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Expression of the cell adhesion molecule, L-selectin, on polymorphonuclear leukocytes during and after… Van Eeden, Stephanus Frederick 1994

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EXPRESSION OF THE CELL ADHESION MOLECULE, L-SELECTIN, ON POLYMORPHONUCLEAR LEUKOCYTES DURING AND AFTER THEIR RELEASE FROM THE BONE MARROW  by STEPHANUS FREDERICK VAN EEDEN  MB.ChB. The University of Stellenbosch, South Africa, 1975 M.Med. The University of Stellenbosch, South Africa, 1984 FCP. The College of Physicians, South Africa, 1984 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES (Department of Experimental Medicine)  We accept this thesis as conforming to the r  uired standard  THE UMVERSfl* OF BRItISH COLUMBIA  August 1994 ©Stephanus Frederick van Eeden, 1994  _ ___ ___ ___  ts for an advanced partial fulfilment of the requiremen Library shall make it British Columbia, I agree that the ission for extensive and study. I further agree that perm by the head of my scholarly purposes may be granted ood that copying or her representatives, It is underst t my written gain shall not be allowed withou cial finan for s thesi this of publication permission.  In presenting this thesis in degree at the University of freely available for reference copying of this thesis for department or by his or  Department of  4!,S4j%A’  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  e  U  ABSTRACT The emigration requires  of polymorphonuclear  leukocytes  a series of leukocyte-endothelial  sequential  steps.  These include,  interactions  tethering  activation  of leukocytes;  leukocytes  out of the vessels into inflamed  firm adhesion  molecules are involved in the tethering expressed on nearly all leukocytes sites of inflammation  (PMN) to sites of inflammation which can be divided in four  of flowing  leukocytes;  to the endothelium tissue.  of leukocytes  including  and;  PMN. The recruitment  into the blood is less clear.  the expression  marrow into the circulation Immunocytochemical  expression  of  of the PMN to  is highly dependent on the expression of this molecule on their  bone marrow  respectively.  emigration  and one of these, L-selectin, is  However, the role of L-selectin in the trafficking  the expression  or  The selectin family of adhesion  cell surfaces.  determine  triggering  The objective  of the PMN from the of this thesis  was to  of L-selectin on PMN during their release from the bone and their eventual  and immunohistochemical  removal into the tissues. techniques  were used to determine  of L-selectin on the PMN in cytological and histological Indirect immunofluorescent  of L-selectin on circulating  specimens  flow cytometry was used to determine PMN.  Changes  the  in L-selectin expression  on  PMN during their release from the bone marrow was studied during cardiopulmonary bypass in humans.  These studies demonstrated  the mature segmented  that the expression of L-selectin on  PMN in bone marrow is higher than on circulating  that some of this L-selectin shed when PMN cross from the bone marrow  PMN and  m hematopoietic  compartment  into the bone marrow venous sinusoids.  However, L  selectin expression on PMN in the venous sinusoids remains high and this results in  an increased expression of L-selectin on circulating release.  This means that circulating  PMN during active bone marrow  PMN expressing the highest levels of L-selectin,  have been recently released from the bone marrow. A new method for labelling PMN in vivo  using  demonstrate  the  thymidine  that circulating  analogue,  5’bromo-2-deoxyuridine  PMN continuously  was  used  to  lose L-selectin while they remain in  the circulation. These studies explain the variable expression of L-selectin on the circulating with the newly released PMN expressing levels of L-selectin.  the highest and the older PMN the lowest  This implies that the expression of L-selectin on circulating  is not just a marker  of cell activation  opportunity  to study  the functional  populations  of the circulating  PMN.  PMN  state, but also of cell age. capabilities  PMN  This creates the  and the trafficking  of different  lv  TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  vii  List of Abbreviations  X  Acknowledgements  xiii  Dedication  xiv  1) General Introduction a) Distribution  1  b) Distribution  of PMN in the bone marrow. of PMN in the vascular space.  c) Distribution  of PMN in the tissue.  2) Bone marrow  of PMN (review) relationship a) The structural-functional marrow-blood barrier.  3 7 10  release  12 of the bone  13 b) Developmental changes and leukocyte egress from the bone marrow. 17 19 c) Factors that cause release of PMN from the bone marrow. 20 d) Adhesion events and egress of PMN from the bone marrow. 27  3) Selectins L-selectin  28  1) Structure of the L-selectin protein 2) Expression of L-selectin on leukocytes 3) Regulation of L-selectin expression  28  4) Ligands for L-selectin  35  5) Functions of L-selectin  36  4) L-selectin  expression  Introduction Working hypothesis Specific Aims  during active bone marrow  32 33  release  of PMN  39 42 42  V  Model of bone marrow  5) Changes  release  of PMN  43  Methods  45  Results  62  Discussion  82  in L-selectin  on circulating  PMN  Introduction  94  Hypothesis  97  Specific Aims  97  Model of in vivo labelling  101  Results  111  Discussion  122  expression  on PMN during their lifespan  in the circulation  7) References  98  Methods  L-selectin  6) Summary  of PMN  127  Methods  130  Results  139  Discussion  147  and future directions  154 159  vi LIST OF TABLES Table I.  The  selectin  group  of cell adhesion  molecules:  Nomenclature,  expression and function (p-29). Table 11.  Clinical characteristics  Table III.  Changes  in the peripheral  cardiopulmonary Table IV.  counts  in patients  Changes in the L-selectin expression on circulating  Changes patients  Table VI.  leukocyte  bypass patients  during  PMN in patients  bypass (p-69).  in L-selectin expression during cardiopulmonary  Comparison  (p-63).  bypass (p-64).  during cardiopulmonary Table V.  of cardiopulmonary  on PMN in the bone marrow in bypass (p-Ti).  of the number of white cells, PMN and BrdU labelled  PMN recovered in three different preparations  of blood from rabbits  labelled in vivo with BrdU (p-i 13). Table VII.  Half-lives  of BrdU labelled  PMN infused  as either  whole blood,  leukocyte rich plasma or purified PMN (p-i 17). Table Vifi.  Half-lives  of BrdU  Chymotrypsin  labelled  PMN  infused into recipient  treated (p-146).  with  and  without  vu LIST OF FIGURES Figure 1: The stages of maturation and flux of polymorphonuclear bone marrow, circulating blood and tissue compartments (p-5). Figure 2: Schematic  presentation  leukocytes in the  of the bone marrow-blood barrier (p-l5).  Figure 3: Domain compositions of the three known selectins  (p-31).  Figure 4: Immunocytochemical grading system used to evaluate staining of PMN in the circulation (p-55). Figure 5: The disector method used to quantify marrow compartment (p-57).  the intensity  of  the number of PMN in each bone  FIgure 6: Photomicrograph of a bone marrow sinusoid demonstrated with a Toluidine Blue 0 stain and the corresponding serial section stained for the presence of L selectin (p-58). Figure 7: Variability specimens (p-66).  in grading the expression of L-selectin on PMN in cytological  Figure 8: The correlation between the visual and Infrascan® grading systems (p-67). Figure 9: The difference between L-selectin expression on PMN in the circulating blood (cytospins) and the bone marrow (bone marrow smears) (p-68). Figure 10: Immunocytochemical determined L-selectin expression on circulating PMN at the start and the end of 5 normothermic and 5 hypothermic cardiopulmonary bypass procedures (p-7O). Figure 11: The expression of L-selectin on segmented bone marrow and the circulating blood (p-72).  PMN and band cells in the  Figure 12: Expression of L-selectin on PMN during bone marrow release of PMN in a normothermic and hypothermic patient as measured by flow cytometry (p-73). Figure 13: Expression of L-selectin on circulating PMN as measured by flow cytometry at the different time intervals in 5 normothermic and 5 hypothermic CPB procedures (p-74). Figure 14: Immunohistochemical determination of L-selectin on PMN in bone marrow hematopoietic and sinusoid compartments at baseline. Combined baseline data for both normothermic and hypothermic groups (p-76).  vu’ Figure 15: Changes in the number of L-selectin negative graded PMN in the bone marrow sinusoids at the start (BM1) and at the end (BM2) of CPB bypass (p). 78 Figure 16: Flow cytometric data concerning the expression of L-selectin and CD18 on PMN incubated at 37, 27 and 4°C in vitro and stimulated with increasing concentrations of zymosan activated plasma ranging from 0 to 5 % (p9). 7 Figure 17: Immunocytochemical changes in the L-selectin negative graded PMN incubated at 37, 27 and 4°C stimulated with increasing concentrations of zymosan activated plasma (p-81). Figure 18: Time course of BrdU incorporation into circulating infused with 25 mg/kg/day of BrdU for 7 days (p-112).  PMN donor rabbits  Figure 19: Immunocyto- histochemical detection of BrdU labelled cells in the circulating blood, spleen and pneumonic lung of recipient rabbits (p-i 14). Figure 20: BrdU labelled PMN in recipient’s circulation as a fraction of the number of BrdU labelled PMN infused after the infusion of whole blood, LRP or purified PMN (p-li5). Figure 21: Semi-quantitation of BrdU labelled DNA in recipient organs 24 h after the transfusion of whole blood and LRP (p-i 19). Figure 22: Immunological detection of BrdU labelled in DNA extracted from organs of one of the recipient rabbits after the transfusion of whole blood from a donor rabbit (p- 120). Figure 23: Immunocytochemical detection of BrdU labelled PMN in cytospin prepared from LRP of the donor blood. Double immunolabelling of PMN on cytospins for both nuclear BrdU (blue) and surface L-selectin (red) using a double alkaline phosphatase technique (p-i37). Figure 24: L-selectin changes on PMN during time spent in the circulation BrdU labelled PMN were infused as whole blood or purified PMN (p-i40).  when  Figure 25: The effect of chymotrypsin on the expression of L-selectin and CD18 as measured with flow cytometry (p-141).  Figure 26: BrdU labelled PMN in the circulation of recipients after the infusion of purified PMN treated with either denatured chymotrypsin or chymotrypsin (p-i43). Figure 27: BrdU labelled L-selectin positive and negative PMN in the circulation of recipients after the infusion of purified PMN treated with either denatured chymotrypsin or chymotrypsin (p-i44).  Ix  Figure 28: Changes lifespan (p-i55)  in the expression  of L-selectin on PMN during their normal  x LIST OF ABBREVIATIONS Microgram(s) il 1  Microliter(s)  60.3  Anti-CD18 MoAb  ACCR  Adventitial  APAAP  Alkaline phosphate  ARDS  Adult Respiratory  BM  Bone marrow  BSA  Bovine serum albumin  BrdU  5 ‘bromo-2-deoxyuridine  CD  Cluster of differentiation  CR  Complement  CAM  Cell adhesion molecule  CPB  Cardiopulmonary  DREG-200  Anti-L-selectin  ECM  Extracellular  EGF  Epidermal  E-selectin  Endothelial-selectin  g  Gram  G0-G4  Grades 0-4 (see grading system figure 4)  G-CSF  Granulocyte  colony stimulating  GM-CSF  Granulocyte  monocyte colony stimulating  cell covering rate anti-alkaline  phosphate  Distress Syndrome  regulatory  designation domain  bypass MoAb  matrix growth factor domain  factor factor  xi GMA  Glycolmethacrylate  h  Hour(s)  ICAM  Intercellular  kg  Kilogram  LPS  Lipopolysaccharide  LRP  Leukocyte rich plasma  L-selectin  Leukocyte-selectin  MFI  Mean fluorescence  MoAb  Monoclonal antibodies  mg  milligram(s)  mm  minute(s)  P-selectin  Platelet-selectin  PBS  Phosphate  PMN  Polymorphonuclear  RGD  Arginine-glycine-aspartic  s  second(s)  SD  Standard  deviation  SE  Standard  error of the mean  sLex  Sialylated  Lewis x antigen  sL-selectin  Soluble L-selectin  TBS  Tris buffered saline  TBO  Toluidine Blue  adhesion molecule  intensity  (also on endothelial  cells)  buffered saline leukocyte acid attachment  peptide  xli  VCAM  Vascular  ZAP  Zymosan activated  cell adhesion molecule plasma  xffl ACKNOWLEDGEMENTS I wish to acknowledge  my supervisor, Dr James Hogg, for supporting  to do a Ph.D. in the Pulmonary leukocyte kinetics.  me in my quest  Research Laboratory and stimulating  I am fortunate  to have trained  a academic mentor and friend of outstanding  my interest in  under James and I consider him  value. Special thanks  are extended to  my committee members Dr’s Keith Walley, Graham Dougherty and Donna Hogge for their patience  and constructive  criticism.  I also want to thank  Jenny  Hards for  sharing with me her time and knowledge on immunocyto- and histochemistry. always  be grateful  for her kindness  and friendship.  To Dr’s Alan Burns,  Doerschuk and Blaire Walker I owe a special debt of gratitude discussion on adhesion molecules. The animal experiments  I will Claire  for many fruitful  were greatly facilitated  by the surgical expertise of Dean English and I am in debt to Simon Bicknell for his help in the animal  theatre  experiments.  Dr Robert Miyagashima  staff deserves a special acknowledgement  and all the operating  for their willingness  and support  in collecting the bone marrow specimens. I would like to acknowledge the assistance of Dr Lawrence Haley and the technical  staff in Immunology  for helping  with the  flow cytometry. I would also like to acknowledge the help of the technical staff of the PRL, Harvey analysis),  Joe Comeau  (photography). encouraging financial  Coxson (stereology), (computers)  and  and Barry  the always  Wiggs (statistical  available  Steward  Greene  Finally, I am grateful to Prof’s James Joubert and Attie de Kock for me to pursue a academic career and I would like to acknowledge  support  I received from the Medical Research  Faculty of Medicine University University  Lorri Verburgt  of British  of Stellenbosch,  Council of South Africa,  Dancun Flockhard,  Columbia and B.C. Lung Association.  the  fellowship from  xiv DEDICATION To my wife, Christelle,  and my family for their continuous  this work a joy to complete.  support that has made  1 1) GENERAL  INTRODUCTION  The control of the number of circulating controversy  for almost a century.  leukocytes has been a topic of interest and  This is especially  true for polymorphonuclear  leukocytes (PMN) because they are implicated in the pathogenesis several diseases (Cochrane et aL, 1965; Hammerschmidt Cochrane,  1965; Phelps and McCarthy,  Neutrophilic  leukocytosis  inflammatory  diseases  is a feature  of tissue injury in  et al., 1980a; Kniker and  1966; Stetson,  1951;  Winn et aL, 1973).  common to many types of infectious  and serves to control the expansion  and  and dissemination  of  microbial or viral infection by killing or removing the pathogenic organisms. In other clinical settings  such as in adult respiratory  al., 1979; Hammerschmidt 1982; Tate and Repine, ischemia-reperfusion  et at., 1980a;  emphysema  of tissue  granulocyte  aggregation  instrumental  in the pathophysiology  a number  and  of activated  of interlocking  fibrinolysis  cascade,  metabolism  (Rinaldo,  kinin  failure,  (Phelps and McCarthy, 1966) and (Janoff, 1983), PMN are thought to  damage.  For example,  intravascular  cell  complement-induced  activation  are  of ARDS (Tate and Repine,  considered  1983b; Rinaldo,  PMN in the lung (Weiland et al., 1986) perpetuates  inflammatory system,  pathways  complement  1986; Hammerschmidt  Activated PMN damage the pulmonary  including pathways,  4) by the generation  the coagulation  and  and arachidonic  acid  et al., 198Gb; Tracey et al., 1988).  endothelium:  1) directly; 2) by the release of  proteases (elastase and collagenase); 3) by the simultaneous and fibrinolysis;  1981; Meguire et al.,  in multi-organ  resulting  tissue injury, gouty arthritis  to the extent  1986). Entrapment  Heflin and Brigham,  1983a), sepsis syndrome  chronic tissue injury in pulmonary contribute  distress syndrome (ARDS) (Brigham et  of arachidonic  activation  acid metabolites;  of coagulation 5) and by the  2 release of oxygen-free radicals (Brigham,  1977; Brigham and Owen, 1975; Cochrane  et al., 1983; Rinaldo, 1986; Tate and Repine, 1983b). Evidence for PMN participating in the development  of ARDS is substantial  (Tate and Repine,  depletion  has been shown to prevent  activators  in a variety of animal models (Heffin and Brigham,  has been described in neutropenic that  develop in these patients  1983b), and PMN  ARDS-like injury after the injection of PMN  patients,  1981). Although ARDS  PMN reconstitution  (Rinaldo and Borovetz,  1985).  worsens the ARDS  pathology in tobacco smoke related lung disease is located in the peripheral (Hogg et al., 1968) and is thought  to be due to an inflammatory  PMN (Wright et aL, 1988; Bosken et al., 1992) initiated (Neiwoehner et al., 1974). Baseline circulating  the  Furthermore,  airways  process involving  by the cigarette  smoke  leukocyte counts have been shown to  be elevated in chronic smokers (Corre et al., 1971) and these PMN passing through the pulmonary  capillary bed are exposed to components of cigarette smoke that delay  (Bosken et al., 1992) and activate  them (Klute et al., 1993) which could release  proteases and reactive oxygen intermediates  that damage the pulmonary endothelium  from within the vascular space. Regulation  of the circulating  important maintaining  in mounting  a response  other inflammatory  The total number  to bacterial  infections  PMN levels is therefore and in controlling  or  states.  of PMN in the body can be broken down into those in the bone  marrow, the circulating,  the marginated  and tissue pools. The number of PMN that  circulate can be influenced by several factors including: the rate of production in the marrow; the rate of release from the marrow; the exchange between the circulating and marginated  pool of intravascular  PMN; and their rate of permanent  removal  3 from the circulation stressful  into the tissues.  The PMN that enter the circulation  stimuli come from either a pool of cells marginated  during  along the vessel walls  within the vascular space and/or from the bone marrow (Doerschuk and Allard, 1989; Muir et aL, 1984). Mobilization of PMN from the marginated increase the total number of PMN in the vascular system.  pool does not effectively Alternatively,  the release  of PMN from the bone marrow increases the total number of PMN in the circulating and marginated  poois and therefore in cells that may participate  in the inflammatory  response.  