease i n thymidine kinase a c t i v i t y (95). A second d i f f e r e n c e between the two vi r u s e s i s t h e i r e f f e c t on host c e l l s y n t h e s i s . While MCMV has l i t t l e or no e f f e c t on c e l l u l a r DNA sy n t h e s i s , HCMV stimulates host c e l l DNA synthesis and induces DNA dependent RNA polymerase a c t i v i t y (62,127). Assembly of the nucleocapsid of HCMV takes place i n the nucleus by the t h i r d day fol l o w i n g i n f e c t i o n (87), although v i r u s does not appear i n the supernatant f o r an a d d i t i o n a l 2-5 days (131). The nucleocapsid moves to the cytoplasm by budding through the nuclear membrane, acquiring an envelope i n the process. A second coat may be acquired by budding into lysosomes i n the cytoplasm (120) . V. Pathogenesis a.) Human Cytomegalovirus High percentages of seropositive individuals i n the population refle c t the prevalence of HCMV i n society. In some parts of the world the proportion of seropositive adults may approach 100%, although i n North America the percentage is probably somewhat less (137) . The adult population may therefore act as a reservoir of latent virus. Several modes of transmission are thought to exist including venereal transmission, oral transmission, transplacental passage from mother to fetus and transmission during passage through the birth canal (109,137). HCMV is also transmitted during blood transfusions and organ allografts when the donor i s harbouring latent virus (36,78). Although most HCMV infection are asymptomatic and never develop into c l i n i c a l disease, a small proportion of individuals are susceptible to and develop c l i n i c a l symptoms. These individuals can be divided into three groups: a.) young adults who develop infectious mononucleosis, b.) fetuses and newborn children, and c.) 8 i n d i v i d u a l s predisposed by immunological d e f i c i e n c i e s i n c l u d i n g genetic d e f e c t s , immunosuppressive therapy, patients with leukemia or lymphomas and patients s u f f e r i n g from acquired immunodeficiency syndrome (25,33,40,44,46,67,69). Disseminated cytomegalic i n c l u s i o n disease i s cha r a c t e r i z e d by l a r g e , swollen, cytomegalic c e l l s which i n d i c a t e the presence of the v i r u s . C l i n i c a l symptoms vary but may include hapatosplenomegaly, thrombocytopenia, jaundice and pneumonia. Congenital i n f e c t i o n s frequently i n v o l v e the c e n t r a l nervous system and may r e s u l t i n mental r e t a r d a t i o n , microencephaly, deafness or blindness (47,88,137). Disseminated HCMV i n f e c t i o n has a l s o been c o r r e l a t e d with the occurence of secondary i n f e c t i o n s , suggesting that the v i r a l i n f e c t i o n predisposes the patient to secondary i n f e c t i o n s (107,110,111). b.) Murine Cytomegalovirus The s u r v i v a l of mice experimentally i n f e c t e d with MCMV i s dependent upon a number of factors i n c l u d i n g i n o c u l a t i o n dose, route of i n o c u l a t i o n , s t r a i n of v i r u s , and the age and genotype of the mouse (17,61,91,101,114). Those mice which die generally do so withi n 8-9 days f o l l o w i n g i n o c u l a t i o n . The a c t u a l cause of death i s unknown. 9 Experimental i n f e c t i o n s r e s u l t i n death or a chronic or l a t e n t i n f e c t i o n f o l l o w i n g an i n i t i a l acute phase of i n f e c t i o n (101). Moribund mice s a c r i f i c e d before death show a viremia with necrosis and edema of many ti s s u e s and organs i n c l u d i n g the l i v e r , spleen, lungs, kidneys, s a l i v a r y glands, heart, and b r a i n (90,91). E s p e c i a l l y prominant i n t h i s group are the l i v e r and spleen, both of which are thought to be e a r l y s i t e s of MCMV r e p l i c a t i o n and i n f e c t i o n , a f t e r i n o c u l a t i o n by e i t h e r the i n t r a p e r i t o n e a l or intravenous route (48,91). Splenomegaly i s a common symptom of MCMV i n f e c t i o n with the organ often doubling i n s i z e . I n f e c t i o n of the spleen i s i n i t i a t e d i n the p e r i f o l l i c u l a r regions and then spreads i n t o the f o l l i c l e s and red pulp; peak t i t e r s of v i r u s occur around days 3-5, a f t e r i n f e c t i o n (82,90,91). Latent v i r u s can be recovered by c o c u l t i v a t i o n with feeder c e l l s , although the a c t u a l c e l l type responsible f o r harbouring the v i r u s i s unknown. Recent studies have suggested the macrophage as a candidate which i s permissive to v i r a l r e p l i c a t i o n and which i s harbouring l a t e n t v i r u s (82,91). within the l i v e r there i s a random d i s t r i b u t i o n of l e s i o n s throughout the lobules , from which i n f e c t i o u s v i r u s can be recovered. The t i t e r of recovered v i r u s depends upon the route of i n f e c t i o n and the dose of v i r u s given. Parenchymal hepatic c e l l s and Kuppfer c e l l s both become i n f e c t e d with the v i r u s , however 10 l a t e n t v i r u s or v i r a l DNA has not been detected l n these c e l l s a f t e r the acute stage of i n f e c t i o n (101,114). Thus, i t appears as i f the l i v e r i s important i n the acute stage of i n f e c t i o n but may not be involved i n the chronic or l a t e n t stage. A viremia i s present from the time of i n o c u l a t i o n u n t i l approximately 8 days post i n f e c t i o n . A f t e r t h i s time no free v i r u s can be recovered from the serum of these animals , however v i r u s can be recovered at l a t e r times by c o c u l t i v a t i o n of blood lymphocytes with a l l o g e n i c or syngeneic feeder c e l l s (141) . Although there i s current controversy over the p a r t i c u l a r type of c e l l which i s i n f e c t e d by the v i r u s , s e v e r a l studies have demonstrated the presence of l a t e n t v i r u s i n p e r i t o n e a l macrophages fo l l o w i n g i n t r a p e r i t o n e a l i n o c u l a t i o n . I t has a l s o been reported that macrophages become a c t i v a t e d during the acute stage of i n f e c t i o n and that macrophages are important i n p r o t e c t i n g mice against MCMV (11,91,113,114). A f t e r the acute stage of i n f e c t i o n the v i r u s can go i n t o e i t h e r a chronic stage or a l a t e n t stage. The fa c t o r s determining which of these occurs are not presently known, but i t i s the r o l e of the immune system to prevent r e a c t i v a t i o n of the v i r u s . When l a t e n t l y i n f e c t e d mice are treated with immunosuppressive agents, an acute and disseminated i n f e c t i o n reoccurs (39,66). 11 VI. Immunological Interactions a.) Human Cytomegalovirus HCMV has a complex r e l a t i o n s h i p with the immune system of i t s h ost. The response of the host v a r i e s with the type of i n f e c t i o n , the age and immune competence of the host and the s e v e r i t y of the i n f e c t i o n . The v i r u s can p e r s i s t l a t e n t l y i n human leukocytes and has been i s o l a t e d from washed leukocytes as w e l l as from the blood of patients (37,76). Drew e t at were able to grow HCMV i n a l v e o l a r macrophages (32). Thus, HCMV may p e r s i s t i n c i r c u l a t o r y monocytes or lymphocytes or both. What e f f e c t t h i s may have on the fu n c t i o n of these c e l l s i s unknown. Normal humoral responses to HCMV are seen i n most i n f e c t e d i n d i v i d u a l s and include production of Ig M, Ig G, and Ig A (41,129). In a small number of patients with HCMV i n f e c t i o n , the ; absence of antibody production can be c o r r e l a t e d with severe i n f e c t i o n s which eve n t u a l l y become l e t h a l . Whether t h i s i s a r e s u l t of the i n f e c t i o n or whether the s e v e r i t y i s a r e s u l t of the patient's i n a b i l i t y to produce antibodies i s not c l e a r (26,97). C e l l mediated immunity i s much more s e n s i t i v e to HCMV i n f e c t i o n and appears to be suppressed i n v i r t u a l l y a l l patient groups with a c t i v e HCMV i n f e c t i o n s (49). A r e v e r s a l of normal T helper: T suppressor c e l l r a t i o s may be responsible f o r the decreased c e l l mediated immunity i n patients with i n f e c t i o u s mononucleosis (14,100). T c e l l r a t i o s i n other types of HCMV i n f e c t i o n have not been examined. One consequence of the immunosuppressive e f f e c t of HCMV i n f e c t i o n i s a p r e d i s p o s i t i o n to s u p e r i n f e c t i o n by other organisms. Concurrent i n f e c t i o n s from o p p o r t u n i s t i c organisms are not uncommon i n transplant patients or c h i l d r e n with dessiminated HCMV i n f e c t i o n s (20,107). b.) Murine Cytomegalovirus The immune response of the mouse e l i c i t s a complex response d i r e c t e d toward the i n f e c t i n g v i r u s . Within 3 days, both humoral and c e l l mediated immune responses are present, and prot e c t i v e antibodies and c y t o t o x i c T lymphocytes can be found i n the blood. Also at t h i s time, antibody dependent c y t o t o x i c c e l l s have been observed and may play a r o l e i n immunity to the v i r u s . Natural k i l l e r c e l l a c t i v i t y may begin as e a r l y as 10 hours a f t e r i n t r a p e r i t o n e a l i n o c u l a t i o n . Thus, a complete response toward the vi r u s appears to be present (2,6,52,85,106,124). Both the n a t u r a l k i l l e r c e l l and antibody dependent c y t o t o x i c c e l l a c t i v i t y , as w e l l as the p r o t e c t i o n mediated by macrophages have been c o r r e l a t e d with the major h i s t o c o m p a t i b i l i t y region (H-2) genotype of the mice used (6,17,85,114). 13 Despite the response toward the virus, widespread suppression of immune responses toward other immunological targets occurs during the acute stage of the infection. Early experiments demonstrated suppressed humoral responses to Newcastle disease virus and sheep erythrocytes in MCMV infected mice (102). The suppressive effect lasted 9-10 days after inoculation, after which there was a gradual return to normal. This period coincides with the course of the acute stage of infection (83). Hamilton et^ al^ i l l u s t r a t e d the v a l i d i t y of these observations to the i n vivo situation by coinfecting mice with MCMV and a second infectious agent (P. aeruginosa, C. albicans or S. aureus). The result was a striking increase i n l e t h a l i t y and multiplication of the bacteria (or yeast) i n the blood and organs of the mice. In controls inoculated with bacteria (or yeast) only, the infectious agent was rapidly cleared from the body and the infection was non-lethal (47,48). Howard et al_ presented evidence that MCMV suppresses both humoral and c e l l mediated immunity. Depression of humoral immunity was determined by depressed hemagglutinin antibody response to an intraperitoneal injection of sheep erythrocytes. Both primary and secondary antibody responses could be suppressed by the virus, but a normal response was seen i f the mice were previously vaccinated to MCMV. A suppressed c e l l mediated response was il l u s t r a t e d by delayed allograft rejection and decreased responses i n mixed lymphocyte reactions and phytohemagglutinin mitogen stimulation 14 (55,56). Lymphocytes i n f e c t e d i n v i v o or i n v i t r o have a suppressed response to s t i m u l a t i o n by e i t h e r T c e l l or B c e l l mitogens (9,10,82,115). C o n f l i c t i n g reports e x i s t as to the population of c e l l s responsible f o r mediating the immunosuppression, and f o r the type of c e l l i n which the v i r u s r e p l i c a t e s . Wu and Ho reported the recovery of v i r u s from T lymphocytes but not B lymphocytes (141), and Selgrade et_ al^ found no suppressive e f f e c t mediated by plastic-adherent c e l l populations (115). Contrary to these reports are the observations of Loh and Hudson. They found the plas t i c - a d h e r e n t population of spleen c e l l s to mediate immunosuppression and a l s o to r e p l i c a t e v i r u s (81,82,83). Maximum t i t e r s were found i n the macrophage r i c h , adherent population and the number of i n f e c t i o u s centers i n the spleen was proportional to the degree of suppression. Infected adherent c e l l s mixed with uninfected non-adherent c e l l s r e s u l t e d i n a population of c e l l s which had a suppressed mitogen response. Thus, the adherent population of spleen c e l l s , and p o s s i b l y the macrophage c e l l s p e c i f i c a l l y , i s responsible f o r mediating the observed immunosuppression. 15 VII. Purpose of the Study The purpose of the present study was to i n v e s t i g a t e the mechanism by which MCMV induced immunosuppression takes p l a c e . Since t h i s i s a part of the pathogenesis of MCMV, i t should be in v e s t i g a t e d i n order to a t t a i n the goal of a complete understanding of the pathogenesis of t h i s v i r u s . A l s o , v i r u s induced immunosuppression i s a phenomenon which occurs i n se v e r a l human v i r u s i n f e c t i o n s and therefore i s a rele v a n t medical problem. I t i s a l s o hoped that the r e s u l t s of t h i s study w i l l provide some i n s i g h t i n t o the r e g u l a t i o n of the immune response. CHAPTER II MATERIALS AND METHODS I. MATERIALS 1. Mice SWR/J mice were obtained from Jackson Laboratories, Bar Harbour, Maine and were maintained by the UBC Animal Care Center. The mice were allowed free access to mouse chow and water during their maintenance and during experiments. Random bred pregnant Swiss white mice for the purposes of mouse embryos were bred by the UBC Animal Care Center. 2. Cells Mouse embryo c e l l s were prepared from pregnant random bred Swiss white mice on approximately the fourteenth day of gestation. Spleen c e l l s were prepared from SWR/J mice indiscriminately of age or sex. BHK-21 c e l l s , a continuous c e l l l ine of baby hamster kidney ce l l s were obtained from Dr. D. Vance, Dept. Biochem., UBC. 3. Viruses The Smith s tra in of murine cytomegalovirus (MCMV) was obtained from the American Type Tissue Col lec t ion , and was propagated i n ter t iary mouse embryo c e l l s . This s tra in was used for the experiments involving MCMV. Sindbis virus was obtained from Dr. D. Vance, Dept. Biochem., UBC, and was propagated i n BHK-21 c e l l s . Bacteriophage TA was obtained from Dr. A. Warren, Dept. Micro . , UBC. Bacteriophage PM2 was obtained from Dr. U . Kuhnlein, B r i t i s h Columbia Cancer Research Center. 4. Tissue Culture Medium Dulbecco's modified Eagle medium (MEM) and RPMI 1640 were obtained in powder form from Grand Island Bio logical Company and dissolved in deionized water. MEM A was supplemented with 3.7 g/1 NaHC03, MEM B with 1.5 g/1 NaHCO^, and RPMI 1640 with 2.0 g/1 NaHC03. Media were f i l t e r s t e r i l i z e d and supplemented with 50 ug/ml gentamicin before use. 5• Fetal Bovine Serum Fetal bovine serum was obtained from Grand Island Biological Company and stored at -20°C u n t i l used. 6. Solutions a. ) Hanks' balanced salt solution (HBSS) consisted of: NaCl 0.14 M KCl 5.40 mM MgS04.7 H20 0.81 mM CaCl 2 1.26 mM NaHPO. .7 H„0 0.61 mM 4 2 KH-P0. 0.44 mM 2 4 Glucose 5.56 mM Phenol red 0.02 g/1 b. ) Elution buffer for Sephadex G-25 column chromatography consisted of: NaCl 0.07 M KCl 2.70 mM MgSO^ .7 H20 0.41 mM 19 CaCl 2 0.63 mM NaHP04.7 H20 0.31 mM KH 2P0 4 0.22 mM c.) Phosphate buffered saline consisted of: NaCl 0.13 M KCl 2.70 mM Na2HP04.7H20 8.10 mM CaCl 2 1.00 mM MgCl2 0.50 mM KH 2P0 4 1.50 mM d. ) Electrophoresis running buffer (pH 8.3) consisted of: Tris-HCl 0.025 M Glycine 0.192 M Sodium dodecyl sulfate 1.0% e. ) Electrophoresis sample buffer (pH 6.8) consisted of: Tris-HCl 0.0625 M Sodium dodecyl sulfate 2.0% Glycerol 10.0% 2-mercaptoethanol 5.0% Bromophenol blue 0.001% f. ) Overlay medium for plaque assays consisted of MEM A containing 0.5% agarose and 5.0% fetal bovine serum. 7. Chemicals Product Acrylamide Agarose Concanavalin A Gentamicin LB8 Latex Beads N,N'-bis-methylene acrylamide Proteinase K Scintrex Sephadex G-25 3 (methyl- H) Thymidine Trypsin Distributor BioRad Laboratories SeaKem Sigma Chemical Co. Sigma Chemical Co. Sigme Chemical Co. BioRad Laboratories Beckman J.T. Baker Pharmacia Chemicals New England Nuclear Difco Laboratories I I . METHODS 1. Mouse Embryo Cell Cultures Embryos were removed from pregnant, random bred, Swiss white mice at approximately the fourteenth day of gestation. The embryos were minced with scissors and then suspended i n Hanks' balanced salt solution (HBSS) supplemented with 2.5 mg/ml of trypsin and stirred vigorously for 30 minutes. The supernatant was drawn off and centrifuged at 900g x 10 minutes. The undigested tissue fragments were subjected to at least two cycles of trypsin digestion and each successive supernatant was centrifuged at 900g x 10 minutes to pellet the cell s from the solution. The c e l l pellets were combined and resuspended in MEM A, containing 10% fetal bovine serum (FBS), and then plated into petri dishes • The cultures were incubated at 37" C i n an atmosphere of 5.0% C0 2, 95.0% a i r and at high humidity. Mouse embryo ce l l s were passaged by removing the growth medium, rinsing the cel l s with HBSS, then incubating the cel l s for 5 minutes i n HBSS, containing 2.5 mg/ml of trypsin, at room temperature. The HBSS-trypsin was then aspirated and the cells were removed from the surface of the petri dish by pipetting MEM A, containing 10% FBS, vigorously over the surface of the d i s h . The c e l l s were p e l l e t e d by c e n t r i f u g a t i o n at 900g x 10 minutes, resuspended i n MEM A, containing 10% FBS, and plated i n p e t r i dishes . 2. BHK-21 C e l l Culture The continuous c e l l l i n e of baby hamster c e l l s was c u l t u r e d i n p e t r i dishes using MEM A, containing 10% FBS, as a growth medium. Incubation was at 37 C i n an atmosphere of 5% CO2, 95% a i r and at high humidity. The c e l l s were passaged by t r y p s i n i z a t i o n and r e p l a t i n g as was described for mouse embryo c e l l s . 3. Spleen C e l l Cultures Spleen c e l l c u l t u r e s were prepared from adult SWR/J mice. Spleens were removed from the mice and placed i n p e t r i dishes containing 10 mis of HBSS . The spleens were bisec t e d and the c e l l s gently teased out from the spleen using forceps. The suspension was passaged through a syringe to break c l u s t e r s of c e l l s . The c e l l s were p e l l e t e d by c e n t r i f u g a t i o n at 900g x 10 minutes and then resuspended i n 0.17 M NH^Cl f o r 5-7 minutes . The c e l l s were again p e l l e t e d by c e n t r i f u g a t i o n and the resuspended i n RPMI 1640. Viable c e l l s were assessed by counting f o r trypan blue excluding c e l l s i n a hemocytometer and the c e l l suspension was then d i l u t e d with RPMI 1640 to y i e l d the desired concentration of c e l l s . A l l incubation of spleen c e l l s was a t 37 C, i n an atmosphere of 5% CX"^ , 95% a i r and a t high humidity. 