a) Distribution  of PMN in the bone marrow  Polymorphonuclear hematopoietic  leukocytes  are produced  factory weighing approximately  in the bone marrow  which  is a  2600g, or 4.5 % of the body weight of  a normal adult. The bone marrow is widely distributed  throughout  the skeleton and  as a single organ is larger than the liver (which weighs ± 1500g). About 55 %-60 % of the bone marrow is dedicated to the production of a single cell type, the neutrophil, with a normal ratio of neutrophils contribute  to erythrocytes  of 2:1 to 3:1. Other granulocytes  about 3 %-5 % of the bone marrow. The cellularity  varies with age and accounts  of the bone marrow  for 75 % of the marrow in the young, 50 % in young  adults and as low as 25 % in the elderly (Hartsock et al., 1965). There is, however, a great variability  between individuals  of differentiation  in the bone marrow have been distinguished:  compartment  of pluripotent  of any given age. Three major compartments  stem cells; 2) committed cells and; 3) maturing  normal adults, the life of PMN is spent in three environmentsblood and tissue.  1) the most primitive  Bone marrow is the site of proliferation,  cells. In  the bone marrow,  terminal  differentiation  4 and maturation  of neutrophilic  granulocytes.  Proliferation  consists of approximately  5 divisions and only takes place during the first three stages of PMN development (myeloblast, promyelocyte and myelocyte stages). After the myelocyte stage the cells become “end cells” that are no longer capable of dividing (Bainton et aL, 1971). They then enter a large storage pool where they mature to segmented  PMN for ±5 days  before being released into the blood (Bainton et at., 1971; Bainton, illustrates  the PMN lifespan and stages of maturation  1980; Wintrobe, myeloblasts,  1981). Of every 100 nucleated  5 % promyelocytes,  and 20% mature segmented  1980). Figure 1  (Bainton et aL, 1971; Bainton,  cells in the bone marrow, 2% are  12 % myelocytes, 20% metamyelocytes  and band cells,  PMN, yielding a total of ±60% developing  PMN.  The length of time that PMN spend in each stage and in various compartments obtained mainly by radioisotope-labelling et aL, 1964). available,  When the radioisotope  techniques diisopropyl  Cartwright  et al., 1964; McAfee and Thakur,  labels all the granulocyte mature granulocytes.  fluorophosphate  32 became [ PJDF  isotope Cartwright  by the bone  (Athens et al., 1961; Athens et at., 1959; 1976). This isotope given systemically,  series of cells and labelling  cells ex vivo labels only the  This difference has been used to estimate the rate of generation  of PMN in the bone marrow and their lifespan  in the circulation.  et at (1964) show that the circulation  with a myelocyte generation to label cells engaged determine  (Athens et al., 1959; Cartwright  it provided a tool to estimate both the rate of PMN generation  marrow and their lifespan in the circulation  was  half-life of PMN is ±7 hours  time of ±3 days. Utilizing tritiated  in DNA synthesis  By using this  thymidine  in vivo, it was shown that  TDR) 3 (H one could  the time from the last division in the myelocyte stage in the bone marrow  5  Figure 1: The lifespan and stages of maturation of polymorphonuclear leukocytes. The times for the various compartments were obtained by isotope-labell ing technique. The ordinate shows the flux of PMN through each compartment and the abscissa shows the time spend in each compartment. Note the stepwise increas e in cell numbers in the compartments where cell division took place. No mitose s occur after the myelocyte stage. The size of the tissue pooi of PMN is unknown.  6 to the appearance  of labelled PMN in the peripheral  blood (Bryant and Kelly, 1958;  Fliender et al., 1964; Maloney, and Patt, 1968). Some similarities  exist in the pattern  of release of PMN for different species; however, there are temporal differences. The emergence  time for segmented  hours in humans  PMN into the peripheral  blood ranges from 96-144  (Fliender et aL, 1964), 102±13.8 hours in dogs (Maloney and Patt,  1968) and as short as 24 hours in mice (Bryant and Kelly, 1958). These emergence times (myelocyte-to-blood could be explained  transit time) may vary considerably  by either a longer maturation  between patients  and  time from the myelocyte to the  segmented PMN or a delay in the release of mature cells from the bone marrow. The work of Cronkite et al (1960) and Maloney and Weber (1963) suggest that there is a random release of cells into the blood from the marrow segmented contrast  to first-in  first-out  kinetics.  Shorter  emergence  times  infections  suggest  immature,  compatible with the “shift to the left” or cytoplasmic  that segmented  seen on differential  counts  released into the circulation unknown.  PMN in these conditions  (Fliender  et al., 1964). Whether  leukocytes  with  “toxic granulation” these  younger  properties  spend only a small proportion  lifespan in the vascular space and circulating replace the circulating  in patients  are younger or more  have different kinetic and functional  Polymorphonuclear  pool of PMN in  cells is still  of their  PMN turn over ±2.5 times per day to  pool. To accomplish this the bone marrow produces ±850 x 106  cells/kg/day in humans. These cells come from the postmitotic granulocyte  pool in the  bone marrow (5.6 x iO cells/kg) which is the largest pool of PMN and is ± 10 times the size of the intravascular  pool (Finch et al., 1977). Inflammation  increase the rate of PMN production time, decrease the time mature  from the precursors,  shorten  and stress  the maturation  PMN reside in the marrow, and stimulate  mature  7 and immature  PMN to enter the circulation (Cronkite, 1988). The myelocyte-to-blood  transit time may be as short as 48 hours during infection (Fliedner et aL, 1964b). The turnover of PMN may increase from 100 billion per day at baseline to over a trillion per day during a serious infection (Walker, and Willemze, 1980; Malech, 1988).  b) Distribution  of PMN in the vascular  space  The fact that PMN can leave the circulation yet remain in the vascular space so that they can be rapidly mobilized into the circulation  in times of stress was recognized  more than a century ago by Virchow (1856), Cohnheim (1867) and Limbeck (Limbeck, 1889). These early observations distribution leukocytes  of leukocytes  set the stage for a systematic  in the vascular  can assume a marginal  the low blood velocity relationship  system.  Cohnheim  in the peripheral  compared  to the central  demonstrated  to The for  in some vascular regions and to be dislodged from others. The  (Andrewes,  that the redistribution  pool in the lung with the intravenous typhoid  vessels.  provides the mechanism  importance of the lung as the major site of PMN margination Bruce and Andrewes  that  established  position in the venules, which was attributed  between blood velocity and margination  leukocytes accumulate  of the  investigation  19 lOb; Andrewes,  was first recognized by  1910a;  Bruce,  of PMN from the circulation  1894). They  to the marginated  injection of peptone (Bruce, 1894), tubercie and  bacilli as well as Escherichia  coli (Andrewes,  191Gb; Andrewes,  1910a).  Goldscheider and Jacobs (1894) reached similar conclusions at about the same time concerning  the importance  of the lung as a site where PMN could withdraw  marginal position. In the 1950’s, successful catheterization to establish  that PMN present in the vascular  to a  of the great vessels helped  system could marginate  along the  8 vessel walls in the lung with various stimuli that increase or decrease the number of cells marginating several  investigators  physiological the  the pulmonary  within  established  that  feature in the pulmonary  Valsalva  and  Mueller  margination  respectively  demarginate  leukocytes  vasculature.  sequestration circulation.  manoeuvres  pulmonary  leukocytosis  sequestration  or decrease  such as  leukocyte infusion  blood flow are an important  factor  of leukocytes. The concept that catecholamines  by decreasing  cells was suggested  the adhesive forces between PMN and the  by Ahlborg et a! (1970) in a study showing that the  of exercise can be blocked by the B-blocker, propanolol. However, this  B-blockers and emphasized  challenged  by Foster et a! (1986) who showed no effect of  the importance  lung. A study by Thommasen  of bloodflow on PMN margination  et a! (1984) of the pulmonary  of PMN in dogs showed that PMN retention concept of a close relationship  between  arterio-venous  is flow-dependent,  in the  difference  which supports the  blood flow and PMN margination  in the  circulation.  The lung has unique anatomical determining  movements,  from the lung (Ahlborg and Ahlborg, 1970; Bierman et al.,  finding was subsequently  pulmonary  increase  is a normal  (Bierman et al., 1952a). Exercise and epinephrine  may influence margination endothelial  of leukocytes  Respiratory  either  1952b) which suggests that changes in pulmonary influencing  Using these techniques,  and physiological  features  that are important  whether PMN will move through the microvasculature  in  or be retarded.  In the larger conducting vessels, the PMN travel in the centre of the flowing stream and have few opportunities alveolar  to interact with the endothelial  surface. However, in the  wall the PMN make close contact with the endothelial  cells because the  9 mean diameter capillary  of the PMN (mean diameter  segments  (mean diameter  7-8 microns)  is larger  than  5-6 microns) (Schmid-Schonbein  Weibel, 1963). The PMN must pass through  many  et aL, 1980;  an average of 100 capillary  segments  from the arteriole to the venule and have been shown to deform as they pass through the capillary endothelial  vessels. This results  in intimate  contact between  the PMN and the  membrane during their journey through the lung and in the PMN having  a much longer transit  time than the red blood cells. This anatomical  between PMN and capillary  diameter  was illustrated  found that  in dogs, 80 % of the PMN are removed  pulmonary  capillaries  in the first pass through  discrepancy  by Martin et al (1982) who from the circulation  the lung.  Similar  by the  findings  reported by Muir et al (1984). They injected labelled PMN and erythrocytes  were  (RBC) in  upright awake humans, and found that ±20% of the labelled PMN failed to negotiate the pulmonary  vascular bed in the same amount of time as the erythrocytes.  The transit time for PMN is longer than RBC and longer in the upper regions of the lung (Hogg et aL, 1985; Wagner et al., 1982). If the capillary pathways  are the same  (Staub and Schultz, 1968), the longer transit time in the flow independent  lung areas  means that the velocity of blood is slower in the upper regions of the lung, supporting the concept that forces tending to propel the PMN and those retarding shifted by changing  blood velocity. This results  in greater  retention  turnover of PMN in the upper regions of the lung. This retention exposure of PMN to airborne activating in the typical distribution 1957).  stimuli,  of smoke related  and a slower  may cause a longer  such as cigarette  lung emphysema  them can be  smoke, resulting  (Leopold and Gough,  10 Weibel et al (1963) has estimated  that there are ±296 x 106 alveoli and 277 x iO  capillary segments in the human lung; therefore there are ±1000 capillary segments per alveolus.  This capillary  without interfering  network allows large numbers  with the majority of available pathways  the pressure drop across the pulmonary  microvascular  of PMN to marginate and with little effect on  bed. The pulmonary  capillary  bed can accommodate  large numbers  times the circulating  PMN pool (Doerschuk et al., 1987a; Staub et al., 1982).  When radioisotopes used extensively  of cells and calculations  became available  to advance  with modern imaging  the understanding  range from twice to 6  techniques,  of both the production  they were and the  intravascular  behaviour of PMN. Most of the studies using isotopes have confirmed  the findings  of many  older studies  concerning  the distribution  circulation  inthat  circulation  results in delay in the lung with gradual accumulation  injecting  labelled  of PMN in the  PMN into the venous or arterial  in the liver and  spleen over time (Martin et al., 1982; Muir et al., 1984; Saverymuttu The influence of the ex vivo labelling  techniques  PMN was recognized as an important  factor in the discrepancies  studies (Haslett  et aL, 1985; Saverymuttu  c) Distribution  of PMN in the tissue  The final tissue destination Some PMN may migrate  et al., 1983).  of the reinjected between different  et al., 1983).  of most PMN in the normal host has not been resolved. to areas of low-grade inflammation,  crevasses  around  important  in host defense mechanisms  the teeth, upper respiratory  that the gastrointestinal  on the behaviour  site of the  such as the gingival  tract and bladder,  where they are  (Van Dyk et aL, 1985). Earlier studies suggest  tract is the most important  site for PMN egress in normal  11 individuals  (Murphy, 1976), but this finding has been questioned  seen in this tissue (Jamuar  in the absence  and Cronkite,  of infection  or other inflammatory  1980). The reticuloendothelial  et aL, 1987a; Doerschuk  people do not undergo  and Allard,  PMN from the circulation  1989), however  major change in the number  studies have proposed that permanent  processes  tissue in the spleen and the  liver may play a key role in the removal of senescent (Doerschuk  as few PMN are  of circulating  splenectomized PMN. Several  removal of PMN from the intravascular  space  into the tissues is a random process (Carper, 1966; Fliender et al., 1964; Raab et al., 1964), and that PMN never return to the circulation the signal for permanent  again. Like the site of removal,  removal of PMN from the circulation  is unknown.  The size  of the tissue pool of PMN depends on the time they survive in this pooi before they are processed. The fate of PMN after they have migrated unclear but they are reported to be capable of surviving 1980).  into the tissues  is still  for several days (Bainton,  12  2) BONE MARROW RELEASE The continuous production  supply of PMN to the circulating  (hematopoiesis)  (hematoegression). granulocytes  OF PMN:  and their subsequent  Under  normal  blood depends on their rate of release into the marrow sinuses  circumstances  only differentiated  cross the sinus wall to gain access to the circulation.  which cells gain access to the circulation only mature  postmitotic  The process by  is selective in several ways; for example  cells that are at the end of the production line and prepared  functional  role, egress from the bone marrow.  immature  white blood cells in situations  1979), the administration 1979; Spertini  Moreover, an increased appearance  such as infections  et al., 1991b) implies unique or separate  mechanisms  production and release (Broxmeyer et al., 1974). The mechanisms release of PMN from the bone marrow are poorly understood. of immature  of mature  into the vascular  leukocytes  1988); the development  deformabiity  of transient  the  Several factors probably  include: the anatomical  into the  localization  of the  holes or pores in the venous sinuses  to allow  changes in the nucleus and  PMN such as increased motility (Lichtman and Weed, 1972a),  (Lichtman,  into  that control  cells adjacent to the vascular space (Lichtman and Erslev,  that facilitate translocation compartment  for neutrophil  space and out of the circulation  egress of marrow cells (Weis, 1983); and developmental cytoplasm of maturing  (Cronkite,  cells in the bone marrow and the selective egress  tissues. Those that have been suggested developing hematopoietic  of  (Boggs, 1967; Cronkite,  of growth factors (Platzer, 1989) or corticosteroids  regulate the retention  for their  the  1970), chemotactic of terminally efferent  ability (Giordano and Lichtman,  differentiated  vascular  space.  1973),  PMN from the hematopoietic Releasing  factors  such  as  13 glucocorticoid  and  (Kampschmith,  androgenic  steroids  1984), components  Muller-Eberhard,  1979),  (Deinard  of the complement  GM-CSF  and  G-CSF  implicated  in the initiation  mechanism  of action is still largely unclear.  a) The structural  and  of PMN release  and functional  Page, system  (Platzer,  1974),  endotoxins  (Ghebrehiwet,  1989) have  also been  from the bone marrow  relationship  of the bone  and  but their  marrow-blood  barrier The major supply of blood to the bone marrow is derived from the nutrient penetrating  periosteal  arteries.  bone gives off radial arteries,  The ascending  becomes fine arterioles  open into the bone marrow venous sinusoids. surface and are fed primarily through the medullary  and descending  central artery in the  and ends in capillaries  from intracortical  These branching  capillaries.  These vessels course  and eventually  drain into a  vascular sinuses (1O-3Om in diameter)  may coalesce and form larger collecting sinuses (50-75 jAm) before entering central sinus. The sinusoids are surrounded the hematopoietic  tissue  located  venous sinuses. The hematopoietic  Cells egress  in cordlike  anastomosing  bands between  these  activity of the marrow takes place in the centre begin to move towards the sinusoids  and enter the postmitotic pool (Weiss and Chen, 1975).  from the hematopoietic  general circulation  into the  by fibroblastic stroma (Lewis, 1982) with  of these cords and the cells that are generated after they have differentiated  that  These sinusoids begin at the endosteal  cavity of the bone, anastomose,  large central venous sinus.  artery and  compartment  of the bone marrow  into the  by crossing the sinus wall.  The sinus wall is a trilaminar  structure  (figure 2) consisting of a continuous  layer of  14 endothellal  cells, a discontinuous  adventitial fenestrae  reticulum  basement  membrane  and abluminal  cells (Weiss, 1970). The endothelial  that are spanned  by a diaphragm  covering  cells have several types of  that is thought  to open transiently  to  allow cells to move into the sinusoids (Muto, 1976). This suggests that the egressing cells pass through the endothelial Shaldai  cell and supports the observations  (1979) who noted the absence of tight junctions in bone marrow endothelial  sinuses. The fenestrae by regulating (Apkarian  are thought  to be dynamic structures  factors such as phorbol esters  and Curtis,  IV collagen  is discontinuous  fenestrations  (Campbell,  cover the abluminal  granules containing  membrane  and completely  matrix  containing lacking  mainly laminin and type at the site of endothelial  (ECM), collagen  cells are fibroblastic  cells that  in nature  type I and Ill, reticular  cell is also rich in microtubules  to retract their extensions and change the percentage (called the adventitial  and  fibres and  (Weiss, 1976) that allow them covering of the endothelial  cell cover rate or ACCR). The normal  (Weiss, 1970) and altered in situations  cells  ACCR is 60 %-65 %  of increased marrow cell egress such as after  (Weiss, 1970), post-phlebotomy  (Tavassoli,  1977) or in myeloid  (Leonardi, and Manthos, 1989; Nagaoka et al., 1986). Egressing blood cells  cannot pass through the adventitial (pericelluar passage). Although regulating  cytoskeleton  material resembling the ECM (Bentley and Foidart, 1980; Weiss,  1976). The adventitial  endotoxin treatment  et al., 1987), and ACTH  1972; Muto, 1976; Weiss, 1970). The adventitial  surface of the endothelial  produce extracelluar  (Lombardi  that can be modulated  1986) and may be controlled by the endothelial  (Steffan et aL, 1987). The basement  leukemias  of Tavassoli and  cells but have to move between or around them  the functional  significance  cell egress is still unclear it has been suggested  of the adventitial  cells in  that these cells regulate  “5  Bone Marrow Blood-barrier Adventitial Cell  Mature PMN  I BasaJL 0  r  Fenestra  Endotheijal Cell  Figure 2: Discontinuous bone marrow-blood barrier. Mature PMN egressing from the hematopoietic compartment into the bone marrow venous sinusoids have to pass through three layers (adventitial cell layer, basal lamina, and endothelial cell).  