4. Propagation of Murine Cytomegalovirus The Smith s t r a i n of MCMV was propagated i n t e r t i a r y mouse embryo c e l l s from the embryos of random bred Swiss mice. T e r t i a r y mouse embryo c e l l s were grown to confluency i n r o l l i n g b o t t l e s i n MEM B, supplemented-with 5% FBS before being i n f e c t e d with MCMV at a low m u l t i p l i c i t y of i n f e c t i o n (<0.1 P F U / c e l l ) . Virus was harvested a f t e r 3-4 days incubation at 37 C during which time the r o l l i n g b o t t l e s were rotated at a speed of 0.25 rev o l u t i o n s per minute. The medium was c o l l e c t e d and cent r i f u g e d at 4000g x 10 minutes to remove c e l l u l a r d e b r i s . The supernatant was then c e n t r i f u g e d a t I9,000g x. 4 hours to p e l l e t the v i r u s . The v i r a l p e l l e t was resuspended i n a small volume of MEM B, containing 5% FBS, and dispensed i n t o one ml al i q u o t s and stored u n t i l use at -70 C. 5. Propagation of Sindbis Virus Sindbis v i r u s was propagated i n p e t r i dishes of BHK-21 c e l l s i n MEM A, containing 10% FBS. P e t r i dishes of confluent BHK-21 c e l l s were infected by removing the medium and inoculating the c e l l s with a v i r a l suspension for one hour, a f t e r which the inoculum was removed and the c e l l s were overlain with fresh MEM A, containing 10% FBS. After completion of the l y t i c c y c l e , the medium was removed and centrifuged at 900g x 10 minutes to remove the c e l l u l a r debris. The supernatant containing the virus was then centrifuged at 36,400g x 3 hours to p e l l e t the v i r u s . The virus p e l l e t was resuspended i n a small volume of MEM A, containing 10% FBS, and stored at -70 C. 6. Plaque Assay of Virus Virus preparations were t i t e r e d by plaque assay. MCMV and Sindbis virus were t i t e r e d on t e r t i a r y mouse embryo and BHK-21 c e l l s r e s p e c t i v e l y . Confluent p e t r i dishes of c e l l s were inoculated with appropriate d i l u t i o n s of the v i r u s preparation i n MEM A, containing 10% FBS, by removing the medium from the c e l l s and replacing i t with the v i r a l inoculum. The c e l l s were incubated for one hour at 37 C, 5% C0 2 > 95% a i r and at high humidity, before the inoculum was removed and replaced by semi-solid overlay medium. The c e l l s were then incubated u n t i l plaques became v i s i b l e and were counted. 7• C o l l e c t i o n of Mouse Serum Serum was co l l e c t e d from SWR/J mice by heart puncture immediately following s a c r i f i c e of the mice. Infected mouse serum (IMS) was coll e c t e d on the fourth day following an int r a p e r i t o n e a l i n j e c t i o n of 5 x 10^ PFU of MCMV. Normal mouse serum (NMS) was co l l e c t e d from uninfected mice. After the blood was co l l e c t e d from the punctured heart, i t was pooled and allowed to c l o t at 4 C. The unclotted f r a c t i o n was removed and centrifuged at 500g for 10 minutes to remove any res i d u a l c e l l s . The supernatant was then centrifuged at 60,000g x 90 minutes to remove any infectious v i r u s from the serum. The supernatant was taken and stored at 4 C u n t i l used. 8. Preparation of Conditioned Media Spleen c e l l s at a concentration of 5 x 10^ c e l l s / m l were infected with MCMV i n v i t r o at m u l t i p l i c i t y of i n f e c t i o n (MOI) of 10 PFU/cell and incubated at 37 C i n RPMI 1640, containing 2% FBS, for 24-48 hours. The medium was removed, centrifuged at 900g x 10 minutes to remove the c e l l s and then centrifuged at 60,000g x 90 minutes to sediment any infectious v i r u s . The supernatant was taken and stored at 4 C u n t i l used. This material was termed conditioned medium. Control conditioned medium was produced i n an i d e n t i c a l manner except the spleen c e l l s were unin f e c t e d . Conditioned media from Sindbis v i r u s , T4, and PM2 i n f e c t e d spleen c e l l s were prepared i n an I d e n t i c a l manner, each using an MOI of 10 PFU/cell of the appropriate v i r u s ; LB8 conditioned medium employed l a t e x beads with a diameter of 0.8 um to mimic i n f e c t i o n at a dose of 10 l a t e x p a r t i c l e s / c e l l . 9 . Mitogen Stimulation Test F r e s h l y prepared spleen c e l l s were assayed f o r t h e i r response to the T c e l l mitogen, concanavalin A (Con A) i n the presence or absence of various substances. Spleen c e l l s at a concentration of 5 x 10^ c e l l s / m l were dispensed i n t o 96 w e l l Lindbro plates at a volume of 0.2 ml/well. Con A was added to a concentration of 2.0 ug/ml to wells which were used to measure mitogen s t i m u l a t i o n . Wells containing unstimulated c e l l s received no Con A. A f t e r incubation f o r 24 hours at 37 3 C, 2.5 uCi of methyl- H-thymidine were added to each w e l l . The plates were incubated f o r an a d d i t i o n a l 18-24 hours before the c e l l s were processed onto glass f i b e r f i l t e r s and washed four times with equal volumes of phosphate buffered s a l i n e (PBS) using a MASH II Harvester ( M i c r o b i o l o g i c a l A s s o c i a t e s ) . The f i l t e r d i s c s were a i r d r i e d and r a d i o a c t i v i t y was counted by l i q u i d s c i n t i l l a t i o n counting. The effects of various conditioned media, extracts , column fractions and sera on the stimulation of spleen cells was determined by i n i t i a l l y suspending the spleen c e l l s in media containing each substance. This was achieved by adjusting the concentration of spleen c e l l s to 5 x IO"7 cells/ml and then diluting the c e l l s to 5 x 10^ cells/ml with the substance to be tested. 10. U l t r a f i l t r a t i o n of Conditioned Media U l t r a f i l t r a t i o n of conditioned media was performed using an Amicon Stirred U l t r a f i l t r a t i o n C e l l , Model 12, with Diaflo membrane f i l t e r s YM2 and YMLO (molecular weight exclusion limits of 1000 and 10,000 daltons respectively). The assembled c e l l was s t e r i l i z e d by passing 70% ethanol through the f i l t e r , followed by a volume of s t e r i l e d i s t i l l e d water. U l t r a f i l t r a t i o n was performed by pipettig 10 mis of conditioned medium into the c e l l and f i l t e r i n g under ^ gas pressure u n t i l a small volume remained. The cycle was repeated u n t i l the desired amount of conditioned medium had been passed through the f i l t e r and only a small volume of l i q u i d remained inside the c e l l . The remaining residual l i q u i d contained a concentration of molecules excluded by the f i l t e r and was used as concentrated conditioned medium. The concentrated conditioned medium was removed from the c e l l , measured v o l u m e t r i c a l l y and adjusted to the desire d concentration by d i l u t i n g with RPMI 1640. The f i l t r a t i o n procedure was c a r r i e d out under conditions of up to 50 p s i pressure of ^  gas and with constant s t i r r i n g provided by a magnetic s t i r r e r i n s i d e the c e l l . U l t r a f i l t r a t i o n was performed at 4 C, and the f i l t r a t e s and residues produced were stored at 4 C u n t i l used. 11. E x t r a c t i o n of L i p i d s from Conditioned Media A modified Folch e x t r a c t i o n procedure was used to ex t r a c t l i p i d s from conditioned media (38). Ten mis of conditioned medium were shaken v i g o r o u s l y with 40 mis of a 2:1 chloroform:methanol mixture i n a separatory funnel and allowed to s e t t l e . The upper phase was drawn o f f and discarded. One quarter volume (8.2 mis) of 1:1 d i s t i l l e d water:methanol was added to the lower phase and again the mixture was shaken vi g o r o u s l y and allowed to s e t t l e . The lower phase was drawn o f f and reduced to dryness under r o t a r y evaporation using a Savant Speed Vac Concentrator. The p r e c i p i t a t e was resuspended i n 10 mis of RPMI 1640. This suspension was used as a source of e x t r a c t i n mitogen s t i m u l a t i o n assays. 12. Sephadex G-25 F r a c t i o n a t i o n of Conditioned Media Conditioned media were concentrated 10-fold by u l t r a f i l t r a t i o n through a YM2 membrane f i l t e r p r i o r to f r a c t i o n a t i o n ; serum was used neat. One ml samples of conditioned media (concentrated) or mouse serum were a p p l i e d to a 20 x 1.6 cm Sephadex G-25 column with a bed volume of 36.2 3 cm • A f t e r a p p l i c a t i o n , each sample was run i n t o the matrix bed and e l u t i o n was c a r r i e d out with a s a l i n e e l u t i o n b u f f e r a t a constant flow rate of 0.30 mis/minute. Three ml f r a c t i o n were c o l l e c t e d from the column. The f r a c t i o n a t i o n procedure was performed at 4 C. 13. Assay of Sephadex G-25 Fractions Fractions from the Sephadex G-25 column were assayed for t h e i r e f f e c t on Con A s t i m u l a t i o n of spleen c e l l s by r e s t o r i n g each f r a c t i o n to proper i o n i c strength and n u t r i t i o n a l status for c e l l growth and then using the r e s u l t a n t s o l u t i o n as a growth medium f o r spleen c e l l s i n a Con A s t i m u l a t i o n t e s t . Six u l of 8X RPMI 1640 were added to 84 u l of the sample f r a c t i o n and 10 u l of spleen c e l l s at a concentration of 5 x 10 7 c e l l s / m l i n RPMI 1640. This was used as the c e l l u l a r suspension which was dispensed i n t o Lindbro plates f o r Con A s t i m u l a t i o n t e s t s . 30 14. Proteinase K D i g e s t i o n One ml samples of conditioned media, concentrated 10X by YM2 u l t r a f i l t r a t i o n were digested with 1.0 mg of proteinase K f o r 30 minutes at 37 C. The samples were stored at 4 C u n t i l f r a c t i o n a t i o n could be performed. F r a c t i o n a t i o n by Sephadex G-25 chromatography was performed on the samples as described In s e c t i o n 12. 15. Polyacrylamide Gel Electrophoresis The Laemmli method of polyacrylamide g e l e l e c t r o p h o r e s i s was used f o r the a n a l y s i s of mouse serum conditioned media proteins (75). The gels were cast i n a 25 cm x 15 cm x 1.5 mm v e r t i c a l gel e l e c t r o p h o r e s i s apparatus (BioRad) and contained 0.375 M T r i s - H C l (pH 8.6) and 0.1% sodium dodecyl s u l f a t e (SDS) i n a l i n e a r gradient of 10/0.05 to 20/0.1.