16 the selectivity In addition  of the bone marrow-blood barrier (Petrides and Dittmann,  to the stromal  marrow contains  and accessory cells, the microenvironment  soluble molecules and extracelluar  of collagen type I and ifi (made by fibroblasts), (Reilly et al., 1985), glycoaminoglycans, fibronectin  (Bentley  hematopoietic hematopoietic  egressing neutrophils  for the proliferation  plasticity  (Lichtman,  1981), and  (i.e. that it behaves  mechanically  and glycoproteins between  displaced  cells) such as  the ECM and the  and differentiation  of the  little is known about the interactions  and the ECM and stromal cells. Some have speculated  ECM serves as an anchor reducing marrow  type IV (made by endothelial  proteoglycans  cells (Owen, 1988). Currently  of the bone  matrix. The ECM is composed  et aL, 1981). The interactions  cells are crucial  1990).  the discharge  of immature  that it provides a barrier as a reversible  by the cell having  deformable the capacity  of  that the  cells from the bone  with a high degree of meshwork  that can be  to egress).  Membrane  associated proteins on white cells and their ligands on the ECM could be responsible for these interactions  (see below). Anatomical  pathways for cell movement across the  bone marrow-blood barrier may be responsible for a constant  “leak” of hematopoietic  elements into the sinusoids under stable condition. However there is a growing body of evidence favoring the hypothesis  that the egress of cells from the bone marrow is  an active process rather than a passive leakage of cells through (Petrides and Dittmann, hematopoietic  pores  1990). This active process probably involves changes in the  cells and their microenvironment  to the ECM during differentiation crawl into the sinusoids.  endothelial  that allow immature  cells to attach  and mature cells to detach from the mathx and  17 b) Developmental  changes  and leukocyte  The production of granulocytes factors. Colony-forming and differentiate  fibroblasts  locally  involves a variety of different hematopoietic  units for granulocytes  colony stimulating  and monocytes (CFU-GM) proliferate  T-lymphocytes  course of 5 to 7 days they become committed  stimulating  factor (M-CSF) for monocytes. Hematopoietic  connective  tissue)  stimulating  factor (G-CSF) for neutrophils  a pivotal  role in this  for granulocytes.  colony stimulating  activity itself but, when administrated  on hematopoietic  interleukins,  and  colony  factors either  cells. IL-i, for example, in vivo, universally  has no induces  cells such as fibroblasts  (Lee et al, 1987)  cells (Bagby et a!, 1986). IL-6, secreted by bone marrow macrophages,  and  hematopoietic hematopoietic  of growth  which results from the induction of G-CSF and GM-CSF  expression by other accessory or auxiliary and endothelial  lineages,  stroma (stromal cells and  These cells secrete  or indirectly  leukocytosis,  Over the  or monocyte  factors and monokines that serve as growth and signaling  a neutrophilic  cells,  and monocyte colony  local production  by acting directly  fibroblasts  neutrophil  of growth factors specific for their individual  colony stimulating  factors  and monocytes.  are  these cells are largely regulated  granulocyte  differentiation  by endothelial  to either  At this point in their differentiation  play  growth factors, notably  microenvironment  by circulating  by the local concentration  growth  factor (GM-CSF) and IL-3, which  in the bone marrow  and augmented  differentiation.  from the bone marrow  under the influence of specific hematopoietic  granulocyte-monocyte produced  egress  endothelial cells  but  cells, has functions  no direct  synergistically  growth factors in promoting  1988). This clearly illustrates  effect on proliferating with  hematopoiesis  the the dynamic interactive  many  direct  human acting  (Ogawa et al, Blood Cells relationship  between the  18 bone marrow stromal cells, connective tissue and hematopoietic of  myelopoiesis,  metamyelocyte, divisions  from  the  myeloblast  through  band forms and segmented  cells. The final phase  promyelocyte,  neutrophils  accompany  myelocyte, another  4 to 8  and occur over 7 to 10 days. The growth factors and interleukins  induce proliferation the functional  and differentiation  of granulocyte  activity of their terminally  that  precursor cells often enhance  differentiated  progeny (Clark and Kamen,  1987, Gasson et al, 1984, Mayer et al, 1987), that may include promoting egress from the bone marrow. These hematopoietic the  of maturing  cytoplasm  translocation  growth factors induce developmental  of terminally  cells  which  differentiated  play  changes in the nucleus and  a pivotal  role in allowing  cells from the hematopoietic  the  compartment  to the efferent vascular space. Marked increase in mobility (Giordano and Lichtman, 1973; Lichtman chemotactic  and Weed, 1972b) deformability  ability (Giordano and Lichtman,  the granulocytes.  (Lichtman  1973) occur during the maturation  Active motility is not a feature of immature  the ability of mature  granulocytes  and Weed, 1972a) and  hematopoietic  cells and  to probe by pseudopods probably contributes  their ability to search out the sinus wall and penetrate  cells of different  ability to be filtered through and to be aspirated Lichtman  stages of maturation  micropore membranes  into glass microcapillary  has been examined (Lichtman  of  by their  and Kearney,  tubes (Lichtman  and Weed, 1972b). Mature granulocytes  to  it (Giordano and Lichtman,  1973). The ability of cells to deform relates to their ability to move. Deformabiity hematopoietic  of  1976)  and Weed, 1972a;  can migrate through filters with  1m pores. The biophysical character of maturing  cells is important in marrow egress  because most cells undergo marked deformation  during migration  through pores in  19 the sinus wall. Like motility, rather than immature  chemotaxis  of granulocytes  cells (Giordano and Lichtman,  is a feature  1973). Chemotaxis  of mature is important  because it may underlie the ability of mature marrow cells to accelerate their release in response contribute  to periods of increased to allowing  may  barrier.  The  to make the pores that develop in the endothelial  cell  the PMN to cross the bone marrow-blood  cells are thought  cytoplasm  (Giordano and Lichtman, of endothelial  1973). These holes frequently  occur adjacent to  cells with the majority of cellular traffic migrating  endothelial  cells.  c) Factors  that cause release  Cell releasing  All these abilities  combined  migrating  junctions  demands.  of PMN from the bone marrow  factors could affect the rate of egress of PMN from the bone marrow  in several ways. Firstly, they may act as specific chemoattractants movement of differentiated  the adventitial  structures  cell cover rate  to reduce the impediment  bone marrow  of leukocytes  release  with  (Weiss, 1970). Changes in the  to bone marrow structures  of leukocytes  to egress by  (Weiss, 1970) which demonstrated  endotoxin induced bone marrow release of leukocytes adhesion properties  that accelerate  cells into the marrow sinusoids. Secondly, they could act  on the sinus wall and stromal reducing  through  by factors that induce  may provide new insights  into this complex  problem. Well characterized complement-system androgenic  releasing  factors for granulocytes  (Ghebrehiwet  included:  and Muller-Eberhard,  steroids (Deinard and Page, 1974); endotoxins  components  1979); glucocorticoid (Kampschmith,  et al., 1972); GM-CSF; and G-CSF (Platzer, 1989). Complement  fragments  of the and  1984; Vos that have  20 been shown to induce bone marrow release of leukocytes are cleavage products of C3 such as C3a and C3e (Ghebrehiwet  and Muller-Eberhard,  1979). Purified C3e was  found to mobilize leukocytes from bone marrow upon perfusion of isolated rat femurs. The observation  that an individual  with a leukocytosis  with homozygous C3-deficiency did not respond  during many episodes of infection (Alper et aL, 1972) suggests  that C3 is critical in the complement-dependent marrow. Complement  fragments  release of leukocytes  from the bone  such as C5a, as well as lipopolysaccharides  (LPS)  and GM-CSF, have also been shown to activate PMN and induce shedding of the cell adhesion  molecule,  association  L-selectin,  from the PMN surface  (Griffin et aL, 1990). The  between a possible de-adhesive events such as the shedding of L-selectin  and the bone marrow release of PMN raises the possibility selectin contributes  to the relocation of more immature  marrow to the circulating  d) Adhesion  events  and egress  the surface of hematopoietic  Springer,  the regulation egress  from the bone  of PMN from the bone marrow  cells has emerged  of cell movement and migration 1990b).  granulocytes  blood.  The control of cell-cell and cell-matrix interactions  regulation  that the shedding of L  from the bone marrow  as an important  pathway  are thought  to play a crucial role in  and the direction and control of leukocyte into  for the  (Campbell and Wicha, 1988; Hynes, 1992;  These adhesion interactions  of hematopoiesis  via the cell adhesion proteins on  the circulation  and  migration  traffic,  into tissues  (Campbell and Wicha, 1988; Williams et al., 1991; Simmons et al., 1992; Miyake et al., 1990a). The mechanisms  involved in the coordinated  egress of specific cell types  from the bone marrow require that the barrier be both selective and responsive  to  21 stressful  Both cell-matrix  stimuli.  mediators,  and cell-cell interactions,  as well as soluble  may be involved in the control of this marrow-blood barrier selectivity.  d.1 Cell-matrix interactions: The spatial proximity of hematopoietic be largely regulated stromal  cells and local sources of growth factors may  at the level of cell contacts. The interaction  components  may also be important  marrow. Studies of peripheral  leukocytes  for retaining  of granulocytes  precursors  with  in the bone  have revealed a number of molecules that  mediate cell-cell and cell-mathx interactions.  Recent reports indicate that some of the  same cell adhesion molecules mediate the binding of myeloid progenitor cells to bone marrow  stromal  elements  (Miyake et al., 1990a; Simmons  ample evidence to show that bone marrow cell-extracellular and that  this interaction  Monoclonal antibodies  is important  for hematopoiesis  to CD44 block lympho-hemopoiesis  et al., 1992). There is  matrix interaction (Miyake  occurs  et al., 1990b).  in long-term bone marrow  culture (Campbell et al., 198Th; Wolf, 1979; Dexter, 1982). Whether changes in these adhesive interactions clear. Fibronectin cell membranes  are involved in the egress of cells from the bone marrow is less  is a major component of the ECM that is present on the stromal of the bone marrow (Torok-Storb, 1988; Tsai et al., 1987; Weiss and  Reddi, 1981). Erythroid progenitors and precursors remain bound to the cell-binding domain, the universal  attachment  fibronectin  differentiation  erythroid  throughout progenitors  release of reticulocytes al., 1990). Membrane  peptide arginine-glycine-aspartic  acid (RGD), on  (Tsai et al., 1987). Loss of receptors from the  for the RGD domain on fibronectin  is believed to initiate  the  from the marrow (Pate! and Lodish, 1984; Vuillet-Gaugler associated  chondroitin  sulphate  et  (CS) on hematopoietic  progenitors can also mediate the binding of these cells to fibronectin on stromal cells  22 via the RGD motif (Ruoslahti, have  focused  on  the  microenvironment  role  1989; Minguell et aL, 1992). Although of proteoglycans  to modulate proliferation  most studies  or glycoaminoglygans  of hematopoietic  in  the  cells, at least one study  suggests a role for these ECM components in the adhesion of myeloid precursors (Del Rosso et aL, 1981). This study found that mature granulocytes to preformed marrow stromal cells, but that treatment hyaluronidase  restored  Immature granulocytes  60 % adhesion  on mature  It has been suggested  cells in vitro.  of interrupting  adhesive  and the marrow stroma. However, the  was not strictly evaluated.  that the interaction  (CD44h) with the glycoaminoglycan  of the hematopoietic  hyaluronate  isoform of CD44  to anchors immature  hematopoietic  cells to bone marrow ECM (Carter, 1982; Miyake et aL, 1990a; Stamenkovic 1991). Expression precursors  of CD44 correlates  and primitive  with maturation  granulocytes  1990). Reduced interaction  between  granulocyte  and maturation  cellular  CD44 and ECM hyaluronate  et al., during  may allow these cells to translocate  their surface before egressing into the circulation.  Campbell  among bone marrow  (Kansas et a!., 1990; Lewinsohn  sinus walls in the bone marrow. Mature granulocytes,  mature granulocytes  status  et aL,  cells are CD44M while expression  myeloid progenitors  decreases on more committed  differentiation  with  on their surface and membrane  cells were capable  between the mature granulocytes  specificity of this interaction  of these granulocytes  to marrow stromal  have little glycoaminoglycans  bound glycoaminoglycans interactions  capability  were unable to adhere  however, upregulate  to  CD44 on  The possible role of these  in bone marrow egress is still unknown.  et a! (Campbell  et al., 198Th; Campbell  et al., 1987a) isolated an ECM  23 protein of relative molecular granulocyte  weight  6Okd that mainly attached  lineage and called it hemonectin.  bone marrow, suggesting component  a possible  spleen. The adhesion of immature cells to hemonectin  mechanism  granulocytes  tissue such as the granulocytic  regulated adhesion to hemonectin  of release of granulocytes  et aL, 198Th). The factors regulating  ECM  for granulocytic  bone marrow cells but not circulating  suggests that developmentally  may be involved in the mechanism  hemonectin  molecular  of bone marrow compared with other hematopoietic  predominance  immature  This molecule was found only in the  that it is both an organ-specific and a lineage-specific  providing  and  to cells from the  the interaction  from marrow (Campbell  between  hemonectin  and  in the bone marrow or the counter receptor on these cells for  are currently  being  investigated.  d.2) Cell-cell interactions: Lectins mediated  Lectins  on  cell  complementary various  binding in the bone marrow: mediate  surfaces  carbohydrates  normal  cell-cell  interactions  with  combining  by  on apposing cells and play a key role in the control of processes  and pathological  in living  and  (Brandley  organisms  Schnaar, 1986; Lis and Sharon, 1986; Sharon and Lis, 1989a; Liener et aL, 1986) such as fertilization,  embryogenesis,  In the 1970’s it became  infection.  carbohydrates  covalently  defense,  well-established  are glycoproteins  that  and with speed and reversibility  and  complementary  cell migration that  on their surfaces in the form of glycoproteins  1986). The lectins  Sharon  immune  Lis,  1989a;  Liener  carbohydrates  and  almost  microbial  all cells carry  (Cook,  or glycolipids specifically,  non  (Cook, 1986; Lis and Sharon,  1986;  bind carbohydrates  et al., 1986). Typically,  the lectins  and  the  are located on the surfaces of apposing cells, which  24 may be of the same type or of different types. Furthermore, combine with carbohydrates phenomenon proliferate  of the ECM that promote cell-matrix  of clinical bone marrow transplantation, preferentially  cell surface lectins may adhesion.  The  in which stem cells lodge and  within the bone marrow, results from specific adhesion of  these stem cells to macromolecular  components of ECM or on the stromal cells unique  to bone marrow. This binding of progenitor cells to stromal cells and ECM involves an interaction  between a membrane  glycoconjugate purified  lectin on the one side and a membrane  on the other (Tavassoli  a lectin  homing  carbohydrate-binding this interaction.  protein  qualities,  and Hardy,  1990). Matsuoka  or ECM  et al (1989)  (MW 110,000) from cloned progenitors  suggesting  that a lectin on progenitors  This supports a wealth of recent information  with  is involved in  on membrane  lectins  with biological recognition functions (Hoppe and Lee, 1982; Lehrman and Hill, 1986; Prieels et aL, 1978; Thornburg dependent  et aL, 1980). These membrane  in their binding to carbohydrates  and are known as C-lectins (Drickamer,  1988; Drickamer and McCreary, 1987). Similarities in hematopoietic belonging  progenitors  to the selectins  involved on lymphocyte-  Granulocytopoiesis granulocyte undergo  group of adhesion  segmented  and neutrophil-endothelial  involves  number  granulocytes.  molecules  on lymphocytes (Stoolman  and PMN  et al., 1984;  1990). These molecules have been shown to be  differentiation  cell lineage and maturation  a limited  exist between this homing protein  and the homing receptors  Yednock and Rosen, 1989; Butcher,  lectins are calcium  cell interactions.  of bone marrow  stem  cells into the  of the granulocyte-committed  cells, which  of cell divisions  and finally  enter  the circulation  as  Each step in this process is likely influenced by interactions  25 between  granulocyte  Furthermore, barrier  and  the  are largely  are important  inflammation  marrow  unknown.  Cell-cell  for leukocytes  that are involved and that control via the cell adhesion  interactions  to cross the endothelial  in the systemic and pulmonary  1990; Lawrence and Springer,  1990). The initial cell-cell  is mediated by the selectin family of adhesion  receptors. One of these selectins, L-selectin, is constitutively granulocytes  and interacts  during recruitment Kishimoto  adhesion.  