% acrylamide/N,N'-bis-methylene acrylamide ( B i s ) and 0-3.9% g l y c e r o l . A s t a c k i n g g e l , 3 cm i n height c o n s i s t i n g of 3.0/0.08% acrylamide/Bis, 0.125 M T r i s - H C l (pH 6.8) and 0.1% SDS, preceeded the main separation g e l . Polymerization of the gels was achieved by adding 0.001% tetramethylethylenediamime and 0.0001% ammonium per s u l f a t e to the g e l s o l u t i o n immediately p r i o r to the c a s t i n g of the g e l s . Samples of mouse serum and 10X concentrated conditioned media were d i l u t e d 50% i n el e c t r o p h o r e s i s sample b u f f e r and placed i n a b o i l i n g water bath f o r two minutes. Twenty-five u l samples were loaded i n t o the wells of the gel and ele c t r o p h o r e s i s was c a r r i e d out at a current of 15 mAmps u n t i l the bromophenol blue i n d i c a t o r dye from the sample b u f f e r reached vthe bottom of the g e l . Electrophoresis was performed using e l e c t r o p h o r e s i s running b u f f e r (see s e c t i o n 6 d.) i n the electrode chambers, and the system was water cooled. 16. S t a i n i n g of Gels The u l t r a s e n s i t i v e s i l v e r s t a i n method of Goldman et a l was used f o r the s t a i n i n g of the polyacylamide gels (45). The gels were soaked f o r 30 minutes i n a s o l u t i o n of 50% methanol and 12% a c e t i c a c i d to f i x the p r o t e i n s , and then soaked 18-24 hours i n 10% ethanol and 5% a c e t i c a c i d , u t i l i z i n g s e v e r a l changes of the s o l u t i o n during t h i s time period. The gels were then soaked f o r 5 minutes i n 0.0034 M potassium dichromate and 0032 N n i t r i c a c i d , washed four times f o r 30 seconds each i n deionized water and placed i n 0.012 M s i l v e r n i t r a t e f o r 30 minutes. This was followed by two r a p i d r i n s e s with 0.28 M sodium carbonate and 0.067% commercial f o r m a l i n . The gels were then allowed to develop i n a t h i r d portion of the sodium 32 carbonate-formalin solution, with gentle agitation u n t i l the development had reached the desired intensity. Development was stopped by placing the gels i n a 1% acetic acid solution. 17. Liquid S c i n t i l l a t i o n Counting of Radioactivity The glass fiber f i l t e r discs containing the air-dried samples from mitogen stimulation tests were placed i n s c i n t i l l a t i o n vials and 2 mis of s c i n t i l l a t i o n f l u i d were added. The vials were capped and counted for radioactivity 3 ( H) on channel one i n an Isocap/300 Liquid S c i n t i l l a t i o n System (Searle Analytic, Inc.). Liquid s c i n t i l l a t i o n f l u i d consisted of toluene containing either 4.2% (vol/vol) Spectroflour PPO-POPOP or 5.0% (vol/vol) Scintrex PP0-P0P0P. CHAPTER III  RESULTS I Evidence for the Presence of a Soluble Immunosuppressive Factor The i n i t i a l step in determining the mechanism whereby MCMV induces a state of immunosuppression on the infected host was to differentiate between a mechanism involving exclusively c e l l to c e l l contact and one involving an extracellular effector molecule. Some previous studies have suggested that i t may be the latt e r mechanism (9,10,83); thus, the p o s s i b i l i t y of a soluble immunosuppressive factor was chosen as the i n i t i a l area for investigation i n this study. I. Immunosuppressive Effect of Serum from Murine Cytomegalovirus  Infected Mice Serum collected from MCMV infected mice, four days after an intraperitoneal injection, and from uninfected mice were added i n varying concentrations to Con A stimulated spleen c e l l s . Previous studies have found serum from Infected mice to suppress the response to Con A, although the authors attribute this to the presence of free virus in the serum (9,10). In order to eliminate the poss i b i l i t y of suppression due to free 34 Figure 1: Effect of Normal and Infected Mouse Serum On Con A  Stimulation of Spleen Cells. A mitogen stimulation test was performed to evaluate the effect of various concentrations of normal mouse serum ( • )and MCMV infected mouse serum (—"O ) on the response of spleen c e l l s to stimulation by Con A. Serum from uninfected mice and from mice which had been given an intraperitoneal injection of 5 x 10^ PFU of MCMV 4 days earlier was added to spleen c e l l s at the time of addition of Con A. Values are expressed as percentages of the control which contained no mouse serum, and represent the mean of four samples ± standard deviations. Figure 1; E f f e c t of Normal and Infected Mouse Serum On Con A Stimulation of Spleen C e l l s 7o Mouse Serum LO Ln 36 v i r u s , the serum was centri f u g e d a t 60,000g x 90 minutes. No i n f e c t i o u s v i r u s could be detected by plaque assay on mouse embryo c e l l s a f t e r the u l t r a c e n t r i f u g a t i o n . The r e s u l t s of t h i s experiment are presented i n f i g u r e 1. Both i n f e c t e d and uninfected mouse serum suppressed the response of the spleen c e l l s to Con A, however the MCMV i n f e c t e d mouse serum showed a s i g n i f i c a n t l y greater degree of suppression when present at concentrations of 2% to 15% (Student's T t e s t , p < 0.05). The most marked suppression occured a t 5% and 10% mouse serum. I I . In V i t r o Production of A Virus Induced Suppressor Factor The presence of a suppressive substance i n the serum of i n f e c t e d mice i n d i c a t e d that the immunosuppression observed i n vi v o was probably due to a soluble suppressor f a c t o r . In v i t r o production of such a molecule would g r e a t l y f a c i l i t a t e studies concerning i t s production, source, p h y s i c a l nature and mode of a c t i o n , therefore an attempt was made to induce the production of the putative v i r u s induced suppressor f a c t o r (VISF) in_ v i t r o . Spleen c e l l s were chosen chosen as the c e l l s most l i k e l y to produce VISF in_ v i t r o s i n ce they contain a complete population of leukocytes and the spleen i s a major target of the MCMV i n f e c t i o n . Spleen c e l l s were i n f e c t e d with MCMV at an MOT of 10 P F U / c e l l . The medium was removed a f t e r 24-48 hours and the c e l l s and free v i r u s were removed by u l t r a c e n t r i f u g a t i o n . The supernatant was termed conditioned medium and was used as a source of VISF by t e s t i n g f o r suppressive a c t i v i t y i n a Con A st i m u l a t i o n t e s t . Control and MCMV conditioned mediun were compared with normal, stimulated splenocytes and MCMV Infected splenocytes i n f i g u r e 2. Although the c o n t r o l conditioned medium did not support s t i m u l a t i o n to the degree seen i n the RPMI 1640 only sample, i t i s s t i l l considerably greater than the response of the MCMV conditioned medium sample. MCMV conditioned medium appears to account f o r a l l the suppression observed i n the MCMV i n f e c t e d c e l l s . Table I i l l u s t r a t e s the r e l a t i o n s h i p between unstimulated and stimulated spleen c e l l s suspended i n conditioned media or i n f e c t e d with MCMV. A l l c e l l s used i n the experiment were obtained from a common pool and therefore any suppression seen i n the unstimulated c u l t u r e s must be due to the e f f e c t s of the medium (or v i r u s , i f present). Although the s t i m u l a t i o n i n d i c e s r e f l e c t the general pattern of s t i m u l a t i o n , the suppression seen i n the unstimulated samples masks the degree of suppression which i s present i n the 38 Figure 2: Suppression of Con A Stimulation of Spleen Cells by  Murine Cytomegalovirus and Murine Cytomegalovirus Conditioned Medium Spleen c e l l s were suspended i n RPMI 1640 or conditioned medium and subjected to a Con A stimulation test. MCMV infected spleen ce l l s were infected at an MOI of 10 PFU/cell. Values represent means from quadruplicate samples ± standard deviations. 39 Figure 2; Suppression of Con A Stimulation of Spleen Cells by  Murine Cytomegalovirus and Murine Cytomegalovirus Conditioned Medium 140 • -120 m-100 80 60 40 20 RPMI RPMI 1640 Control M C M V 1640 f M C M V Conditioned Conditioned Medium Medium TABLE I; E f f e c t of Conditioned Media on the Uptake of H-dThd by Unstimulated and Con A Stimulated Spleen C e l l s Medium CPM H-dThd Incorporated Stimulation Uninfected/ 2 3 Unstimulated Con A Index Infected RPMI 1640 20392 ±,2951 109240 ± 32936 5.36 _ RPMI 1640 + -MCMV MOI 10.0 5164 ± 586 6356 ± 1619 1.23 17.19 Control Conditioned 30069 ± 11318 56014 ± 11099 1.86 _ Medium MCMV Conditioned 9847 ± 5088 4895 ± 6 6 2 0.50 11.44 Medium 1. CPM H-dThd incorporated by unstimulated or Con A stimulated spleen c e l l s suspended i n the described medium. Values are means ± standard deviations from quadruplicate samples. 2. Stimulation Index i s the r a t i o of stimulated to unstimulated counts. 3. Ratio of Con A stimulated uninfected counts to in f e c t e d counts. 41 stimulated samples. Direct comparison of the stimulated samples, as seen i n the uninfected to infected ratios or by direct comparison of stimulated CPM (figure 2), provides a more accurate representation of the degree of suppression which i s actually taking place. Therefore, rather than stimulation indices, direct comparisons of the stimulated samples are used throughout this study. To further determine whither the suppressive a c t i v i t y was due to a suppressor factor (VISF) or simply residual undetected virus particles, the conditioned media was tested for suppressive a c t i v i t y at each stage of preparation. In table I I , control conditioned medium and RPMI 1640 are compared with the MCMV conditioned medium before and after the ultracentrifugation step. The ultracentrifuged MCMV conditioned medium gave a significantly greater response than the untreated MCMV conditioned medium (Student's T test, p < 0.025) yet both were sign i f i c a n t l y suppressed when compared with the control medium. Resuspension of the ultracentrifuge pellet produced a medium which was suppressive but not nearly as much so as the ultracentrifuged supernatant. Thus, i t appeared that the suppressive a c t i v i t y i s a soluble quality and cannot be removed by ultracentrifugation. TABLE I I : E f f e c t of the P u r i f i c a t i o n of Conditioned Media on the Suppression of Con A Stimulation of Spleen C e l l s Supernatant Treatment"*" CPM 3H-dThd 3 Incorporated % Suppression RPMI 1640 None 522,513 ± 24,287 0.00 Control CM Ult r a c e n t r i f u g e d 214,996 ± 83,598 58.80 MCMV CM None 19,198 ± 6,749 96.33 MCMV CM Ult r a c e n t r i f u g e d 44,535 ± 17,216 91.48 MCMV CM Resuspended UC P e l l e t 2 125,121 ± 27,987 76.05 1. U l t r a c e n t r i f u g a t i o n was at 60,000g X 90 minutes. 