L-selectin  to foci of inflammation  PMN in synovial  selectins,  moieties on the endothelium  P- and  to initiate  is shed from the surface of leukocytes (Humbria et al., 1994; Julita et al., 1989;  et al., 1989; Porteu and Nathan,  1991b). For example, in patients  expressed by nearly all  with the other inducible  E-selectin, as well as inducible carbohydrate leukocyte-endothelial  barrier to sites of  vascular beds (Butcher, 1991; Butcher,  1991; Yong and Khwaya,  contact between PMN and endothelium  intravascular  microenvironment.  tissue into the bone marrow venous  The cell-cell and cell-matrix interactions  process  molecules  bone  during bone marrow release of PMN, cells have to cross an endothelial  when moving from the hematopoietic  sinusoids. this  precursors  1990; Tedder,  with rheumatoid  et al.,  L-selectin expression on  arthritis,  fluid is much lower than expression  1991; Spertini  on circulating  PMN that  implies shedding of L-selectin when PMN emigrate from the vascular space (Humbria et al., 1994). It has been proposed that L-selectin shedding may be a de-adhesion endothelial  event and a signal for migration  cell barrier and into the underlying  al., 1989; Jutila  during PIvIN activation of the PMN through  the  tissue (Tedder, 1991; Kishimoto  et  et a!., 1989; Smith et al., 1991).  In the bone marrow, L-selectin is expressed on nearly all postmitotic (Griffin et a!., 1990), and Lund-Johansen  et a! (Lund-Johansen  myeloid cells  and Terstappen,  1993)  26 have shown that L-selectin expression increases with granulocyte bone marrow. The fact that the crossing of an endothelial L-selectin shedding  led us to consider the possibility  maturation  barrier is associated  that a similar shedding  selectin occurs when PMN cross the bone marrow-blood barrier.  in the with of L  27 3) SELECT1NS Lectin or carbohydrate  recognition  involved  of biological  in a number  domains  of proteins  events  (Sharon  carbohydrate-mediated  cell  have been shown to be and  Lis, 1989b), but the  relationship  between  inflammation  was not always clear. The discovery of the selectins, a family of three  cell  glycoproteins  surface  inflammation  containing  lectin  adhesion  domains  to be clinically significant. an increasing  coordinated during  that  mediate  responses by recognition of cell-specific carbohydrates,  divergent fields and has given rise to some observations  among  and  manner  lymphocyte  leukocyte  regional  has unified these  that may ultimately  prove  The selectins are a group cell adhesion molecules included  number  of leukocyte  adhesion  molecules  that  act in a  to orchestrate  the migration  of various classes of leukocytes,  recirculation,  inflammation,  metastasis  and other  types  leukocyte traffic (Hemler, 1988; Springer, 1990b). Recent work has demonstrated existence of this carbohydrate endothelial  motif  the  novel family of adhesion molecules that appear to utilize protein  interactions  for specific cell-cell binding  between  leukocytes  and  cells. Molecular cloning revealed the unifying aspect of this new family  of three adhesion molecules to be their common structure, dependent  of  lectin- or carbohydrate  (EGF),  variable  numbers  consisting  binding domain, an epidermal of complement  regulatory  of a calcium  growth factor type moieties  (CR), a  transmembrane  region and a cytoplasmic  1990; Stoolman,  1989; ) (figure 3). The most notable is the C-type lectin motif found  at the N-terminus  of the protein which has a 60 % -70 % amino acid sequence homology  tail (Bevilacqua  et al., 1989; Geng et al.,  between the three different selectins and functions as the principal component in cell adhesion events (Bevilacqua  et al., 1989; Johnston  et al., 1989; Lasky et al., 1989;  28 Tedder et al., 1989; Siegelman,  and Weissman,  l989b;  ). While the lectin domain  seems to be the major determinant  of cell adhesion, its structure  to be conformationally  upon the EGF and the CR domains (Bowen et aL,  dependent  1990; Watson et al., 1991). while E-selectin P-selectin endothelial  and function appear  L-selectin (CD62L) is expressed on nearly all leukocytes  (endothelial-leukocyte  (CD62, PADGEM,  adhesion molecule or ELAM-1, CD62E) and  GMP-140,  CD62P) are inducible  cell surface. Table I summarizes  the properties  molecules  on the  of the selectins.  L-selectin L-selectin is the smallest human counterpart 14 antibody  cell adhesion  molecule of the selectin family and is the  of the murine lymphnode homing receptor identified by the MEL-  (Gallatin  et aL, 1983a).  L-selectin was first identified  cDNA cloning (Bowen et al., 1989; Siegelman located on chromosome  1 at band q23-25. The three selectin genes,  are within 200  that they derived from an  related gene (Collins et al., 1991; Ord et al., 1990; Watson et al., 1990).  It is noteworthy  that the selectins genes map very close to the locus on the genes that  code for complement  activation  proteins (such as complement  receptor 1 and factor  B) with the same complement  repeat motif that is found in the selectins.  that this family of adhesion  molecules  through gene duplication  1) Structure  by  et al., 1989a; Tedder et al., 1989), is  kilobases of one another in the human genome, suggesting evolutionary  structurally  It is likely  evolved from a common progenitor  gene  and exon amplification.  of the L-selectin protein: (figure 3)  The L-selectin on lymphocytes  is smaller (Mr of ±74,000) than the molecule on PMN  2q Table I  Selectins: Nomenclature, expression and function Expression  Proposed function  Name  Cell type  L-selectin  Lymphocytes  Shed after cell  Lymphocyte  LECAM-1  Monocytes  activation(min).  recirculation-PLN.  LAM-i  Neutrophils  Conformational  Leukocyte rolling.  TQ-1  Eosinophils  change(?).  Neutrophil  Leu-8  inflammation.  CD62L MEL-i4 Platelets  Increases upon  Neutrophil/monocyte  PADGEM  (cr-granules)  thrombin,  rolling.  GMP- 140  Endothelium  histamine,  Leukocyte  CD62P  (W-P bodies)  Substance P  inflammation.  P-selectin  activation(min). Endothelium  Increases upon  Neutrophil/monocyte  ELAM-1  transcriptionally  IL-i, TNF, LPS  rolling.  CD62E  activated  activation(hours).  Leukocyte  E-selectin  inflammation.  PLN, peripheral lymph nodes; ELAM-1, endothelial-leukocyte adhesion molecule 1; GMP-140, granule membrane protein 140; PADGEM, platelet activation-dependent granule external membrane protein. TNF, Tumor necrosis factor; LPS, lipopolysaccharide; W-P, Weibel-Palade.  30 (Mr of 90,000 to 100,000) (Griffin et al., 1990) and both molecules  are highly  glycosylated  as L-selectin  translational  processing of the molecule. The molecule has a C-type lectin domain,  a short epidermal  cDNA encodes only a ±37kd, protein  growth factor-like domain, two complement  to those found in C3/C4 binding cytoplasmic  tail (figure 3). The lectin-like  other carbohydrate mannose  proteins,  binding  binding  proteins,  proteins.  a membrane  suggesting  post-  moieties homologous  spanning  domain  and a  domain of L-selectin is homologous with  including  This domain  asialoglycoprotein  contains  residues found in C-type amid lectin carbohydrate  essentially  receptors  and  all the invariant  recognition domains (Drickainer,  1988). Both murine and human homing receptors have been shown to required Ca to function (Stoolman et al., 1987).  The EGF domain contains the 6 Cys-residues 39% identical  to the amino-acid  proteins are involved in mediated  sequence  conserved in all EGF domains and is of EGF. As EGF domains  protein-protein  interactions,  the L-selectin EGF  domain may serve a similar function and may not be merely a structural the molecule (Kansas et at, 1991). The two complement minimum  in several  feature of  motifs in L-selectin are the  protein binding unit in the C3/C4 binding proteins and the conservation  of these moieties signals possible function significance  One striking  feature  between  the lymphocyte  homing  in binding interactions.  molecule MEL-14 and L  selectin on other leukocytes is the homology in the transmembrane  domains and the  cytoplasmic tail, which is 88 % homologous. These regions in E- and P-selectin are not homologous to L-selectin.  L-selectin is shed from the cell surface following cellular  31  L-selectjn N  COO H  P-se led in _f 2 NH -  Co OH  E-selectjn NH2-{___ -  COOH  Figure 3: Domain compositions of the three known selectins. The extracellular portion of each selectin contains an amino-terminal domain homologous to the C-type lectins, an adjacent epidermal growth factor domain followed by a variable number of complement regulatory domains (circles) and transmembrane sequence (black diamond). A short cytoplasmic tail (open rectangle) is at the carboxyl terminus of each selectin.  32 activation,  which makes it likely that this region is of critical importance  phenomenon  and thus in the regulation  of L-selectin contains Phosphorylation  two potential  of L-selectin expression. The cytoplasmic tail  sites for phosphorylation  of the tail has been postulated  the molecule from the surface of leukocytes biochemical stimulate  evidence  of phosphorylation  phosphorylation  in this  by protein kinase C.  to be associated with the shedding of (Kansas et al., 1991). However, direct  has been difficult  because  agents  that  also enhance L-selectin shedding.  2) Expression of L-selectin on leukocytes: The expression  of L-selectin, which is limited to hematopoietic  complex function of lineage, stage of differentiation, location  of the cells. L-selectin  is expressed  activation  cells (Table I), is a  status, and anatomical  on the surface  of most leukocytes  including lymphocytes, neutrophils, monocytes, eosinophils, hematopoietic progenitors cells and immature  thymocytes  (Griffin et aL, 1990; Tedder et aL, 1990a). L-selectin  is expressed late in B-cell development,  is lost after activation,  and is re-expressed on  memory B-cells. Similar patterns hold for T-cells, it is expressed on virgin T-cells, lost during cell activation  and re-expressed  Tedder et aL, 1990b; Kansas progenitors nucleated  by most memory T-cells (Tedder et aL, 1985;  et al., 1985b; Kansas  et al., 1985a). Early erythroid  cells (BFU-E) are L-selectin positive with a lack of expression on mature or non-nucleated  erythrocytes.  Among myeloid cells, L-selectin is expressed by nearly all circulating monocytes, portion  and eosinophils  of L-selectin  neutrophils,  (Griffin et aL, 1990; Tedder et aL, 1990a). The largest  is found on the cell surface  and does not localize in the  33 intracellular  compartment.  monocytes,  express  instability  In contrast  to lymphocytes,  low levels of mRNA for L-selectin,  or different post-translational  mature  neutrophils  which suggests  including  development  differentiation.  of progenitors  traffic into lymphnodes  stages of differentiation  progenitor  unknown.  or inflammatory  bone marrow. Furthermore,  An interesting  or  are known not to  interactions  may  within the  the function of L-selectin on myeloid cells at the different  and possible changes with maturation  is still unclear.  of L-selectin expression: and unique feature of the regulation  of leukocytes  with a variety of stimuli  reduced the cell surface expression  of L-selectin expression following cellular  (Kishimoto  activation.  et al., 1989), such as  tumor necrosis factor, leukotrine of L-selectin  is the  B4 and LPS,  on T- and B-cells as well as on  (Tedder et al., 1990a). A large fragment of L-selectin (Mr ±69,000) can  be immunoprecipitated expression  to the  sites, it is possible that L-selectin  matrix (ECM) or endothelial  phorbol esters, complement fragments,  granulocytes  cell committed  As progenitors  loss of the molecule from the surface of leukocytes Treatment  throughout  The role of L-selectin in the localization  is currently  mediate leukocyte/extracellular  3) Regulation  cells  in the bone marrow (Griffin et al., 1990; Kansas et aL, 1990),  80 % of CFU-GM that is the earliest  granulocyte/monocyte  message  protein processing in myelomonocytic  (Tedder et al., 1989; Ord et al., 1990). L-selectin is expressed continuously myeloid differentiation  and  from  is down-regulated  the  supernatant,  by shedding  distinct from that which down-regulates  demonstrating  L-selectin  that  rather than by internalization,  modulation  which is  of most other surface molecules.  It has been proposed that the shedding of lymphocyte  L-selectin might be necessary  to enable these leukocytes  the endothelium  to transmigrate  through  into sites of  34 inflammation, leukocytes  providing  a rapid  to the endothelium  means  for the adhesion  and  de-adhesion  of  (Kishimoto et al., 1989; Jutila et aL, 1989). Another  possible signal for receptor shedding is ligand binding which would provide a rapid means  for leukocyte  adhesion  to the endothelium  following receptor shedding. The temporal relationship interaction  Although  during inflammation  the mechanism  proposed that a membrane-bound  between leukocyte-endothelial  of L-selectin remains  releasing  it has been  activity  cleaves the  a nearly intact extracellular  not appear to result from the activation as the supernatant  unknown,  protease with chymotrypsin  ligand binding activity (Spertini et al., 1991b; Schleiffenbaum  leukocytes  de-adhesion  and L-selectin shedding has not been established.  of shedding  receptor near the membrane,  with subsequent  domain with  et al., 1992). This does  of induced soluble proteases  released by  from the fluid of cells that have shed L-selectin does  not contain soluble proteases that can alter the expression of L-selectin. Therefore, it is likely  that  enzymatic  cleavage  of L-selectin  may result  from the specific  activation of a membrane bound protease that may be rather ubiquitous array of L-selectin negative cell types transfected  since a broad  with L-selectin cDNA are able to  shed the receptor (Spertini et al., 1991b). The function of L-selectin may be further regulated  by the presence of functional  (Schleiffenbaum consistent  L-selectin in the extracellular  environment  et al., 1992). Soluble L-selectin in normal plasma caused a small but  inhibition  of lymphocyte  attachment  to high endothelial  venules  and  higher concentrations completely inhibited L-selectin-dependent leukocyte attachment to endothelium.  This soluble serum L-selectin may be a protective  reduce random leukocyte  recruitment  during generalized  mechanism  intravascular  to  leukocyte  35 activation.  A small  amount  of this  immunoprecipitated stimulation,  soluble  isoform of L-selectin  from the supernatant  suggesting  that L-selectin  The demonstration  relationship regulating  The relatively  to the circulating  can also be  cultured  without  shed at a slow rate with  of a new receptor (Spertini et al., 1991b).  of sL-selectin in circulating  al (1992) supports the idea that circulating intravascularly.  fluid of lymphocytes  is constitutively  expression kept constant by the re-synthesis  (sL-selectin)  human plasma by Schleiffenbaum  leukocytes  constitutively  et  shed L-selectin  high levels of soluble L-selectin in plasma and the leukocyte count suggest a possible role of sL-selectin  the circulating leukocyte count and leukocyte recruitment  (Schleiffenbaum  et al., 1992).  4) Ligands for L-selectin: The ligand used by lymphocyte L-selectin (MEL-14) to home to peripheral lymphnodes via  high  endothelial  venules  has  recently  been  identified  as  carbohydrate  determinants  Sgp5O and Sgp9O, coined GLYCAM-1 (Lasky et al., 1992b).  carbohydrate  ligand has been shown to be fucosylated,  components essential  sulfated  and sailylated,  suggested  all  for ligand binding activity to L-selectin (Ley et al., 1991a; True  et al., 1990). The ligand(s) or counter receptor for L-selectin in postcapillary of non-lymphoid  This  tissue has yet not been identified.  that the L-selectin on PMN can interact  adhesion molecules by presenting  sialylLewisx  venules  However, in vitro findings have with inducible  endothelial  (sLex) and related carbohydrates  cell as  ligands to the vascular selectins, P- and E-selectin (Picker et al., 1991). Mulligan et  36 al (1993) have demonstrated dependent  lung  injury,  confirming  Moreover, the congenital leukocyte  adhesion  endothelium  that synthetic  sialyl-Lewis  the in vivo importance  of this  stimulated  deficiency  and  an inability  of PMN to roll on activated  has been shown in vitro to interact  human  umbilical  vein endothelium  et al (Norgard-Sumnicht  endothelium  a heparin-like  Baumhueter  L-selectin, like the  with sLex through  et aL, 1991a). Norgard  et aL, 1993) have identified  in non-lymphoid  ligand that binds a L-selectin chimera  et al (1993) established  that  function as a ligand for L-selectin. This is relevant of CD34 with apparent  constitutive  the protein  and translocation  of regulating  L-selectin-CD34  response. It remains cytokines,  to the endothelial interaction  molecule; and  core of Sgp9O is  that a glycoform of CD34 could considering  the broader tissue  expression on the endothelium  diversity of non-lymphoid blood vessels (Fina et al., 1990). Differential glycosylation  its lectin  ligand(s) expressed on cytokine  (Spertini  identical to the sialomucin molecule CD34, suggesting  distribution  interaction.  absence of sLex ligand results in the clinical syndrome of  domain (Foxall et al., 1992), or other still unidentified  recently  protects against P-selectin  (Jutila et al., 1991; Segal et al., 1976). Furthermore,  other selectins,  Sumnicht  X  in a  vessel-specific  surface may be potential pathways  during the acute or chronic inflammatory  to be seen whether CD34 is subject to biosynthetic  control via  as has been reported for the ligand for L-selectin in cultured endothelial  cells (Delia et at, 1993; Spertini  et al., 1991a).  5) Functions of L-selectin: As observed intravital  by Cohnheim  (Cohnheim,  microscopy, leukocytes  1889) more than  begin to interact  a 100 years ago using  with the vessel wall by rolling  37 along the endothelium  to adjacent  response is seen throughout amphibians  the vertebratae,  and in mammals  increases dramatically  (Cohnheim,  after injury. The rolling  in both cold-blooded animals  such as  1889). The number of rolling leukocytes  during the course of an inflammatory  Born, 1972) and is important posteapillary  tissue within minutes  in the accumulation  reaction (Atherton and  and emigration  of leukocytes  in  venules (Fiebig et aL, 1991). The velocity at which cells tumble and roll  in shear flow near the vessel wall is much faster than what is observed for rolling cells on inflamed  endothelium,  suggesting  that an enhanced  occurs between the leukocyte and the vessel endothelium  adhesive  interaction  (Atherton, and Born, 1973).  The selectin family of cell adhesion molecules is specialized to mediate the rolling of leukocytes  and  contributions interaction  rolling  observed  from all three of inactivated  physiological  in  vivo  with  inflammation  In PMN, L-selectin  selectins.  