2. The p e l l e t from the u l t r a c e n t r i f u g a t i o n was resuspended i n an i d e n t i c a l volume of RPMI 1640. 3 3. CPM H-dThd incorporated i n t o Con A stimulated spleen c e l l s suspended i n the appropriate conditioned media. Values are means ± standard deviations from quadruplicate samples. 43 I I I . S p e c i f i c i t y of the Virus Induced Suppressor Factor The p r e v i o u s l y described experiments e s t a b l i s h e d the presence of a v i r u s induced suppressor f a c t o r (VISF), however the s p e c i f i c i t y of VISF was not determined. In f i g u r e 3 the degree of suppression from a v a r i e t y of conditioned media was examined. Spleen c e l l s were incubated with one of seve r a l v i r u s e s at an MOI of 10 PFU/cell or with l a t e x beads at a concentration of 10 l a t e x b e a d s / c e l l , and conditioned media were prepared. PM2 and T4 represent enveloped and non-enveloped p a r t i c l e s , both of which are non-infectious i n the eukaryote c e l l . Sindbis v i r u s i s an enveloped RNA v i r u s and, although i n f e c t i o u s f o r some mouse c e l l s ( i e . f i b r o b l a s t s ) , i t d i d not appear to invoke c y t o l y s i s of the leukocytes. LB8 l a t e x beads were s p h e r i c a l l a t e x p a r t i c l e s approximately 800 nm i n diameter. In f i g u r e 3 each type of conditioned medium was tested a t various d i l u t i o n s . A l l conditioned media supported a decreased response at 90%, when compared to the c o n t r o l medium, however, MCMV conditioned medium supported a much greater degree of suppression than the other four conditioned media. Also, the a c t i v i t y of the MCMV conditioned media was present when d i l u t e d to 20%; the non-MCMV conditioned media displayed no s i g n i f i c a n t suppression when d i l u t e d 44 to only 50%. I f a s i m i l a r suppressor f a c t o r Is present In the other four conditioned media, i t Is at very low concentrations r e l a t i v e to MCMV conditioned medium. IV. Factors A f f e c t i n g the Production of the Virus Induced  Suppressor Factor In order to f a c i l i t a t e the c h a r a c t e r i z a t i o n of VISF I t was necessary to determine the optimal conditions f o r production i n  v i t r o . Previous experiments had determined that a low MOI (< 3 PFU/cell) did not c o n s i s t e n t l y produce a s i g n i f i c a n t degree of Immunosuppression, and that the degree of immunosuppression was pro p o r t i o n a l to the MOI (82). The MOI of 10 PFU/cell was chosen because i t c o n s i s t e n t l y demonstrated considerable suppression and i t was possible to obtain v i r u s i n high enough t i t e r s to c a r r y out such an i n f e c t i o n . To determine whether VISF was produced over a long period of time or produced r a p i d l y over a short period of time, and i f so, at what time a f t e r i n o c u l a t i o n , c o n t r o l and MCMV conditioned media were c o l l e c t e d at various times a f t e r i n o c u l a t i o n . Figure 4 demonstrates the production of VISF over a period of s e v e r a l days. A f t e r 24 hours the MCMV conditioned medium did not demonstrate a furth e r decrease i n s t i m u l a t i o n , thus the production of VISF was completed wit h i n 24 hours a f t e r i n f e c t i o n . The decrease i n s t i m u l a t i o n 45 Figure 3: E f f e c t of Various Conditioned Media On Con A Stimulation  of Spleen C e l l s Conditioned media from uninfected, v i r u s i n f e c t e d and l a t e x bead ino c u l a t e d spleen c e l l s were compared with RPMI 1640 f o r t h e i r e f f e c t on spleen c e l l s t i m u l a t i o n . Conditioned medium was su b s t i t u t e d f o r RPMI 1640 to the percentage described before Con A was added to the samples . Values are expressed as a percent of the RPMI 1640 sample which was a r b i t r a r i l y termed the maximum response. Each value represents the mean from four samples; e r r o r bars were too small to be i n c l u d e d . Figure 3: Effect of Various Conditioned Media On Con A Stimulation of Spleen Cells R P M I Control M C M V S i n d b i s P M 2 T 4 L B 8 . 1640 ON 47 observed i n the c o n t r o l curve was probably due to depletion of nutr i e n t s and exc r e t i o n of metabolic byproducts i n t o the medium. I n t e r e s t i n g l y , the MCMV curve d i d not decrease a f t e r 24 hours although media d e p l e t i o n and rel e a s e of metabolic byproducts must have taken place. This may have been a r e s u l t of degradation of VISF, or VISF may have masked any e f f e c t s of such f a c t o r s present i n the medium. Many c e l l s require serum f o r in_ v i t r o c u l t i v a t i o n and f o r the production of c e l l products. To examine the serum requirements of spleen c e l l s f o r the production of VISF, MCMV i n f e c t e d and uninfected c o n t r o l c u l t u r e s were prepared using varying concentration of f e t a l bovine serum. The r e s u l t s of s t i m u l a t i o n te s t s using the conditioned mdia are presented i n fi g u r e 5. Quite c l e a r l y there i s no dependence of immunosuppressive a c t i v i t y upon FBS concentration i n e i t h e r the c o n t r o l or the MCMV samples. For the production of VISF, a serum concentration of 2% FBS was chosen f o r two reasons. F i r s t , a higher concentration would have made i t more d i f f i c u l t to i s o l a t e and p u r i f y the suppressor f a c t o r . Secondly, the v i r u s preparations used to i n f e c t the spleen c e l l s contained serum, therefore a choice of 0% FBS, or very low l e v e l s of serum would r e s u l t i n an undersirable d i f f e r e n c e between the two media . An incubation time of 24-48 hours was chosen because allowed s u f f i c i e n t time f o r the production of VISF but not s i g n i f i c a n t amounts of suppressive substances i n the c o n t r o l conditioned medium. 49 Figure 4: Kin e t i c s of the Production of the Virus Induced  Suppressor Factor Conditioned medium was assayed for suppressive (VISF) a c t i v i t y a f t e r various lengths of incubation. Control (•—•—) and MCMV (•—O^—) conditioned media were prepared a f t e r varying lengths of incubation time with the spleen c e l l s and assayed for a c t i v i t y by d i l u t i n g fresh spleen c e l l s at 5 x 10 7 c e l l / m l i n conditioned medium to y i e l d 5 x 10^ c e l l s / m l . Con A stimulation tests were then performed on these spleen suspension. Values represent means -standard deviations from quadruplicate samples. Figure 4: K i n e t i c s of Production of the Virus-Induced Suppressor Factor P 24 48 72 96 120 144 Ln O Time (hours) 51 Figure 5: E f f e c t of F e t a l Bovine Serum On the Production of Virus  Induced Suppressor Factor Conditioned media were prepared by incubating uninfected and MCMV i n f e c t e d spleen c e l l s i n RPMI 1640 supplemented with varying percentages of FBS. Production of VISF was measured by suppression of Con A s t i m u l a t i o n of fr e s h spleen c e l l s suspended i n uninfected ( — ) and MCMV i n f e c t e d (—•-O—«•) conditioned media. Values represent the means from quadruplicate samples ± standard d e v i a t i o n s . Figure 5: Effect of Fetal Bovine Serum on the Production of Virus-Induced  Suppressor Factor 2 4 6 8 10 % FBS CHAPTER IV  RESULTS I I Ch a r a c t e r i z a t i o n of the Virus Induced Suppressor Factor The previous chapter presented evidence of immunosuppressive a c t i v i t y present i n v i r u s - f r e e supernatants from MCMV i n f e c t e d mice. This e f f e c t was due to the presence of a putative immunosuppressive molecule termed VISF. The evidence presented demonstrates the a c t i v i t y but does not i n d i c a t e that i t i s due to a s i n g u l a r molecular s p e c i e s . This chapter i s devoted toward presenting evidence f o r the existence of a suppressor molecule and the c h a r a c t e r i z a t i o n of some of i t s p h y s i c a l p r o p e r t i e s . I E f f e c t of Indomethacin on the Production of the Virus Induced  Suppressor Factor Prostaglandins of the E s e r i e s are known to be immunosuppressive, Involved i n immune r e g u l a t i o n and produced by macrophages (42). In a d d i t i o n , a prostaglandin has been im p l i c a t e d as a mediator of dengue v i r u s induced immunosuppression (23), therefore the p o s s i b i l i t y of VISF being a prostaglandin was considered. To i n v e s t i g a t e t h i s p o s s i b i l i t y , conditioned media were 54 prepared from control and MCMV infected spleen c e l l cultures supplemented with indomethacin, an inhibitor of prostaglandin synthesis . As il l u s t r a t e d in figure 6, indomethacin f a i l e d to affect the production of suppressive a c t i v i t y , suggesting that prostaglandins play no role i n the observed immunosuppression unless the mechanism involved the release of preformed prostaglandins. To investigate the p o s s i b i l i t y of preformed prostaglandins being released, a total l i p i d extraction from MCMV and control conditioned media was performed using a modified Folch extraction technique (38). The extracted l i p i d s were resuspended in RPMI 1640 at a volume equivalent to the original volume. Table III demonstrates the effect of the resuspended l i p i d s on the stimulation of spleen ce l l s . No significant difference was observed at either of the two concentrations tested (Student's T test, p 0.05). When tested at a concentration of 80% of the original concentration, the control 3 suspension demonstrated a considerable decrease i n H-dThd incorporation relative to the control conditioned medium. This may have been due to chemical modification of some of the extracted substances to produce toxic or suppressive species. The corresponding MCMV sample gives a very similar value, a value which is s ignificantly greater than that of the MCMV conditioned medium indicating that either the suppressor factor was not extracted or i t s biological a c t i v i t y was destroyed i n the extraction procedure. 