cells  during  cytokine-stimulated  cell adhesion molecules sequentially  cells to support rolling of leukocytes,  PMN L-selectin functional  selectin whose expression Spertini  et al., 1991a;  phenomenon inflammation  to the cells  at  expressed on endothelial  P-selectin being up regulated  plays a central lectin  recognizing  molecules  mediated  Von Andrian  bind  number  stimuli  to inducible  (Picker et al., 1991; that  for PMN emigration  of animal  as a  sLex to both P- and E  et al., 1993). Evidence  by the selectins is essential  comes from a limited  simultaneously  that  presenting  is induced by inflammatory  within minutes  1990a). In this cascade  role, functioning  cell surface ligands, and as a structure  endothelial  contributes endothelial  and E-selectin within hours (Abbassi et al., 1993; Springer,  constitutively  involve  shear stresses (Smith et aL, 1991). The other two selectins, in contrast,  are cytokine-inducible  of events  may  studies  this  rolling  to areas of  (Ley et al., 1991b;  38 Mulligan  et al., 1991; Mulligan  antibodies  to L-selectin  and  et aL, 1994; Watson  a L-selectin-IgG  et aL, 1991). Monoclonal  chimera  mesenteric blood vessels and peritoneal inflammation  and P-selectin  chimera  protect  against  PMN rolling  to E-selectin inhibit PMN  during acute inflammation  complement  mediated  functions  of PMN in conjunction with the I -integrins 2  of PMN L-selectin underline  initial phase of the inflammatory  the importance  initially  derived,  of lymphocytes  which  (Simon et aL, 1992). These of this molecule during the  1964) suggested  led to the identification  venules of peripheral  et aL, 1983a). In addition lymphnodes,  to directing  L-selectin on lymphocytes  selectin on PMN in recruiting Michie et al., 1993).  tissue-specific  that home back to sites from which they were of the lymphocyte  recognized by the MEL-14 mAb that directed lymphocyte high endothelial  intravascular  response.  The work by Gowans et al (Gowans and Knight, adhesive interactions  of the lung;  acute lung injury  (Mulligan et aL, 1993; Mulligan et al., 1994). L-selectin also augmented aggregation  in  (Watson et al., 1991; Ley et al.,  1991b; Von Andrian et aL, 1991); monoclonal antobodies mediated damage to the vascular endothelium  inhibit  lymphnodes lymphocyte  L-selectin  homing to post-capillary  (Gallatin et al., 1983b; Gallatin recirculation  through peripheral  may also function in a similar fashion to L  lymphocytes  to foci of inflammation  (Lasky, 1992a;  39  4)L-SELECTIN EXPRESSION  DURING ACTIVE BONE MARROW RELEASE  OF POLYMOPHONUCLEAR  LEUKOCYTES  Introduction The emigration requires  of polymorphonuclear  intercellular  information  during inflammation  leukocyte  adherence  roll on the luminal  leukocytes  then  perivascular rolling  is observed  and adhere  transmigration.  are  This  distinct  transient  is mediated  1990).  in postcapillary  surface of endothelial more tightly  tissue. Evidence indicates  phenomenon  endothelium  that mediate this leukocyte-endothelial  et aL, 1992; Yong, and Khwaya,  leukocytes  spread  and finally  emigrate  from  the  adhesion  those  required  of leukocytes  cell adhesion  that loosely adhere  followed by the down-regulating  leukocytes  stop. This activates  are transiently  the and  are three  derived cytokines, resulting  molecules  supporting  (Lasky, 1992a). The selectins  on most leukocytes,  that tighten adhesion and direct emigration  into the  activated  L-selectin,  allowing the leukocytes to transiently  The  to regionally  of these selectins,  is expressed  where  for stopping  proteins that interact with cell-surface carbohydrate  them to endothelial  cell  cells before stopping.  that the adhesion molecules  by the selectins  on the endothellum  in  During  venules  related membrane  ligands  increase  has become available (Butcher, 1991; McEver, 1992  1990b; Zimmerman  inflammation  (PMN) at sites of inflammation  In the last 7 years an extraordinary  concerning the mechanisms  interaction Springer,  adhesion.  leukocytes  ligands. One  binds to inducible  to the vascular  wall  the leukocytes by exposing  in up-regulation  -integrins 2 of the 3  (McEver, 1992). During inflammation,  expressed  to recruit  leukocytes.  This is  of adhesive events, which limits and terminates  the  40 recruitment activated  of leukocytes. endothelium  (Kishimoto  Firm adhesion  results in the shedding  of leukocytes  results  1989; Kishimoto  et aL, 1991b; Tedder,  is that chemotactic  of L-selectin  et at, 1991b). Notwithstanding in mediating  homing of leukocytes  factors on the endothelium  occurs when lymphocytes lymphnodes  leukocyte-endothelial to lymphnodes  cross high endothelial  (Kishimoto et at, 1990; Spertini  the increase in information  the bone marrow to the circulating  concerning the role of L  cell interaction  or recruitment  during  in the bone marrow  little is  barrier from  blood.  (Griffin et al., 1990), and a study by Lund-Johansen 1993) shows that  physiological  to foci of inflammation,  In the bone marrow, L-selectin is expressed on nearly all postmitotic  segmented  et at,  or the migration  known regarding adhesion events when leukocytes cross the endothelial  maturation  stimulation  is still unknown.  venules (REV) in homing to peripheral  Terstappen,  event  et at, 1989; Griffin et al., 1990). Whether the process of rolling, the  process itself induce shedding  selectin  through  of L-selectin from the cell surface (Julita  contact of the PMN with chemotactic  shedding  1990; Spertim  proposed for this phenomenon in shedding  of leukocytes  of L-selectin as a de-adhesion  et aL, 1989; Porteu and Nathan,  1991). One explanation  Similar  and emigration  L-selectin  expression  with the highest  myeloid cells  et al (Lund-Johansen increases  expression  with  and  granulocyte  on band cells and  PMN in the bone marrow. However, this study did not directly compare  the expression of postmitotic bone marrow PMN with circulating the crossing of an endothelial  PMN. The fact that  barrier is associated with L-selectin shedding led us to  41 consider the possibility that a similar shedding of L-selectin occurs when PMN cross the bone marrow-blood barrier.  42 WORKING HYPOnIHESIS The evidence that the migration  of PMN through an endothelial  barrier is associated  with the shedding of L-selectin from their surface led to the working hypothesis L-selectin  that  is shed from the surface of PMN as they cross from the bone marrow  hematopoietic  compartment  into the circulating  blood. Therefore, the studies in the  first part of this thesis were designed to measure L-selectin expression on PMN in the hematopoietic  and sinusoidal compartments  of the bone marrow and in the peripheral  blood during an episode of active bone marrow release.  SPECIFIC  AIMS  This working hypothesis 1) To determine hematopoietic (sinusoidal  was examined  the expression  the following specific aims:  of L-selectin on mature  tissue (hematopoietic  compartment)  by pursuing  compartment),  and the circulating  PMN in the bone marrow  the bone marrow venous sinusoids  blood.  2) To study the changes in L-selectin expression on PMN in these three compartments during  active bone marrow  suppressed  of PMN and when bone marrow  release  is  by hypothermia.  3) To determine vitro.  release  the effect of hypothermia  on the shedding of L-selectin from PMN in  43 Model of bone marrow The PMN released  release  of PMN  from the marginated  pool come from organs such as the liver,  spleen and lung with the lung being the major source of marginated and English, during  PMN (Doerschuk  1991; Doerschuk and Allard, 1989; Muir et al., 1984). Previous studies  cardiopulmonary  PMN that is attributed 1981; Haslam  bypass (CPB) demonstrated to the activation  fall in circulating  of the complement system (Chenoweth et aL,  et aL, 1980). This neutropenia  must be due to either a mobilization  an inithi  is followed by a neutrophilia,  of marginated  which  PMN from a pool other than the  lung or to a release of new cells from the bone marrow. As the lung represents major pool of marginated newly released leukocytosis  PMN and as it is removed from the circulation during CPB,  PMN from the bone marrow  after commencing  1985) and these fragments, release  form the largest  observed during CPB. An increase in fragments  to occur minutes  of leukocytes  accumulation  the  CPB (Chenoweth  component  of the  of C3 has been shown  et al., 1981; Quiroga et al.,  such as C3e, have been associated with the bone marrow  (Alper et aL, 1972). Animal  of PMN in the lungs of the animals  studies  have shown that  after complement  activation  the is  twice as many as could be accounted for by the population of PMN in the circulation (Doerschuk and Allard, 1989). As the release of the PMN from the liver or spleen is unlikely  in these circumstances  this suggests activation.  that PMN are released from the bone marrow following complement  The leukocytosis  temperature be prevented  (Doersehuk, and English, 1991; Rosolia et al., 1992),  dependent  associated with CPB-surgery  (Quiroga et al., 1985); that is the rise in circulating  by lowering body temperature  with an opportunity  has also been shown to be  to 27°C. This observation  PMN can  provides us  to study bone marrow release of PMN under conditions of active  44 release and when this release is suppressed by hypothermia.  The fact that L-selectin  is expressed on nearly all myeloid cells in the bone marrow (Griffm et aL, 1990) and that  removal  enzymatic  of L-selectin  cleavage  from the cell surface is postulated  by an unidentified  cell associated  to occur through  protease  that  may  be  temperature  sensitive (Spertini et al., 1991b; Tedder, 1991) led us to consider that the  temperature  sensitive bone marrow release of PMN (Quiroga et al., 1985) is related  to a reduction in cleavage of L-selectin from the PMN surface.  45 METHODS Experimental  procedures:  The goals outlined by the specific aims were pursued in studies on volunteers patients  who were undergoing  normothermic  and hypothermic  and on  CPB-surgery.  In vitro studies: The effect of temperature first determined  in vitro using PMN isolated from the blood of 10 healthy infection-  free laboratory workers. to simulate prepared  on the shedding of L-selectin from the surface of PMN was  Zymosan activated plasma (ZAP) was used to activate PMN  in vivo complement  by incubating  Sigma), 5mg/mi plasma.  human  activation  seen during  CPB-surgery.  plasma combined with zymosan A yeast (Z-4250,  The mixture was incubated  for 30 minutes  down twice at 500 x g for 10 minutes, and used within 30 minutes. leukocyte  rich plasma  ZAP was  (LRP) was prepared  dextrose (ACD) (1:5) as anti-coagulant  In 5 experiments,  from blood collected  and sedimented  at 37°C, spun  in acid citrate  using high molecular weight  (Mw 100,000-200,000: 4%) dextran, final dilution 1.9% dextran (Chemical Dynamics Coop., New Jersey) for 25 to 30 minutes. incubated  Leukocyte  at 37, 27 or 4°C for 15 minutes,  stimulated  0.1 % or 0.01 % dilution of ZAP at the above mentioned cells were fixed with 0.05 % glutaraldehyde Kishimoto shedding (Kishimoto  for 4 minutes temperatures  samples  were  with a 5%, 1 %, and then the  prepared in 0.2M phosphate buffer pH 7.6.  et al have shown that 4 mm of >90 % of L-selectin  rich plasma  of maximal  stimulation  from PMN and significantly  of PMN cause  upregulated  CD 18  et al., 1989). Leukocyte rich plasma prepared from each specimen was  used to make cytospins by cytocentrifugation  at 180 x g on a Cytospin 2 (Shandon  46 Lab Products, Cheshire, England) for 4 minutes onto 3-aminopropyl-tri-ethoxysilane coated slides. Leukocyte rich plasma was diluted with PMN buffer 1:1 (138mM NaC1, 27 mM KC1, 8. 1mM NaIl .2 4 P 7H20, 0 l.5nM KH 4 P0 5mM glucose, pH 7.4) or further 2 , if necessary to obtain a single layer of cells on the slide. The leukocytes on the slides were stained with the APAAP method (Cordell et al., 1984) using anti-Leu-8 (Becton Dickinson, 1mm Systems, CA) as the primary antibody for the presence of L-selectin as described potassium  under  immunocytochemical  ethylenediamine  tetra-acetic  with the same concentrations temperatures  analysis.  In 5 separate  experiments,  acid (EDTA) blood 1 (100 d ) was stimulated  of ZAP (5 %, 1 %, 0.1 % and 0.01 %) at the different  (37, 27 and 4°C) for 4 mm. PMN L-selectin  (using FITC conjugated  anti-Leu-8) and CD18 (using FITC anti-CD 18, Sigma Chem, St Louis, MO) expression was determined  by flow cytometry as described under flow cytometric analysis. These  studies were done to determine whether the shedding of L-selectin under hypothermic conditions is PMN activation  dependent.  In vivo studies: Patient  population:  Ten patients  (3 women and 7 men, mean age 63 years, range 43-76) were recruited  from patients  admitted  either coronary arterial were studied Approval Columbia)  during  to St. Paul’s Hospital, Vancouver, B.C., for elective CPB for bypass and/or valvular replacement hypothermic  from our institution’s human  normothermic  (St. Paul’s Hospital  experimentation  informed consent was obtained  and 5 during  committee  from all patients  surgery.  Five patients  CPB procedures.  and the University  was obtained  of British  for the study  prior to surgery.  and  47 Experimental  protocol  The surgery was performed under general anaesthesia The anaesthetic  drugs used were thiopental,  muscie-relaxants  and oxygen-nitrous  with endotracheal  diazepam,  oxide enflurane  fentanyl,  intubation.  non-depolarizing  or isoflurane  mixture.  Access  to the thoracic cavity was obtained via a median sternotomy  and specimens of bone  marrow were obtained  using a small curette.  from the cut surface of the sternum  Bovine heparin was given intravenously  before the introduction  of the venous and  arterial cannulas using a dose that is required to lengthen the activated clotting time to 450 seconds (Hemochron) and was repeated to keep the activated that range during the CPB procedure.  No immune-modulating  clotting time in  drugs such as steroids  were used before or during the surgery. The CPB apparatus  consisted  of a Sarns roller pump, a cardiotomy  (Bentley® 220f) and a Cobe Optiflow II bubble oxygenator  reservoir  supplied with 98 % oxygen  and 2 % carbon dioxide. The CPB circuit was primed with 5 % Ringers lactate except in one case where packed red cells mixed with a crystalloid instead.  Pulmonary  solution  were used  blood flow was reduced by diverting the venous return from the  heart to the oxygenator pump in steps until complete CPB was achieved and restored at the end of the procedure by increasing Systemic hypothermia 28°C. Patients  venous return to the right heart stepwise.  was used in 5 patients  with a perfusate  were cooled until bladder temperature  and rewarmed to 37°C at the end of the CPB procedure. was maintained  by keeping the perfusate  at 37°C.  temperature  (core temperature) In 5 patients,  of 26-  was 27°C  normothermia  48 Blood and bone marrow  samples  Peripheral  were obtained  blood samples  temperature  from an arterial  at which they were collected. In preliminary  line and kept at the  experiments,  samples were  taken across the CPB apparatus  at different time points during bypass, to determine  the effect of PMN circulating  through  expression.  the bypass apparatus  No notable effect of the apparatus  found. These samples were collected: (baseline),  on PMN L-selectin  on PMN L-selectin expression  before the start of anaesthesia  were  and surgery  after the sternum was split (BM1); before restoring pulmonary blood flow  in the normothermic  group and just before rewarming  in the hypothermic  (BM2); and at the end of the surgical procedure just before the sternum  group  was closed  (BM3).  Bone marrow samples were obtained from the sternum at time points BM 1, BM2 and BM3. Smears were made immediately were processed for histology modification  by embedding  (GMA) using a  analysis (Burgio et al., 1990; Murray and Evens, 1991). Plastic  of tissue is frequently  microscopical  examination  use as an alternative  resolution  of cellular  embedded without decalcification decalcification  in our study; thinner  detail, tissue shrinkage (in preliminary  our antigen of interest).  for light the tissue  sections can be cut with is less and tissue can be  experiments  of bone marrow by either hydrochloric  tissue useless for detecting  to wax embedding  and GMA is the plastic of choice. Embedding  in plastic had a variety of advantages improved  in glycolmethacrylate  of methods described for processing bone marrow tissue in plastic for  immunohistochemical embedding  and allowed to air dry. Bone marrow samples  we demonstrated  acid or EDTA rendered  that the  49 As indicated above, bone marrow specimens were collected from the cut surface of the sternum  with a small curette  and immediately  prepared  with 0.2M phosphate  buffer pH 7.6 for two hours at the temperature  which  the tissue  glutaraldehyde  was collected.  Fixation  with  has been shown in preliminary  and to have the least influence  were then transferred  this  very low concentration  experiments  on the detection  fixation, specimens were transferred  fixed in 0.025 % glutaraldehyde  of our antigen  of interest.  After  to acetone at 4°C for 12 hours. The specimens  A and infiltrated  Ltd.) containing  under vacuum (-75mmHg) using a  Freeze dryer Model FDU 101, Blazer Union, for 2 hours at 4°C. The infiltrated was placed in embedding  of  to preserve morphology  to GMA resin monomer (JB4®; Polyscience,  0.9% benzoyl peroxide-solution  at  molds and GMA chemically  polymerized  tissue  by a mixture  of  200L of JB4® solution B to 5m1 of solution A (JB4®; Polyscience, Ltd.) for 12 hours at 4°C. Polymerized resin blocks were brought to room temperature  and 2m sections  cut with a Sorval® JB 4 microtome fitted with a glass knife (made with a LKB Knife maker Type 7801B, Stockholm, Sweden). Sections were floated on a room temperature water bath, transfered  to glass slides and air-dried overnight at room temperature  insure adhesion of sections to the glass slides during the staining  Hematological Peripheral  procedure.  analysis: blood samples were collected in standard  Dickinson, NJ) containing cell counts were performed were identified  to  tubes (Vacutainer,  potassium EDTA. Blood cell counts and differential with a Sysmex Model E4000 (Toa, Japan).  (College of American  stained blood smears from all samples.  Pathologists,  Becton white  Band cells  1991) and counted on Wrights  The blood smears were evaluated  in a blinded  50 fashion by evaluating  100 PMN in randomly  were corrected for hemodilution  Corrected cell count  L-selectin  selected fields.  