55 Figure 6: Effect of Indomethacin On the Production of Virus Induced  Suppressor Factor Conditioned media were prepared by incubating uninfected and MCMV Infected spleen c e l l s in RPMI 1640 containing 2% FBS, and with or without indomethacin. The production of VISF i n the conditioned medium was assayed by measuring suppression of Con A stimulation of fresh spleen c e l l s suspended i n the conditioned media. Values represent the means from quadruplicate samples ± standard deviations. Figure 6; E f f e c t of Indomethacin On the Production of the Virus-Induced Suppressor Factor Indomethacin (molarity) ' Ln ON 57 I I . Concentration of the Virus Induced Suppressor Factor by  Ultra f i l t r a t i o n A method of concentrating VISF was needed i n order to obtain sufficient quantities of the factor for fractionation studies . Attempts to do so were made by u l t r a f i l t r a t i o n through Amicon membrane f i l t e r s . If VISF was larger than the molecular exclusion limit of the f i l t e r then i t would be retained i n the residual volume and could be concentrated i n this manner. Since the suppressive effect of the MCMV conditioned medium was not due to a prostaglandin, the most l i k e l y alternative was a protein or peptide. If this was the case the molecule would be large enough to be concentrated by u l t r a f i l t r a t i o n . Conditioned medium was passed through membrane f i l t e r s with defined exclusion limits and the residual l i q u i d and f i l t r a t e were analysed for the presence of suppressive a c t i v i t y i n a Con A stimulation test. Using a membrane f i l t e r with a molecular exclusion l i m i t of 10,000 daltons (Amicon YMLO f i l t e r ) , no a c t i v i t y was retained by the f i l t e r (results not shown) , however when an Amicon YM2 f i l t e r (molecular exclusion l i m i t of 1000 daltons) was used the suppressor a c t i v i t y was retained by the f i l t e r and was not present i n the f i l t r a t e (see table IV) . The residual fraction was i n i t i a l l y a ten-fold concentrate of the retained molecules and was 58 TABLE I I I ; E f f e c t of L i p i d s Extracted from Conditioned Media  On the Stimulation of Spleen C e l l s by Con A Medium CPM H-dThd Incorporated Uninfected:Infected Control MCMV RPMI 1640 150,716 24,518 6.15 ±24,516 ±3,998 RPMI 1640 + 80% Extract 36,536 36,138 1.01 Suspension 1 ±5,584 ±5,672 RPMI 1640 + 20% Extract 114,743 96,641 1.19 Suspension 1 ±12,697 ±21,583 1. Extract suspension i s the resuspension of materials extracted from c o n t r o l and MCMV conditioned media by Folch e x t r a c t i o n . 3 2. CPM H-dThd incorporated i n t o Con A stimulated spleen c e l l s suspended i n the described medium. Values represent means ± standard deviations from quadruplicate samples. 3. Ratio of c o n t r o l CPM to MCMV CPM. 59 d i l u t e d to the concentration shown i n table IV. No s i g n i f i c a n t d i f f e r e n c e was observed between the MCMV n o n - f i l t e r e d and the MCMV residue IX medium (Student's T t e s t , p > 0.05), i n d i c a t i n g that a l l the VISF was recovered from the concentrated r e s i d u e . Figure 7 presents a more accurate estimation of the recovery of VISF from the 10X concentrated r e s i d u e . Concentrated residue from the u l t r a f i l t r a t i o n was d i l u t e d to f i n d the end point of suppressive a c t i v i t y , and thereby quantitate the amount of VISF present. The r e s u l t s demonstrate suppressive a c t i v i t y a t d i l u t i o n s as low as 0.4X the o r i g i n a l concentration, but at 0.2X no suppressor a c t i v i t y was detected. U n f i l t e r e d MCMV conditioned medium could be d i l u t e d to 0.2X with detectable suppressor a c t i v i t y (see f i g u r e 3 ) . The r e s u l t s of these experiments Indicated that VISF had a molecular weight between 1000 and 10,000 daltons and could be recovered with a high e f f i c i e n c y and concentrated by u l t r a f i l t r a t i o n . I I I . F r a c t i o n a t i o n of Conditioned Medium by Gel F i l t r a t i o n  Chromatography The r e s u l t s of the u l t r a f i l t r a t i o n provided an important step i n the i s o l a t i o n of VISF. Not only did they define a molecular weight range f o r the f a c t o r but u l t r a f i l t r a t i o n a l s o provided a means of concentrating VISF, allowing f r a c t i o n a t i o n studies by methods which 60 normally Involve a degree of dilution. One such method i s gel f i l t r a t i o n chromatography. The results of the u l t r a f i l t r a t i o n experiments suggested that the molecular weight of VISF was between 1000 and 10,000 daltons, therefore Sephadex G-25 was selected as a gel f i l t r a t i o n matrix. Samples of conditioned medium were concentrated 10X by u l t r a f i l t r a t i o n and were separated on a Sephadex G-25 column. Three ml fractions were collected and assayed for suppressor a c t i v i t y . A comparison of the fractions from control and MCMV conditioned media is presented i n figure 8. Fractions 4 and 13 are significantly lower in the MCMV curve; repeat experiments showed that fraction 4 was not consistently suppressive relative to the control fraction. Fraction 13 was consistently suppressive i n repeat experiments and therefore was considered to contain VISF. The location of i t s fractionation indicated a molecular weight less than 1400 daltons, thus agreeing with the molecular weight provided by the u l t r a f i l t r a t i o n data. TABLE IV: Effect of U l t r a f i l t r a t i o n Through a YM2 Membrane F i l t e r On the A b i l i t y of Conditioned Media to Suppress Con A Stimulation of Spleen C e l l s 6] Conditioned Medium F i l t r a t i o n Fraction Concentration F a c t o r 1 CPM H-dThd Incorporated^ Control MCMV F i l t r a t e 1.0 417,413 + 42,052 Residue 1.0 632,035 + 86y687 Residue 2.0 556,568 + 36,595 Residue 3.0 255,267 + 16,712 Residue 4.0 178,140 + 16,499 Non-filtered 1.0 10,537 + 7168 F i l t r a t e 1.0 154,734 + 22,852 Residue 1.0 12,099 + 4596 Residue 2.0 9,169 + 2798 Residue 3.0 5,831 + 3214 Residue 4.0 4,119 + 1665 1. Concentration factor relates the concentration of the contents of the f r a c t i o n r e l a t i v e to the u n f i l t e r e d conditioned medium. 3 2. CPM H-dThd incorporated into spleen c e l l s suspended i n each f i l t r a t i o n f r a c t i o n . Values represent means ± standard deviations from quadruplicate samples. 62 Figure 7; Effect of the Concentration of the Virus Induced  Suppressor Factor on the Stimulation of Spleen Cells Conditioned medium from uninfected ( ) and MCMV infected ("••••O ) spleen c e l l s was concentrated tenfold by u l t r a f i l t r a t i o n through on Amicon YM2 Diaflo f i l t e r . VISF was retained i n the residual volume. This concentrated medium was added to spleen c e l l s in RPMI 1640 to yield the appropriate concentration factor and Con A stimulation tests were then performed to assay the effect of VISF, at different concentrations, on stimulation of the spleen c e l l s . The concentration factor was the ratio of the YM2 retained molecules to their concentration in unfiltered conditioned medium. Values represent means from quadruplicate samples ± standard deviations. Figure 7; Effect of the Concentration of Virus-Induced Suppressor Factor  On Con A Stimulation of Spleen Cells 0.2 04 0.6 0.8 1.0 1.2 1.6 2.0 Concentration Factor 64 IV. F r a c t i o n a t i o n of Mouse Serum by Gel F i l t r a t i o n Chromatography To compare the in_ v i t r o produced VISF to the suppressor f a c t o r produced i n vivo and found i n the serum of i n f e c t e d mice, samples of serum from MCMV i n f e c t e d and uninfected mice were f r a c t i o n a t e d on the Sephadex G-25 column. Fractions were c o l l e c t e d and assayed f o r immunosuppressive a c t i v i t y . The r e s u l t s presented i n fi g u r e 9 i l l u s t r a t e a complex pattern with many f r a c t i o n s from both sera containing suppressive a c t i v i t y . Of p a r t i c u l a r i n t e r e s t are f r a c t i o n s 8-12 of the uninfected mouse serum and f r a c t i o n s 6-13 of the MCMV i n f e c t e d mouse serum. Fractions 6,7 and 13 demonstrate s i g n i f i c a n t l y greater suppression i n the i n f e c t e d mouse serum curve (Student's T t e s t , p < 0.0005). These f r a c t i o n s may represent a d d i t i o n a l suppressive substances i n the i n f e c t e d mouse serum, or they may represent the t a i l s of a d i s t r i b u t i o n curve of a substance present i n higher quantity i n the i n f e c t e d mouse serum. Although i t was not possible to define the f r a c t i o n responsible f o r the di f f e r e n c e i n immunosuppressive c a p a c i t i e s of the two unfractionated sera (see fi g u r e 1 ) , the data i n f i g u r e 9 are not in c o n s i s t e n t with the l o c a t i o n of VISF i n fi g u r e 8. 65 Figure 8; Sephadex G-25 Fractionation of Control and MCMV  Conditioned Media Conditioned media from uninfected ( — ) and MCMV infected ( o •) spleen c e l l s was concentrated 10X by YM2 u l t r a f i l t r a t i o n . One ml samples were loaded onto a 20 x 1.6 cm Sephadex G-25 column and eluted with a saline buffer at a rate of 0.3 ml/minute. Three ml fractions were collected and assayed i n quadruplicate for suppression of Con A stimulation of spleen c e l l s • Values represent the means from quadruplicate samples ± standard deviations. The small arrows mark the positions of two molecular weight standards, aprotinin (MW 6300), and angiotensin (MW 1400). The large arrow marks the position of VISF. Figure 8: Sephadex G-25 Fractionation of Control and Murine Cytomegalovirus Conditioned Medium 100 Fraction Number 67 Figure 9: Sephadex G-25 Fractionation of Normal and Infected Mouse  Serum Serum was col lected from normal, uninfected mice (—•—•) and from mice infected with 5 x 10 6 PFU of MCMV, at 4 days post infect ion ( O •) • One ml samples were loaded onto a 20 x 1.6 cm Sephadex G-25 column and eluted with a saline buffer at a rate of 0.3 ml/minute. Three ml samples were col lected and assayed for suppression of Con A stimulation of spleen c e l l s . Values represent the means from quadruplicate samples ± standard deviations. Figure 9: Sephadex G-25 Fractionation of Normal and Infected Mouse Serum • N M S 2 4 6 8 10 12 14 16 18 Fraction Number 69 V. F r a c t i o n a t i o n of Sindbis Virus and LB8 Conditioned Media by Gel F i l t r a t i o n Chromatography Two a d d i t i o n a l samples were f r a c t i o n a t e d on the G-25 column to again assess the s p e c i f i c i t y of VISF to MCMV i n f e c t i o n s . The f r a c t i o n a t i o n and assay procedures were i d e n t i c a l to those used i n the preceeding two sections . Conditioned media were prepared from spleen c e l l s i n f e c t e d with Sindbis v i r u s or LB8 l a t e x beads. The conditioned media were concentrated 10X by u l t r a f i l t r a t i o n and fr a c t i o n a t e d on the Sephadex G-25 column. No low molecular weight, suppressive f r a c t i o n s were observed i n e i t h e r curve and values f o r f r a c t i o n 13 were approximately the same as the c o n t r o l ( f i g u r e 10). These data suggested that the immunosuppressive a c t i v i t i e s of these conditioned media ( f i g u r e 3) were not due to the same molecular species which was found i n f r a c t i o n 13 of the MCMV conditioned medium. VI. Proteinase K Digestion of the Virus Induced Suppressor Factor The m a j o r i t y of soluble immunoregulatory molecules are proteins or peptides, although other cl a s s e s of immunoregulatory molecules have been described (15,28,42). The molecular weight range i n d i c a t e d was cons i s t e n t with i t being a peptide. To determine whether or not t h i s was the case, s u s c e p t i b i l i t y to degradation by 70 the proteolytic enzyme, proteinase K was examined. When control and MCMV conditioned media were digested with proteinase K, the resulting solution supported no stimulation of lymphocytes by Con A, therefore the effect of the enzyme on VISF could be determined. To overcome this problem the digested conditioned medium was fractionated on the Sephadex G-25 column and the fractions were assayed for suppressive a c t i v i t y ; the results are presented in figure 11. The proteinase K digestion resulted i n a number of strongly suppressive fraction (fractions 2-7) possibly due to the production of smaller suppressive fragments from the digestion of some of the larger molecules present i n the conditioned medium. Fraction 13, the fraction shown to contain VISF (figure 8), displayed an increased support for Con A stimulation of spleen cells , presumably due to proteolytic degradation of VISF. Table V presents the numerical values for the undigested and proteinase K digested fractions 13. No significant difference was observed between the proteinase K treated and untreated controls , however the digested MCMV fraction was significantly greater (Student's T test, p < 0.005) than the undigested fraction suggested that VISF i s l a b i l e to proteinase K digestion and i s therefore a peptide. 71 Figure 10: Sephadex G-25 Fractionation of Sindbis Virus and LB8  Conditioned Msdia Conditioned media from Sindbis virus ( — A — ) and LB8 latex bead ( 'A ) treated spleen c e l l s were concentrated tenfold by u l t r a f i l t r a t i o n . One ml samples were loaded onto a 20 x 1.6 cm Sephadex G-25 column and eluted with a saline buffer at 0.3 ml/minute. Three ml samples were collected and assayed for suppression of Con A stimulation of spleen c e l l s . The large arrow indicates the fraction i n which VISF elutes; the small arrow indicates values of this fraction from uninfected ( • ) and MCMV infected ( O ) conditioned media. Values represent the means from quadruplicate samples ± standard deviations. Figure 10: Sephadex G-25 Fractionation of Sindbis and LB8 Conditioned Media 73 Figure 11: Sephadex G-25 Fractionation of Proteinase K Digested  Control and MCMV Conditioned Medium Conditioned media from uninfected (•—-•—) and MCMV infected ( -O ) spleen c e l l s were concentrated tenfold by u l t r a f i l t r a t i o n . One ml samples were digested with 1 mg of proteinase K for 30 minutes at 37 C. The samples were then loaded onto a 20 x 1.6 cm Sephadex G-25 column and eluted with a saline buffer at 0.3 ml/minute. Three ml fractions were'collected and assayed for suppression of Con A stimulation of spleen c e l l s . The large arrow indicated the value from the fractionation of undigested MCMV conditioned medium. Values represent the means from quadruplicate samples ± standard deviations. Figure 11; Sephadex G-25 Fractionation of Proteinase K Digested Control and Murine Cytomegalovirus Conditioned Medium Fraction Number TABLE V: Effect of Proteinase K On the Fractionation of VISF by Sephadex G-25 Chromatography Conditioned Medium Treatment CPM H-dThd Incorporated Control None 282,493 ± 17,937 Control Pro K 259,625 ± 37,467 MCMV None 56,493 ± 23,160 MCMV Pro K 134,311 ± 38,707 1. Conditioned medium was concentrated 10X and fractionated on a Sephadex G-25 column. 2. Samples treated with Proteinase K were digested p r i o r to fra c t i o n a t i o n . 3 3. Values represent CPM H-dThd incorporated into spleen c e l l s i n a Con A stimulation t e s t . C e l l s were suspended i n Fraction 13 from each e l u t i o n . Values are means ± standard deviations from quadruplicate samples. VII. Electrophoresis of Mouse Serum and Conditioned Medium Proteins and peptides are resolvable by electrophoresis when present i n sufficient quantity to allow detection. Therefore an attempt was made to resolve VISF into a detectable band by electrophoresis• Samples of normal and infected mouse serum, and MCMV and control conditioned media were dissolved i n an SDS sample buffer and aliquots of each sample were applied to an SDS-polyacrylamide gel. Following electrophoresis of the samples, the gels were stained by an ultrasensitive s i l v e r stain capable of detecting proteins at 3 concentrations as low as 0.01 ng/mm . In neither the serum nor the conditioned media samples was a protein band, specific for the MCMV infected sample, observed i n the appropriate molecular weight range to be considered as candidate for VISF. A possible explanation for this i s very low concentrations of VISF in both the serum and conditioned medium. CHAPTER V DISCUSSION Many viruses have been implicated as immune modulators, most commonly as suppressors of the immune system (116). In most cases the extent of suppression has been characterized by testing the response of infected animals or leukocytes to a series of immune challenges. In contrast, very few data have accumulated to present a mechanism for the observed immunosuppression. As a result, this aspect of the pathogenesis of the v i r a l infection i s poorly understood. The object of the present study was to investigate the mechanism whereby MCMV can induce a state of immunosuppression i n infected mice. The results presented i n the preceeding two chapters indicated the presence of a soluble immunosuppressive factor, termed VISF, which i s released during MCMV infection of spleen c e l l s . Previous studies have presented some evidence to suggest the presence of a soluble immunosuppressive factor. Booss and Wheelock presented evidence that serum from infected mice was immunosuppressive but were unable to conclude whether or not this was due to free virus in the serum (9,10). Loh and Hudson presented evidence for immunosuppression i n spleens with as few as 100 infectious centers per spleen (82) . They also showed a correlation between infectious centers and the degree of immunosuppression. The low number of infectious centers indicated that a c e l l to c e l l contact event was probably not the mechanism of immunosuppression; an alternative explanation was the release of a lymphokine or monokine-like factor. The results in chapter III demonstrate that a immunosuppressive factor i s present i n both the serum from infected mice as well as supernatants (conditioned media) from i n vi t r o cultures of infected spleen c e l l s , both of which were free of infectious virus. The effect of infected mouse serum on Con A stimulation of spleen c e l l s , shown i n figure 1, demonstrates a suppressed response when 2-10% mouse serum was added to the cultures• Higher or lower percentages of mouse serum resulted i n values which were not significantly different from those using normal mouse serum. Suppression by high concentrations of normal mouse serum was presumably due to immunoregulatory