All leukocyte counts  by means of the hematocrit;  =  Observed cell count  boseline hematocrit observed hematocrit  determination:  a) Evaluation  of L-selectin expression on PMN in the circulating blond  All blood specimens were kept in an incubator  and processed at the temperature  at  which they specimen was collected. Leukocyte rich plasma (LRP) was prepared from  blood collected in acid citrate dextrose as an anti-coagulant slides as has been previously described.  Slides were air-dried and stained within 7  days. All cytospins prepared from the peripheral minutes  prior to immunocytochemical  1) Immunocytochemical  staining  The alkaline phosphatase detect the presence incubated  blood were fixed in acetone for 10  staining.  for L-selectin:  anti-alkaline  of L-selectin  with 5 % rabbit  phosphatase  (APAAP) technique  on cells (Cordell et al., 1984).  serum for 15 mm  secondary linking antibody. This incubation  minutes  was used to  Specimens  to block non-specific  were  binding  of the  was followed by the application  of the  primary antibody anti-Leu-8 (5g/ml) at room temperature 60 minutes.  and cytospun on coated  in a humidity chamber for  All antibodies were prepared in Tris Buffered Saline (TBS) pH 7.6 for 60  and 1 ±30 i l of the antibody  were determined  empirically  solution was used per slide.  by preliminary  titration  Antibody dilutions  experiments  and found to be  51 saturation  at 5gIm1. Non-immune  primary antibody dilution  were used as  of rabbit  anti-mouse  mouse IgG (5 Jhg/ml) and the omitting  negative  controls.  As a linking  IgG (Z259 DAKO, Denemark)  antibody  a 1/20  was applied  for 45  minutes; and finally, the anti-mouse conjugated with alkaline phosphatase (D651 DAKO, Denemark)  in a 1/50 dilution was applied for 45 minutes.  washed in TBS for 5 to 10 minutes following each antibody application. phosphatase  was developed  containing  by a final incubation  50mg naphthol-AS-BI-phosphate,  of the  of 20 minutes  complexes Slides were  The alkaline in a substrate  in 0.6ml N-N-dimethylformamide  added  to 0.5m1 of 4% sodium nitrite solution with 0.2m1 fuschin (Merck 4040: 5g in lOOmi 2M HCL) and lOOmi of TBS pH 8.7. Endogenous by adding 17.5mg levamisole filtered Harris-hematoxylin mounting  medium  to the solution. for 60 seconds.  and evaluated  alkaline  phosphatase  was blocked  The slides were counterstained  with  All slides were mounted in an aqueous  on a Zeiss Universal  Research  light microscope  (Model 1W, West Germany) at 400X magnification. 2) Immunofluorescent Immunofluorescent of L-selectin cytometry. prepared  flow cytometry staining of circulating  with FITC-conjugated  PMN was done to determine the expression  anti-Leu-8  A whole blood method for preparing  (Becton Dickinson,  CA) using flow  specimens was used. The cells were  from EDTA blood using a commercially  available  kit (Coulter Clone®,  Coulter Electronics, Florida). Briefly, 100d of EDTA blood was incubated PBS buffer and 0.24g FITC-conjugated in the dark.  anti-Leu-8 for 10 mm  with 1 200 . d  at room temperature  For each blood sample, a negative control was done using non-immune  mouse FITC-conjugated  IgG2a (Becton Dickinson, CA). After washing the cells twice,  the red blood cells were lysed (Immuno-lyse,  Coulter  Clone®).  The remaining  52 leukocytes were fixed with 1 % paraformaldehyde  and stored at 4°C. Flow cytometry  was performed on the specimens within 24 hours (Model; Profile EPIC II, Coulter Electronics,  Florida).  distinctive  Analysis  gates  forward and side scatter  evaluated  per specimen  fluorescence intensity  b) Evaluating  for the PMN were established profiles.  and are presented  using  the  A total of 3000 gated cells were  as either percent positive cells, mean  (log), or histograms.  L-selectin expression on bone marrow specimens  1) Bone marrow smears were made when bone marrow samples were collected, airdried and stored at room temperature,  and stained within 7 days using the method  described for cytospin specimens. 2) Immunohistochemical presence  of L-selectin  phosphatase buffered  staining of plastic embedded sections of bone marrow for the was performed  by a three  layered  biotin-avidin  alkaline  technique. Sections were allowed to hydrate for 15 minutes in 0.5M Tris  saline,  pH 7.6 (Sigma Chemical  Co, St.Louis, MO), trypsinized  with a  solution of 0.05% trypsin (Sigma Chemical Co, St Louis, Mo) prepared in TBS at 37°C for 5 minutes.  Endogenous  minute incubations  avidin binding  activity  was blocked by successive 20  of sections with avidin 0.1 % and 0.01 % biotin (Dako, Denemark)  (Woods, and Wanrke, 1981). The monoclonal antibody anti-Leu-8 (Becton Dickinson, CA) was used to label L-selectin. Each section was incubated temperature  in a humidity  chamber with lOOd of a 0.5g/ml solution of anti-Leu-8  prepared with 1 % BSA TBS. Non-immune MO) was used in a similar dilution Omitting  the primary  for an hour at room  antibody  mouse IgG (Sigma Chemical Co, St.Louis,  as the primary  antibody  was done as an additional  as negative  control.  control. Slides were  53 incubated  for 30 minutes  in biotin conjugated  goat anti-mouse  IgG (Fc receptor  specific) 15g/ml. A goat serum blocking step was omitted as goat sera do not react with Fe receptors of human leukocytes incubated  for 30 minutes  (Alexander and Sanders,  with avidin (4gIm1) conjugated  1977). Slides were  with alkaline phosphatase  (DAKO, Denemark).  All slides were washed twice in TBS for 15 mm  antibody application.  The alkaline  phosphatase  was developed for 15 minutes in the  dark with a commercially available kit (Histomark® Red, Kirkegaard Gaithersburg, producing  MD). This is a new Fuchsin-naphthol  a red-scarlet  counterstained  reaction  product.  with Mayer’s-hematoxylin  between each  and Perry Lab,  AS-BI phosphate  The sections  were washed  system  with TBS,  for 60 seconds, air-dried and mounted with  Coverbond®.  Immunocyto-  and histochemical  grading  On the cytospin and bone marrow smears, according to the intensity  systems PMN and band cells were evaluated  of staining of the cell surface using an arbitrarily  designed  grading system, grading PMN from negative (GO) to highly positive (deep red staining of more than 75 % of the PMN surface area, G4) (figure 4a-f). The slides were coded and examined  hypothermic  without  knowledge  CPB group.  Fields were selected in a systematic  and 100 cells were evaluated were evaluated  of their origin from either the normothermic  per specimen.  All cells of interest  except if the cell was broken or overlapping  randomized  or  fashion  in a selected field with other cells, as  overlapping  cells tend to trap stain between them. The inter-observer  determined  by having two observers grade 10 randomly selected slides, and the intra  observer variability  by having one observer repeating  variability  the measurements  was  3 weeks  54 later without knowledge of the origin of the slides or the initial results. The intensity  of staining  of PMN on the histological  less than on the cytological  specimens  were graded from negative  specimens of bone marrow was  (cytospins and BM-smears); therefore,  or background  (GO) to highly positive (deep red staining  of more than 50 % of the PMN surface, G3). Inter-observer system  was tested  photomicrographs variability  by having randomly  3 observers selected  evaluate  from  variability  of the grading  100 cells from a total of 15  10 patients  and  the  intra-observer  by one observer evaluate the same cells 3 weeks apart without knowledge  of the initial results. tested against  The reproducibility  a computerized  of the visual grading  high resolution  system (BioviewsInfraScan®)  analysis  PMN  intensity,  saturation  randomly  selected PMN were evaluated  system was further  true colour (1024 x10 , 24bit) image 24  used in our laboratory  that determines  and hue of a colour in an area that is digitized.  the  One hunderd  for the fraction of surface area that stained  intense red and were compared to the visual grading system in a blinded fashion.  Quantitative  histology  The L-selectin expression on PMN in sinusoids and in the hematopoietic  tissue of the  bone marrow were quantified  using standard  (Cruz-Orive  and Weibel, 1981; Gundersen,  1977; Steno, 1984). Toluidine Blue 0 (TBO) was used  to delineate  the vascular  structures  staining of bone marrow endothelial  morphometric  in the bone marrow.  markers  resulted  immunocytochemical  Immunocytochemical  cells (factor 8 and CD3 1) was used to confirm the  presence of vessels in the bone marrow identified vessel  techniques  in background  staining  by TBO. However, both of these of PMN  labelling of sections difficult to interpret.  that  made  double  Therefore, L-selectin  55  Figure 4: Photomicrographs (A) to CE) represent the range of staining of PMN in the circulation. PMN were graded according to the intensity of staining from: (A) GO (negative): (B) Gi (positive andJor <25% deep red stain of the cytoplasm): (C) G2 (between >25 and <50% of deep red stain of the cytoplasm): (D) G3 (between >50 and <75% of deep red stain of the cytoplasm): to CE) G4 (highly positive, more than > 75% deep red stain of the cytoplasm). Cells excluded were broken cells, overlapping cells and mononuclear cells. Both circulating PMN prepared from leukocyte rich plasma and bone marrow smears were stained by the APAAP method (see text). Photomicrograph (F) demonstrates the range of staining of PMN typically seen in a single field (from highly stained cells to negative cells). The bars represent a length of 5gm.  56 on PMN and the vasculature  of the bone marrow were stained on two adjacent serial  sections. The number of PMN per unit volume of bone marrow was estimated  using  the disector method (Steno, 1984). Five 2m thick serial sections of bone marrow were cut and labelled. Sections two (reference section) and four (lookup section) (4gm apart) were stained for the presence of L-selectin, and sections number one and five with TBO to delineate the vasculature. and eosin and used to identified which were used as the marrow  sinusoids  Section three was stained with hematoxylin  cells and structures  between slides two and four  disectors (figure 5). In five randomly  were identify  selected fields bone  in section one. The same sinusoidal  identified in sections 2, 4 and 5 and all areas of interest photographed Universal  research lightmicroscope  Gold (ASA 100) film and printed magnification  of photographs  of hematopoietic  as 100 x 150mm  estimated  by using standard  vessels,  such as bone spicules  Weibel, 1981; Gundersen, in the reference photograph  with a final  on photographs  of  of sections 1 and 5 (figure 6a). Volume fat spaces and bone trabeculae  point counting techniques  counting chambers placed over corresponding 4 using landmarks  photographs  of 3,630X. Vessels were delineated  tissue,  with a Zeiss  (Model 1W, West Germany) on 35mm Kodak®  sections 2 and 4 (figure 6b) using photographs fractions  vessel was  with quadrilateral  areas on photographs and megakaryocytes  were  unbiased  of section 2 and (Cruz-Orive,  and  1977). All PMN as well as band cells within the chamber (section 2) that did not appear in the lookup photograph  (section 4) were marked. Mature PMN were identified by their unique nuclear shape, dense chromatin with no nucleoli and granular cytoplasm if visible. All marked PMN were graded according to their intensity  of staining as mentioned previously. Section  1, 3 and 5 were used to verify the nature of any doubtful cells. Photographs  were  5.7  Serial sections for Disector Section 1  TBO  2  L-selectin  3  Spare (H&E)  4  L-selectin  5  TBO  <  Reference  Look-up  Figure 5: The disector method to quantify the number of PMN in each bone marrow compartment. Five 2m thick serial sections were cut and numbered 1-5. Sections 1 and 5 were stained with Toluidine Blue 0 (TBO) to delineate the vasculature and section 2 (reference section) and 4 (look up section) for the presence of L-selectih. Section number 3 was stained with hematoxylin and eosin and used to identify cells and structures between slides 2 and 4.  5S’  Figure 6: Photomicrograph (a) shows a bone marrow sinusoid demonstrated with a Toluidine Blue 0 stain of a 2m thick section of bone marrow embedded in glycolmethacrylate and photomicrograph (b) the corresponding serial section stained for the presence of L-selectin using the avidin-biotin alkaline phosphatase technique (see text) on PMN (arrow). The broken line represents the vascular margin of the sinusoid in the reference section. These PMN were graded from negative (GO) to highly stained (G3) when >50% of the PMN surface area stained deep red. The bars represent a length of 10im.  59  evaluated  in a single blinded fashion without knowledge of the patient from which  they came. A calculated  total of 0.03 135mm  of bone marrow tissue was examined  from each specimen. This was calculated:  VBM  where VEM represents  =  Lx Wx D  the total volume of bone marrow examined for each specimen,  L the length, W the width and D the depth of the disector. The length and width measured  on the microphotograph  final magnification unit  volume  calculated.  were converted to real length by dividing by the  of the tissue on the microphotographs.  ) of bone marrow 3 (lxmm  sinusoids  The number of PMN per  and hematopoietic  tissue  was  For example the number of PMN in the sinusoids was calculated:  =  N  where N,vSflUSQd  the number of PMN in the sinusoids of a particular  is  one cubic millimetre  specimen,  Vvsinusoid x VBM  of bone marrow,  the number  the volume fraction of sinusoids  specimen per  of PMN counted  in the specimen  and VBM the  volume of bone marrow examined in each specimen. Similar calculations for the hematopoietic  in the  were done  tissue and the number of cells in each grading category. All the  results were expressed as the number of PMN in each grade per cubic micrometer bone marrow compartment.  of  60  analysis:  Statistical Statistical  analysis was performed using SYSTAT® Version 5.1 software (Systat, Inc.,  Evanston,  IL) (Wilkinson,  1990).  The corrected white blood cell, PMN and band cell counts at each time point were  between  compared  groups and over time using  measurements.  repeated  a generalized  Differences in L-selectin expression  linear  model for  on PMN after ZAP  stimulation  were evaluated  temperature.  Differences between PMN L-selectin expression on circulating  using  a two-way  analysis  of variance  for each versus  bone marrow cells and differences between time points within groups and between groups  were analyzed  corrections  and  percentage  of baseline  using  testing  tested for statistical times. Individual  a two-way  for multiple  time points were considered statistically  Immunocyto-  presented  time points were for different  different from baseline if  variables  between groups.  to be significant.  The  data  A probability are  t-test of less  expressed  and histochemical  as  by calculating  scoring systems  variations in the immunocytochemical the scoring differences  grading system were  noted by two observers or by one  observer grading slides 3 weeks apart without knowledge of the identity Differences  as a  error except when otherwise mentioned.  Inter- and intra-ob server evaluated  data  Bonferroni  for that time point did not include 100%. Students  considered  was  mean± standard  With  with  significance by comparing 95 % confidence intervals  was used to compare demographic 0.05  comparisons.  of variance  (MFI), differences between experimental  a 95 % confidence interval  than  analysis  within  the range of the 95% confidence intervals  of slides.  (mean±2xSE)  were  61 considered  to represent  acceptable  reproducibility.  The inter- and intra-observer  variation of the immunohistochemical  scoring system were evaluated  the Pearson coefficient of mean-square  contingency(R  2 as a fraction of the maximum R coefficient of mean-square (the null hypothesis  2)  by calculating  for each grade and expressing  possible value, R 2 (Sachs, 1982). The Pearson  contingency  is an extension of the Pearson chi-square test  being that the rows and columns of a matrix are independent).  In the immunohistochemical  grading  score, 2 R m ax would represent  the value of the  Pearson  chi-square  between  and within observers: thus R= ±0.82. For analysis of both the inter- and  the intra-observer acceptable  coefficient  variation,  reproducibility.  visual grading  if there  is 100% agreement  an R IR ratio >0.75 2  A Spearman  was considered  rank correlation  system with the computerized  in the grading  score  to represent  was used to compare the  image analyzer.  62 RESULTS:  Surgical  procedure:  Table II shows data concerning the patient population studied and variables in the surgical procedure.  Although the total duration of the surgical procedure was longer  (p<O.Ol) in the normothermic  group, the time between the collection of BM1 and  BM2 and the pump times were not significantly  different between the two groups.  The longer surgical time was because more of the patients in the normothermic  group  required combined procedures (coronary bypass and valve replacements).  Hematological  results:  The total white blood cell count, PMN cell count the beginning  and band cell count rose from  (BMI) to the end (BM3) of CPB surgery  in both groups (Table Ill).  These data also show that the increase in white cell, PMN and the peripheral  blood  band cell counts  group  observed  between  BM1 and BM2 in the normothermic  (p <0.01) was prevented by hypothermia.  Rewarming  the patient after the completion  of surgery was associated with a rise in both the percentage  of penpheral  blood PMN  and band forms to levels that is comparable to the length of the surgery. The absolute number consistent  of these  cells were lower than  with the total duration  Immunocytochemical Examples  grading  of the staining  immunocytochemistry  in the normothermic  group, which was  of the procedure.  system:  achieved for L-selectin in cytological specimens using  are shown in figure 4a-f.  The inter- and intra-observer  differences were less than 5 % for grading both negative (GO) and highly positive PMN  ‘3  TABLE II Characteristics of Cardiopulmonary Bypass Patients  Normothermic  Hypothermic  n=5  n=5  Age (years)  67.2±8.3  57.4±10.8  Sex (MJF)  4/1  1/4  Coronary Bypass  3  2  Valve Replacement  0  3  Both  2  0  Pump time (mm)  120±27  68±26  Time cooled at 27°C  0  21±13.5  Surgery time (mm)  160±36  93±55*  Surgery:  with paired T-test  64 Table ifi Peripheral leukocyte counts during Cardiopulmonary Bypass surgery  Normothermic n=5 Corrected White Cell Count: Baseline 6.9±0.48 BM1 6.4±0.59 BM2 16.7±3 BM3 21.8±3.25’ Corrected PMN Cell Count: Baseline 4.9±1.52 4.6±0.47* BM1 BM2 13.1±2.12 BM3 18.7±2.2’ Corrected Band Cell Count: Baseline 0.25±0.07 0.18±0.03* BM1 BM2 2.98±0.56 BM3 6.24±1.06’  Hypothermic n=5 4.8±0.51 4.7±0.42 3.1±0.81 11.8±4 2.9±0.33 2.8±0.29 1.9±0.65 9.1±3.15 0.26±0.09 0.15±0.04 0.59±0.28 2.67±0.83  All leukocyte counts were corrected for hemodilution using the hematocrit and expressed as number x10 /L. Baseline, BM1, BM2 and BM3 are time points when 9 blood was collected (see text). Note that hypothermia prevented the rise in the white cell, PMN and band cell counts that occurred in the normothermic patients between BM1 and BM2. These counts was partially restored when the patients were rewarmed to normothermia at the end of surgery (BM3). Difference between BM1 and BM2 in the normothermic group, <0.05. Difference between baseline and BM3 in the normothermic group (‘p <0.01) and hypothermic group ($p <0.05).  65 (G4). Because intra-observer  variation  in grading the intensity  of staining  of PMN  was the smallest for the negative (3 ± 2.1, mean ± 2xSE) and highly positive (grade 4+) cells (4± 2.9), these observation  grades were used by preference  when comparing  differences between time points and groups (figure 7). Similar results were obtained in histological  sections with all the 2 R / R values  >0.75 with the highest value  being in the GO grade (0.91). There was a good correlation between the visual grading system and the computerized  image analyzer  (Infra-Scan)  grading  PMN (R=O.9)  (figure 8).  Immunocytochemistry  Table IV shows that the segmented PMN and band cells in the bone marrow stained strongly  for L-selectin with the majority of these cells faffing in the G4 category.  Under baseline conditions the peripheral  blood PMN stained less intensely  groups with more GO and Gi cells and less G4 cells (p< 0.01) (figure 9). normothermic  conditions  when there  was bone marrow  between blood and bone marrow become smaller hypothermic  release,  in both Under  this difference  (see table IV). However, under  conditions, where bone marrow release was slowed, the difference in GO  and G4 cells between blood and bone marrow persisted (p<O.Ol) (table IV). Figure 10 demonstrates  the changes in PMN L-selectin during cardiopulmonary  bypass (BM1  to BM2). Over this time period (BM1 to BM2), the number of highly positive PMN(G4) increased  significantly  in the circulation  of the normothermic  36 ± 6.6 %, p <0.03) but not in the hypothermic  group (9±3.3 to  group(10 .7±4.7 to 18 ± 5.7%, p =NS)  (table IV). The expression of L-selectin on band cells was marginally segmented  lower than on  PMN in the bone marrow smears (figure 1 la) but significantly  higher  40-  27  ()  I  14  ci) ci) ‘4-  ‘4-  U  1  1  1.  -12  -25 0  I  I  I  GO  Gi  G2  —  I  I  G3  G4  Grade Figure 7: Variability in grading the expression of L-selectin on PMN in cytological specimens. Slides were coded and examined without knowledge of their origin and in randomized fields on each slide a 100 segmented and non-segmented PMN were graded (see grading system figure 4). Grades are expressed as a fraction of the 100 cells counted. The graph demonstrates the inter-observer variability when one observer graded 10 slides 3 weeks apart without knowledge of the origin of the slides or the initial results. The y-axis represents the mean difference between the two observations and the error bars are two standard errors. The variability in grading negative (GO) and highly positive (G4) cells was small (<5%).  ‘7  100R=’0.91 80 C) .  60  -C  ct  1....  0  C)  HD  40Cl)  0 -  ‘4-  20-  C  0 GO  I  I  Gi  G2  I  G3  I  G4  Visual grading system  Figure 8: The correlation between the visual and Infrascan® grading systems. A 100 PMN on five randomly selected slides. Cellsd were stained (red) for the presence of L-selectjn with the APAAP technique. PMN were graded from negative (GO) to highly stained (G4) visually (see grading system, figure 4) and compared to the computerized high resolution true color image analysis system determining the area of the cell surface that stained intense red (y-axis is area of intense red). There was a good correlation (Spearman rank correlation, R=O.91) between the two grading systems.  a S  Go-.  •  )  Figure 9: The difference between L-selectin expression on PMN in the circulating blood (panel a) and the bone marrow (bone marrow smears, panel b). In the circulating blood a wide range of staining intensity (GO to G4) was seen in contrast to the bone marrow where the majority of PMN were highly stained (G4) (see table IV). Bar is 1Otm  ‘ci Table IV Changes in PMN L-selectin expression during cardiopulmonary bypass Gi  G2  G3  G4  37.4±6.4 1.8±0.9  17.8±2.5 14.6±2.6  9.4±2.4 35±3.4  9±3.3t 47±4.3  Blood 8.2±5.2 Bone marrow 0.7±0.4  12.8±2.68 5.1±1.6  16±3.1 18.5±1.5  27.4±3.6 31±4.2  36±6 45±2.7  Blood 4.4±2.4 Bone marrow 1.7±1.4  12.2±3.6 4.5±1.5  18.8±4 18.3±5.1  31.8±3 26.5±3.6  32.6±6.4 49±8.3  Ilypothermic: Blood* BM1: 28±10.1 Bone marrow 0.2±0.1  27.2±3.7 4.2±1.1  17.7±3.1 15.8±4.3  15.7±4 22.6±3  10.7±4.7 57.4±6.3  Bloods 17.8±11 Bone marrow 0.4±0.4  16.8±4.2 9.6±1.7  18.6±3.6 21.2±2  29.4±8 26.4±5.9  18±5.7 42.2±6  Blood 8.2±4.8 Bone marrow 1.4±1.4  18.6±6.4 7.2±3.3  16±1.9 15.4±2.1  24.2±4.2 28.6±3  32.6±8.6 47.4±4  Grades  GO  Normothermic: Blood* BM1: 26.2±6.5 Bone marrow 0.4±0.2 BM2:  BM3:  *p <0.01, $p <0.05; Difference between L-selectin expression on bone marrow and circulating PMN. tp < 0.03; An significant increase in highly stained PMN with bone marrow release in the normothermic group (BM1 to BM2) which was suppressed in the hypothermic group. Grades; On bone marrow smears and PMN in circulating blood, a 100 PMN were graded from negative (GO) to highly positive (G4), see text for grading system (figure 4). BM1,BM2 and BM3 referred to time points blood and bone marrow specimens were collected, see text.  70  a *  z 0 C  LJ  C.)  b  GO  Gi  G3  02  04  Grades BM1  BM2  Figure 10: Immunocytochemical determined L-selectin expression on circulating PMN at the start (BM1 before bypass, solid bars) and the end (BM2 end of bypass, hatched bars) of normothermic cardiopulmonary bypass surgery (a). With bone marrow release of PMN in the circulation the number of strongly positive (G4) cells increased and negative (GO) cells decreased (*p <0.01). However, when active bone marrow release was suppressed by hypothermia (b), L-selectin expression on circulating PMN remaines the same. One hundred PMN were counted for each specimen and the y-axis represents the fraction of those PMN. Values are the mean±SE of 5 patients.  71 than segmented  PMN in the circulation  (figure 1 lb).  Flow cytometry: Figure 12 is an example of the changes in L-selectin expression PMN as measured hypothermic obtained Intensity,  by flow cytometry  (figure  during  12d-f) cardiopulmonary  by flow cytometry  normothermic  bypass.  Figure  on circulating  (figure  12a-c) and  13 shows the data  where the expression of L-selectin (Mean Fluorescence  MFI) on circulating  PMN in the normothermic  group increased  stepwise fashion from 10.8±4.4 at baseline to 12.2±4.9. This represents increase in MFI(log) of circulating  in a  a 16±9.8%  PMN L-selectin expression at the end of the bypass  procedure (p <0.05). In the hypothermic  group, on the other hand, the MFI changed  little over the same period and tended to fail at the end of the surgical procedure.  Morphometric  studies:  The staining intensity  of PMN in the histological  specimens of bone marrow was less  than that achieved on bone marrow smears due to the light glutaraldehyde step  required  to preserve  glycolmethacrylate.  morphology  and  the processing  of the  fixation  tissue  into  In each group one patient was excluded from the morphometric  analysis because of inadequate  (hypoplastic) bone marrow tissue. At baseline in both  groups (figure 14), a significant  difference in the expression of L-selectin on PMN was  seen between the sinusoids negative  and hematopoietic  PMN the highest in the sinusoids  and L-selectin  positive  compartments  with the fraction of  (24.7±3.5 versus 10.3 ±2.5 %, p <0.004)  PMN (G1-G3) higher  in the hematopoietic  compartment  (88 ± 3.1 versus 69.5 ±4.8%, p <0.005). The expression of L-selectin on PMN in the  72. 70  -  a 60 50  C  40  F  C  :U  30 2O 10-  0  —  GO  Gi  G2  —  G3  G4  Grades  b  ‘-4  C C  T  U  0  I GO  Gi  G2  G3  G4  Grades  Segm PMN  Bands  Figure 11: The expression of L-selectin on segmented PMN (solid bars) and band cells (hatched bars) in the bone marrow (a) and the circulating blood (b). Note the small difference in L-selectin expression between segmented PMN and band cells in the bone marrow but a significant difference between these cells in the circulation during bone marrow release of PMN. Slides were randomly selected from time points (BM2 and BM3) with peripheral band cell counts >10% of total PMN counts, allowing 100 band cells to be evaluated per slide. Values are the mean±SE of 13 time points.  73  BM1  BM2 a  D 0 C.)  2  BM3 b  C  2  2  i.._ :d..  A G)  —a  e  f  C)  .  :  1 —  Mean Fluorescence Intensity Figure 12: Expression of L-selectin on PMN during bone marrow release of PMN in a normothermic (panels a to c) and hypothermic (panels d to f) patient as measured by flow cytometry. Note the increase in L-selectin expression in the normothermic patient during bone marrow release of PMN (BM1 to BM3). The two populations of cells seen in panel (b) may represent a population of PMN that have shed their L selectin with intravascular cell activation (left) and a population of PMN released from the marrow with high levels of L-selectin (right). No evidence of an increase in L-selectin during bypass was seen in the hypothermic patient (BM1 to BM2). In each specimen 3000 PMN were evaluated and expressed as mean fluorescence intensity on a Log scale (x-axis).  74  140  124  N=5 *  108  -  Norm Hypo  CtJ  92  76  60  Baseline  BM1  BM2  BM3  Time Intervals Figure 13: Expression of L-selectin on circulating PMN as measured by flow cytometry at the different time intervals in 5 normothermic and 5 hypothermic CPB procedures. In each specimen 3000 PMN were evaluated and expressed as mean fluorescence intensity (MFI) on a Log scale. Values in this graph are expressed as a percentage of baseline MFI values (obtained before anaesthesia and surgery start). All values are the mean±SE of 5 specimens. In the normothermic group the percentage changes in the MFI over time increased significantly (*p <0.05) whereas hypothermia was associated with either no change or a slight decrease in MFI at the end of surgery. Norm=normothermic group Hypo =hypothermic group  75 hematopoietic  compartment  did not change over the study period (Table V). Figure  15 shows that with bone marrow release of PMN in the normothermic selectin  negative  PMN (GO) increased  from  5.75±1.1 x 10 at baseline  14.6±2.8 x iO PMN/mm 3 at the end of bypass hypothermic  group it stayed  a gradient  in the  the same (6.2 ± 1.2 to 6.3 ±0.82 x iO, p =NS).  from 24 to 46 % of all the PMN in the sinusoids  demonstrate  (BM 1) to  (BM2), P <0.03, whereas  increase in the GO PMN in the sinusoids in the normothermic increase  group, the L  of PMN L-selectin  This  group represents  an  (Table V). These data  from the hematopoietic  tissue  to the  sinusoids at baseline that increased during active bone marrow release of PMN and remained  the same when marrow release was prevented  In vitro studies  by hypothermia.  of L-selectin:  Figure 16 shows the flow cytometry data concerning the expression of L-selectin and  CD18 on PMN incubated at 37, 27 and 4°C stimulated  with increasing concentrations  of ZAP ranging from 0 to 5%. At 37°C, PMN shed L-selectin and upregulated in a dose dependent  manner.Incubation  the effect of ZAP stimulation  of the cells at 27°C (figure 1 6b) suppressed  except at the highest dose and incubation at 4°C (figure  16c) eliminated  the effect of ZAP even at the highest dose. PMN incubated  and stimulated  with low concentrations  reflected by the increased  CD18 expression.  of CD18 upregulation  PMN incubated  However, little changes  staining  and L-selectin shedding  at 37°C. These findings  at 27°C  of ZAP (0.01 and 0.1 %) were activated  selectin expression was seen at these low concentrations dissociation  CD18  were supported  and grading of PMN on cytospins of peripheral  as  in PMN L  of ZAP stimulation.  This  was not apparent  with  by immunocytochemical blood, which showed no  76  100-  *  80-  GO  z a  #  60-  G1  0 0  G2  40-  C, I.  G3  IL  20-  0  Hematopoietic  Sinusoids  Figure 14: Combined baseline data for both normothermic and hypothermic groups. The fraction of cells negative for L-selectin (GO) was greater in the bone marrow sinusoids than in the bone marrow hematopoietic tissue in both groups of patients (*p <0.004), and the fraction of positive PMN (sum of Gi, G2 and G3) was greater in the hematopoietic compartment (‘p < 0.005). Values are the mean±SE with n=8.  14955 1653  815  2049  828  6761  12023  4081  1232  6330  14294  6217  1727  1739  2896  342  13141  14623*  15429  2959  1153  6238  10992  G,  5750  0 G  Venous sinusoids  4175  7636  2014  5159  1532  3671  573  573  932  1597  2164  2927  2 G  3917  6602  2108  6352  2087  4748  3436  3436  877  2415  1974  3500  3 G  3156  7303  585  4961  1293  5229  5258  6534  3247  7104  1829  4763  0 G  11082  34635  5294  27799  4909  35404  3091  28457  2591  22546  14801  38733  S, 2 G  2084  5880  1167  11471  1323  10269  7503  10746  5888  8850  2551  3992  Hematopoietic tissue  L-selectin expression on PMN in the bone marrow  The values represents the number of 3 PMN/mm in each bone marrow compartment. The values in italics are the standard error of the mean of four patients. *Represents a significant increase of GO graded PMN from BM1 to BM2 (p<O.03).  BM3  BM2  BM1  Hypo  BM3  BM2  BM1  Normo  Grade  TABLE V  8362  17848  12286  23207  3501  13146  6939  16329  5640  9504  4365  6832  3 G  7g  20  *p<003  *  $ .—  0  100  z w  z 0 BM1  Hypothermic  B2  Normothermic  Figure 15: Changes in the number of negative graded PMN (GO) in the marrow sinusoids at the start (BM1) and at the end (BM2) of bypass. With bone marrow release of PMN in the normothermic group (hatched bars), the number of GO PMN increased significantly (p < 0.03). However, when bone marrow release was prevented by hypothermia, the number of GO cells in the sinusoids remained the same. The y axis represents the number of PMN per cubic millimetre of bone marrow. Values are the mean±SE of four patients in each group.  37°C 7’?  0%  0.01%  0.1%  1%  300-  5%  27°C  *  T  4-I  0 C)  240-  4-I  C) 0 C) C) 0 C) I  *  180-  120-  0  LI ci C)  80-  0 0.01%  0.1%  1%  5%  4°C  0%  0.1% 1% 0.O% Zymosan Activated Plasma  Figure 16: Flow cytometry data concerning the expression of L-selectin and CD18 on PMN incubated at 37, 27 and 4°C in vitro and stimulated with increasing concentrations of zymosan activated plasma (ZAP) ranging from 0 to 5%. The solid bars represent L-selectin and the hatched bars CD18 expression. Note that at 37°C, PMN shed L-selectin and upregulate CD18 in a dose-dependent manner. However, these changes are suppressed at 27°C and abolished at 4°C. At 27°C a dissociation between L-selectin shedding and CD18 upregulation was seen with stimulation of PMN with low concentrations (0.01 and 0.1%) of ZAP. Values of mean fluorescence intensity are the mean±SE of 5 experiments with all values expressed as a fraction of baseline.  05 *p<O.  $p <0.02  80 change in the number of negative PMN when stimulated ZAP at 27°C in contrast to a significant  with low concentrations  increase at 37°C (figure 17).  of  8I  *  z 0 ci)  4°C  >  C)  27°C  C  —  4-’  C)  P01  a) C) 0  37°C  IT  0  0% 0.1% 5% Zymosan Activated Plasma Figure 17: Changes in the negative graded PMN (GO) incubated at 37, 27 and 4°C stimulated with no(control or 0%), 0.1% and 5% zymosan activated plasma. PMN on cytospin specimens were stained for L-selectin by the APAAP method in 5 experiments. Note the significant increase of L-selectin negative (GO) cells with a mild stimulus (0.1% ZAP) in the 37°C incubated cells that was not seen in the 27°C incubated cells.  *p<o. 05 05 #p<O.  82 DISCUSSION This  study  confirms  leukocytosis  several  during CPB-surgery  that hypothermia  a temperature  reports  (Chenoweth  showing  that  there  is systemic  et al., 1981; Quiroga et aL, 1985) and  prevents this leukocytosis by suppressing  of PMN. The quantitative  sensitive  previous  the bone marrow release  histology of the bone marrow and the in vitro studies link  sensitive L-selectin loss from the surface of the PMN to a temperature  release of these cells from the bone marrow hematopoietic  marrow sinusiods. They further demonstrate bone marrow and the circulating  tissue into the  a PMN L-selectin gradient between the  blood that decreases  with active bone marrow  release of PMN.  Evidence for bone marrow release of PMN: The PMN storage pool in the bone marrow is estimated  to be 5.6 x iO cells/kg with  ± 40 % of these cells being mature segmented neutrophils  and ± 60 % being band forms  and metamyelocytes  (Boggs, 1967). Under stable conditions, the PMN released from  bone marrow are mainly mature segmented PMN and these cells form the bulk of the so-called marginated the circulation  pool of cells. The marginated  with moderate stress such as exercise (Muir et al., 1984; Foster et al.,  1986) whereas more severe stress associated activation circulation.  cells can be rapidly mobilized into  is required Although  to release  with trauma,  band cells and rarely  the ratio of segmented  infection or complement metamyelocytes  to band cells released from the bone  marrow is not known and probably varies with the type and the intensity stimulus,  an increase in the circulating  into the  of the  band cell count provides definitive evidence  that PMN are being released from the bone marrow.  Our study shows an increase  83 in circulating  PMN during  either the intravascular  CPB-surgery  marginated  and the source for this increase  is from  or the bone marrow pools. As bypass of the  pulmonary circulation removes a major source of marginated  cells (Peters et aL, 1985)  the release of cells from the bone marrow is the most likely source of the increased number of circulating marginated  leukocytes  in these studies.  Furthermore,  release from the  pool is characterized  by an increase  in circulating  PMN without  a  concomitant  increase  Hetherington  and Quie, 1985). Release from the bone marrow, on the other hand, is  characterized  by an increase in the number of circulating  in the  number  blood (Boggs, 1967; Hetherington  of circulating  band  cells  (Boggs,  1967;  band cells in the peripheral  and Quie, 1985; Marsh et aL, 1967; Jagels  and  Hugli, 1992). In this study, the band cells increased from 5 % of the total PMN count at baseline  to more than 25 % of the circulating  groups (see table Ill). The fact that hypothermia cells in the circulation  and rewarming  pool at the end of surgery in both suppressed the total number of band  restored band cell counts to normothermic  levels confirms the previous report from our laboratory that the bone marrow release of PMN is a temperature  dependent  phenomenon.  