factors such as normal immunosuppressive protein, immunoregulatory alpha globulin, C reactive protein and alpha fetoprotein (15). The inablibty of Booss and Wheelock to show consistent suppression due to IMS may have been due to low levels of suppressive a c t i v i t y i n the serum or use of too low or too high a concentration of mouse serum for optimal results. Assessment of suppressive a c t i v i t y from i n vitro cultures of infected spleen c e l l s proved to be much less complicated than the studies on infected mouse serum. MCMV conditioned medium showed 79 s i g n i f i c a n t suppression r e l a t i v e to the control conditioned medium at concentrations of 20% or greater and evidence was presented that the observed immunosuppression was not due to free virus i n either the serum or the conditioned medium (figures 1,2; table I I ) . Two parameters of the production of VISF which were examined were the e f f e c t of the concentration of f e t a l bovine serum on production of VISF, and the time a f t e r i n f e c t i o n at which VISF was produced (figures 4,5). Although f e t a l bovine serum content had no effe c t on the production of VISF i n v i t r o , 2% FBS was chosen for the standard production of VISF i n order to keep the serum content low, but also to prevent the serum present i n the virus inoculum from making a q u a l i t a t i v e or large quantitative difference between the MCMV and c o n t r o l conditioned media. Figure 4 indicates that VISF i s produced w i t h i n 24 hours of i n f e c t i o n . This r e l a t i v e l y short period of production suggests that the mechanism does not involve c e l l to c e l l Interactions to trigge r the production of VISF. Although changes i n the r a t i o of T helper c e l l s : T suppressor c e l l s have been associated with virus induced immunosuppression, including HCMV mononucleosis (3,14,108,128), the short time period required for the production of VISF would not allow time for the necessary interactions and d i f f e r e n t i a t i o n to take place for changes i n c e l l r a t i o s . I t i s therefore concluded that the production of VISF i s not related to changes i n T c e l l r a t i o s , although such changes may occur i n vivo concurrent with or as r e s u l t of the production of VISF. 80 Previous studies have implicated the macrophage c e l l as playing a major role i n harbouring latent virus and being permissive for MCMV replication (11,81). These studies have shown that the macrophage i s principally responsible for the replication of MCMV in spleen c e l l populations and most of the input virus i s taken up by macrophages during infection. This information, coupled with the time of production of VISF suggests that VISF i s a product of the macrophage which i s released a short time after the uptake of the virus by the macrophage• Many immunoregulatory factors have been reported, and macrophage i s responsible for the production of many of them (28,132). In addition, Loh and Hudson have demonstrated that the adherent population of spleen c e l l s mediate MCMV induced immunosuppressi on of Con A and LPS stimulation of spleen c e l l s (82); Bixler and Booss have reported a similar finding using hemolytic plaque assays to test immune function (8). Although-many viruses may induce immunosuppression (116), this observation i s not universal for a l l viruses nor i s i t a result of simple phagocytosis of particles, v i r a l or otherwise, as i s demonstrated by the results of figure 3. The production of VISF appears to be specific for MCMV, although i t i s possible that certain other viruses may also induce i t s production. Figure 8 demonstrated that VISF was in fact a molecular entity by isolating i t i n a single fraction from the Sephadex G-25 column. 81 Similar fractions from Sindbis virus and LB8 la t e x bead conditioned media showed no degree of immunosuppression, thus reemphasizing the s p e c i f i c i t y of VISF production for MCMV (figure 10). I t i s possible however, that the small degree of immunosuppression observed i n the non-MCMV samples i n figure 3 was due to low quantities of VISF which were not detected i n figure 10. Wainberg and I s r a e l have reported the production of a soluble immunosuppressive factor from lymph node c e l l s infected with any of a va r i e t y of viruses (64,133). Their factor was apparently a product of the macrophages but d i f f e r e d from VISF by the n o n s p e c i f i c i t y of the virus needed to stimulate i t s production. Although many viruses induce immunosuppression, the mechanisms indicated for d i f f e r e n t virus-host systems are va r i e d . Included among the mechanisms which have been reported are alt e r a t i o n s i n T c e l l r a t i o s (3,108,111), production of prostaglandins (21), production of a nonspecific suppressor factor (64,133), i n h i b i t i o n of helper factor production (13) and c e l l associated suppressor mechanisms (18). I t may w e l l be that immunosuppression i s simply a common outcome of a va r i e t y of dif f e r e n t virus-host i n t e r a c t i o n s . The position of VISF on the Sephadex G-25 fr a c t i o n a t i o n (figure 8), and the retention of VISF during u l t r a f i l t r a t i o n define a size range of 1000-1400 daltons for VISF. Additional information to the physical nature of VISF was i t s l a b i l i t y to the proteolytic enzyme, proteinase K, suggesting that the molecule i s , at least i n part, a 82 peptide. Whether or not i t contains other moieties i s yet unknown. This description does not correspond to any factor produced i n other systems of virus induced immunosuppression with the exception of the factor described by Wainberg and I s r a e l (64,133), on which no information i s available on the physical or chemical nature of the molecule, and therefore the two cannot be compared. Numerous immunosuppressive factors have been described from systems not inv o l v i n g v i r u s e s , some of which are comparable to VISF. Cultures of activated macrophages have been reported to secrete a d i a l i z a b l e suppressor factor which was not a prostaglandin, c o r t i c o s t e r o i d , cAMP or thymidine (72,79,134,139). These cultures could be activated with a v a r i e t y of substances including t h i o g l y c o l l a t e and Corynebacterium parvum. Studies of MCMV i n f e c t i o n of macrophages have indicated that the virus activates macrophages and this i s an important step i n c o n t r o l l i n g the spread and r e p l i c a t i o n of the viru s (91,113). Several alternatives to a macrophage produced factor e x i s t including the low molecular weight peptide which was found i n T c e l l lysates and i n h i b i t e d mitogenesis (98), the spleen derived immunosuppressive peptide found i n bovine spleen homogenates (34,80), thymic hormones (4,5) and immunoregulatory alpha globulins (93,128). Because of the incomplete characterization of these molecules and VISF, i t i s not possible compare or contrast them further. 83 The in vitro studies have demonstrated the presence of a low molecular weight peptide produced i n low quantities by MCMV infected spleen c e l l s . This peptide, VISF, inhibits the incorporation of 3 H-dThd into unstimulated and Con A stimulated spleen c e l l s (figure 2, table I ) . Con A stimulation of spleen c e l l s was chosen as a test for immune competence throughout these studies, although i t is not necessarily indicative of many other immunological functions. The Con A stimulation test was chosen for i t s consistency and rapidity. Many previous studies on MCMV induced immunosuppression have also used the Con A stimulation test (9,10,82,83,115) although other studies have u t i l i z e d a variety of other immune challenges (8,47,55-57,68,102). Since v i r t u a l l y a l l immune functions are impaired, It was assumed that one mechanism was responsible, rather than different mechanisms for different functions. Suppression of unstimulated spleen c e l l s by MCMV conditioned medium (table I) supports the one mechanism theory by il l u s t r a t i n g that the suppressive a c t i v i t y of VISF i s not restricted to Con A stimulation, however complete biological characterization of the a c t i v i t y of VISF should be carried out after the complete purification of VISF has been achieved. The studies presented i n figures 1 and 9 attempt to demonstrate the relevancy of the i n vitro studies with the i n vivo situation. Figure 1 demonstrates the presence of a soluble suppressor factor i n the serum of infected mice. Attempts to isolate this factor in.a 84 pa r t i c u l a r Sephadex G-25 f r a c t i o n were complicated by the presence of normal suppressive substances present i n the serum of both infected and uninfected mice (figure 9). Fraction 13 of the infected mouse serum was suppressive r e l a t i v e to the normal mouse serum f r a c t i o n and therefore i s consistent with the position of VISF i n f r a c t i o n 13 i n figure 8. The presence of other suppressive fractions i n figure 9 complicate the p i c t u r e , making i t impossible to implicate the contents of f r a c t i o n 13 as being responsible for the suppressive capacity of the infected mouse serum seen i n figure 1. Further studies are necessary to determine whether or not VISF i s present i n the infected mouse serum. One of the goals of th i s study was to use the MCMV system as a model to provide i n s i g h t i n t o the pathogenesis of HCMV i n f e c t i o n . I t i s d i f f i c u l t to rel a t e the findings of th i s study to the observed suppression of immune responses i n HCMV infected humans because l i t t l e work has been done on the mechanism of suppression i n HCMV i n f e c t i o n s , except HCMV mononucleosis (14,111). 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