Evidence for L-selectin loss when PMN cross the bone marrow-blood barrier The release peripheral (Athens  of bone marrow neutrophilia  induced  et al., 1961; Kajita  intravascular  complement  cause morphologic  stores of PMN appear  lived. It seems unlikely  accounts  for the  by many factors known to promote leukocytosis  and Hugh,  activation.  and adherent  to largely  1990; Jagels  Chemotactic  properties  and Hugli,  factors that induce neutrophilia  of the stimulated  that PMN recruitment  1992) including  cells that are short-  from the bone marrow represents  a  84 chemotactic  response per  Se,  at all. However, stimulation  because a gradient would exist for only a brief period if of bone marrow cells by chemotactic  both morphologic and cytoskeletal and reorganization either  changes within the PMN, as well as modifications  of cell surface molecules involved in adherence between PMN and  endothelium  elements  (Griffin et al., 1990; Von Andrian  et al., 1991) or stromal  of the bone marrow.  L-selectin  is expressed  throughout  myeloid  Lewinsohn  et al (1987) demonstrated  by nearly  all circulating  differentiation  PMN more or less continuously  in the bone marrow that  (Griffin et al., 1990).  in mice mature  expressed higher levels of L-selectin (MEL-l4) than peripheral we know of no previous quantitative PMN in the bone marrow expression  of L-selectin  bone marrow circulating  PMN  PMN, but  study comparing the expression of L-selectin on  compartments  was highest  (figure 14) in the bone marrow significant  factors may induce  of humans.  Our data  on PMN in the hematopoietic  and that this compartment  show that  the  compartment  did not change  to a  extent over the study period (see Table V). PMN in the bone marrow  sinusoids expressed less L-selectin than those in the hematopoietic  compartment  with  more L-selectin negative and less L-selectin positive PMN. The baseline difference in L-selectin  expression  hematopoietic  compartment  the bone marrow-blood  between  several explanations.  and  those  in the  (figure 14) suggests that L-selectin is shed when crossing  endothelial  from sinusoids into the circulation. L-selectin expression  the PMN in the sinusoids  barrier or gradually Our observation  lost as PMN are released  that there is a difference in PMN  across the bone marrow- blood barrier at baseline could have The immature  myeloid cells in the bone marrow make close  85 contact with the processes of adventitial  reticular cells and may be anchored to these  cells or the matrix through pro-adhesive lectin-like adhesion molecules. of these  molecules  movement  towards  Alternatively,  during  maturation  the sinus  under  stable  walls and eventual  conditions  release  the process of crossing the endothelial  Gradual loss might  permit  into the circulation.  barrier may result in a loss of  L-selectin from the PMN surface similar to that which occurs with recruitment PMN into an area of inflammation. band forms that have entered marrow endothelium entry  the marrow  with an overshoot  are anchored  cleavage  leukocytosis  resulting  to the  in a rapid  such as that  seen in  bypass surgery.  L-selectin is expressed on granulocytes during cell activation and regulation  is that the mature PMN and  microvasculature  by L-selectin with subsequent  into the circulation  cardiopulmonary  A third possibility  of  (Smith et al., 1991; Tedder, 1991).  of shedding  by a membrane  and is known to shed from the surface of PMN  is unknown,  bound protease  enzymatic  Although the mechanism  cleavage of the surface receptor  with chymotrypsin  activity  is the most likely  possibility (Spertini et aL, 1991b; Tedder, 1991). Cell activation  is thought to induce  changes in the conformation  of the L-selectin protein and expose nascent  are susceptible  cleavage.  to enzymatic  bone marrow during rewarlning from 35-37°C temperature  (Quiroga  sensitive  after CPB occurs over a narrow temperature  negative  the possible  range  role of a  cleavage of L-selectin in the release of PMN from  the bone marrow. Our data demonstrate L-selectin  The fact that the release of PMN from the  et al., 1985) led us to consider  enzymatic  sites that  an increase in the number and fraction of  PMN in the bone marrow venous sinusoids  with active bone  86 marrow release of PMN (figure 15 and Table V) which was also accompanied  by an  increase in the total number of PMN in the sinusoids  (Table V). This suggests that  the leukocytosis  not only released  induced  by complement  activation  sinusoids but also involves egress of PMN from the hematopoietic  PMN the  compartment  to the  venous sinusoids.  This increase was not seen under conditions where the bone marrow release of PMN was suppressed. responsible  The in vitro  studies  suggest  for L-selectin cleavage from the activated  by reducing the temperature  of PMN activation  when L-selectin  shedding  of the enzyme  PMN surface was suppressed  to levels that prevent marrow release.  in L-selectin shedding was independent CD18 was upregulated  the activity  that  This reduction  by ZAP stimulus because  was suppressed  at 27°C.  This  functional dissociation between PMN activation and L-selectin shedding suggests that the enzyme responsible  for L-selectin cleavage is functionally  disabled at 27°C and  totally inactive at 4°C. It also suggests that the enzyme responsible  for the cleavage  of L-selectin during marrow release may not be one of the granular proteases released from activated  cells.  The concept that bone marrow release is related to the loss of L-selectin is supported by the previously  reported observation  that myeloid cells with low expression of L  selectin tend to be released from the bone marrow into the circulation 1990).  It is also of interest  hyperplasia  that patients  receiving  with an increased number of circulating  (Griffin et al.,  GM-CSF develop marrow  progenitor cells (Platzer, 1989)  and that GM-CSF induces shedding of L-selectin from all myeloid cells including  the  87 progenitor cells. These observations  are consistent with the hypothesis that cleavage  of L-selectin from the cell surface contributes  to the relocation of progenitor cells from  the marrow into the blood (Griffin et al., 1990). shown to induce complete shedding hour of administration,  GM-CSF has been  of L-selectin from circulating  PMN within one  with low L-selectin expression on the PMN persisting  to 6 days (Demetri and Antman, following  Furthermore,  1992).  GM-CSF administration  This implies that the resulting  may be related  to cleavage  for up  leukocytosis  of L-selectin  from  myeloid cells in the bone marrow. Spertini et al (1991b) have demonstrated  that acute  myelomonocytic  express  leukemia  and chronic myeloid leukemia  selectin, in contrast to their normal counterparts. on these  cells could explain  their  relocation  cells rarely  L  The lack of L-selectin expression from the bone marrow  into  the  bloodstream.  The present studies show that shedding of L-selectin from the surface of PMN can be inhibited  by hypothermia,  baseline  conditions,  hematopoietic  both in vivo and in vitro.  the expression  compartment  of L-selectin  They also show that under  is greatest  on the PMN in the  of the bone marrow and that this expression is decreased  in the bone marrow sinusoids.  Although  several possible mechanisms  could explain  this result, the data clearly show that L-selectin expression is reduced as the PMN leave the marrow  and that  suppressed  by hypothermia.  Regulation  of cell-matrix  promoting  this change  is prevented  and cell-cell interactions  receptors on the surface of leukocytes  when  marrow  via the expression  release  is  of adhesion  has been proposed as a possible  88 mechanism  for controffing  Wicha, 1988; Hynes, 1990b; Williams  1992; Miyake et a!., 1990a; Simmons  et al., 1991).  receptors for the universal on erythroid marrow  the egress of PMN from bone marrow (Campbell,  progenitors  extracellular  matrix, hemonectin,  is that the loss of  peptide, arginine-glycine-aspartic  is believed to initiate  (Tsai et aL, 1987).  et aL, 1992 Springer,  An example of this mechanism  attachment  Another  protein  binds more specifically  anchor them in the marrow until full maturation  acid (RDG),  the release of reticulocytes  adhesion  and  from the  in the bone marrow  to myeloid cells and may  (Campbell et al., 1987a).  However,  the nature of this protein, the receptor on the myeloid cells for hemonectin  and the  factors that control this adhesion event are not known. In this study we have linked the release of PMN from the bone marrow to the shedding  of L-selectin from their  surface. This adds to the body of evidence that the adhesion promoting molecules play a critical role in the egress of leukocytes shedding  from the bone marrow. We speculate  of L-selectin from the surface of PMN may be an important  that  step in their  release from the bone marrow.  Evidence of increase in expression of L-selectin on circulating PMN during active bone marrow release L-selectin is expressed by nearly all circulating  PMN and post-mitotic  myeloid cells  in the bone marrow (Collins et al., 1991; Griffin et al., 1990; Spertim et al., 199 ib). Lewinsohn  et a! (1987) demonstrated  in mice that  mature  expressed higher levels of L-selectin (MEL- 14) than peripheral a reduced  expression  of L-selectin  (Lewinsohn et al., 1987). Lund-Johansen  bone marrow circulating  (IVIEL-14) on more immature  PMN  PMN, with  myeloid  cells  et al (1993) have also recently demonstrated  89 in that humans L-selectin increases on bone marrow PMN with cell maturation.  We  know of no other study directly comparing the expression of L-selectin on PMN in the peripheral  blood and bone marrow compartments  of humans  during  active bone  marrow release of PMN.  Evaluating  bone marrow smears showed at baseline the expression of L-selectin was  higher on bone marrow PMN than on cirrculating and hypothermic  PMN in both the normothermic  groups and that it did not change to a significant  study period (Table IV). There was less L-selectin on circulating  extent over the  PMN (more negative  or Gi cells and fewer highly positive or G4 cells) than on the bone marrow PMN in both groups of patients  under baseline conditions  (Table IV). Furthermore,  as the  PMN on bone marrow smears are diluted by blood from the bone marrow venous sinusoids,  our estimate  of the L-selectin expression  compartments  is probably  hematopoietic  compartment  on PMN in the hematopoietic  low, and the PMN L-selectin and the circulating  gradient  between  the  blood is probably even larger than  observed. The difference in L-selectin expression  between bone marrow PMN and  PMN in the circulation under baseline conditions suggests that L-selectin may be lost as PMN cross the bone marrow-blood barrier or that circulating lose L-selectin during their intravascular mixture of cells from the hematopoietic  PMN progressively  life. As bone marrow smears contain a  tissue and bone marrow venous sinusoids, the  reduction in PMN L-selectin crossing the bone marrow-blood barrier is unlikely to be the sole reason for the difference in L-selectin expression we observed between bone marrow smears and cytospins. Loss of L-selectin from the PMN during their lifespan in the general circulation  is a likely explanation  and this hypothesis will be explored  90 in the second part of this thesis.  L-selectin  expression  expression  on bone marrow cells during normothermic  remained  on circulating  PMN increased  and approached  the level of  CPB surgery, but it always  lower than on bone marrow PMN (Table IV). As this increase  occurred  when the PMN were being released from the bone marrow, it is consistent with new cells containing  more L-selectin entering the circulation.  blood PMN expressing  the highest  release in the normothermic selectin expression that notwithstanding  The increase in peripheral  levels of L-selectin  group supports  in the circulation  (G4) during  bone marrow  this concept. The increase in PMN L  during bone marrow release of PMN suggests  the lost of L-selectin in the bone marrow during PMN release,  the released PMN expression of L-selectin is still higher than PMN in the circulation under became  baseline  conditions.  more permeable  This may imply that during  active release  the bone marrow-blood with less shedding  barrier  of L-selectin  crossing the barrier. The bone marrow-blood barrier has been described as becoming more permeable during active bone marrow release of PMN (Weiss, 1970, Tavasolli., 1977).  The alternate  possibility  while they are in the circulation either  that PMN upregulate  is much less likely because there are no reports of  in vitro or in vivo upregulation  activation.  Furthermore,  of L-selectin  the low levels of L-selectin  suggest either message instability  their L-selectin expression  in mature  mRNA in circulating  or low levels of translation  of PMN was suppressed  cell cells  and processing (Tedder  et al., 1989) and imply that L-selectin is unlikely to regenerate  When the bone marrow release  PMN during  on circulating  by hypothermia,  PMN.  the L  91 selectin expression on circulating However, restoration an increase selectin.  Assuming  of bone marrow release by rewarming  in the number  of peripheral  These observations  circulating  PMN is highly that  PMN remained close to baseline levels (figure lOb).  the majority  establish  the patient resulted  blood PMN expressing  in  high levels of L  that the level of L-selectin expression on the  dependent  on their  release  from the bone marrow.  of band cells in the circulation  have been recently  released from the bone marrow, our finding that band cells in the circulation  express  higher levels of L-selectin than segmented PMN (figure 1 ib), supports the hypothesis that peripheral  blood PMN expressing  high levels of L-selectin have been recently  released from the bone marrow.  Interestingly,  the expression of L-selectin on band cells in the bone marrow was lower  than on segmented  PMN, which suggests  that the mature  bone marrow contain the most L-selectin. This observation Lund-Johansen  segmented  PMN in the  supports the findings of  et al (1993) that L-selectin expression increases with PMN maturation  in the bone marrow. The segmented shown to be more deformable  mature  PMN in the bone marrow have been  and reactive to chemotactic  stimuli  than immature  myeloid cells (Lichtman,  1970). These are the cells released from the bone marrow  under baseline conditions  (Rosolia et al., 1992; Ulich et al., 1989). With a stimulus  for bone marrow  of PMN, the mature  circulation.  release  With a more intense  or prolonged  metamyelocytes  appear in the circulation  Muller-Eberhard,  1979; Kampschmith,  activates  intravascular  PMN are the first to enter stimulus,  the  band cells and rarely  (Deinard and Page, 1974; Ghebrehiwet  and  1984; Platzer, 1989). As such a stimulus  also  PMN to shed L-selectin,  our finding  of an increase  in L  92 selectin  on circulating  activation  PMN shows that  the cumulative  effect of complement  during CPB surgery favoured bone marrow release.  The significantly  lower L-selectin expression on circulating  PMN can be partly explained  marrow-blood increased  barrier.  PMN than bone marrow  by the loss of PMN L-selectin when crossing the bone  Notwithstanding  this loss, L-selectin  on circulating  with bone marrow release of cells. Therefore, the wide distribution  selectin expression on circulating  PMN can be explained  PMN of L  as a mixture of cells with  high L-selectin expression that have recently been released from the bone marrow and a population  of older PMN that have lost some or all of their L-selectin while  they are in the circulation. of PMN expressing circulating  Cardiopulmonary  bypass results in bone marrow release  high levels of L-selectin,  PMN expressing  which increases  the population  of  high levels of this cell adhesion molecule.  Summary  1) We have shown that under baseline greatest  conditions  the expression  of L-selectin is  on bone marrow PMN and that there is a stepwise decrease in expression  from the hematopoietic  compartment  in the bone marrow  to the bone marrow  sinusoids, and a further decrease to the circulation. 2) The shedding hypothermia,  of L-selectin  from the surface  of PMN can be inhibited  by  both in vivo and in vitro.  3) Our data further support the concept that L-selectin is partly shed from the PMN as they cross from the hematopoietic sinusoids with a stimulus  compartment  into the bone marrow venous  for bone marrow release such as complement  activation.  93 4) With bone marrow release of leukocytes, L-selectin expression on circulating increased,  supporting  the concept that circulating  PMN  PMN expressing high levels of L  selectin are cells recently released from the bone marrow.  94 5) CHANGES  IN L-SELECTIN  ON CIRCULATING  PMN  Introduction The selectin family of adhesion-promoting are found on endothelial  molecules, including L-, E- and P-selectin,  cells (E- and P-selectin), platelets  (L-selectin). They mediate leukocyte rolling and margination  (P-selectin) and leukocytes  of PMN in post capillary  or collecting venules (Ley et al., 1993; Ley et aL, 1991b; Von Andnan et aL, 1992; Von Andrian selectin  et aL, 1991), the earliest  manifestation  has been shown to be essential  of leukocyte  recruitment.  in the leukocyte-endothelial  interaction  cascade of events in vitro and in vivo that result in PMN recruitment inflammation  L  to foci of  (Butcher, 1991; Ley et aL, 1991b; Mulligan et aL, 1994; Von Andrian  et al., 1991; Springer,  1 990b). Recruitment  of PMN to the inflamed peritoneal  of mice can be blocked by blocking antibodies  cavity  to L-selectin or L-selectin-Ig chimera  (Jutila et aL, 1989a; Jutila et al., 1989b; Watson et al., 1991). In two models of PMN dependent acute lung injury in rats (intrapulmonary intravascular documented important  complement  activation),  the requirement  (Mulligan et aL, 1994). These data suggested role in the recruitment  vessels.  The selectin-dependent  adhesive  initial  of L-selectin  that L-selectin plays an  interaction  between  -integrins 2 of the f3  well as ICAM-1 and ICAM-2 on the endothelium that accompanies  eventual  transmigration  reaction  the leukocytes  is followed by the second phase of leukocyte recruitment,  to depend on the engagement  has been  of PMN during the acute inflammatory  in the systemic and pulmonary  endothelium  deposition of Ig-G complexes and  which appears  (CD 11/CD 18) on the leukocytes for the firm adhesive  into the extravascular  and  as  interaction  space (Butcher,  95 1991; Larson and Springer, 1990; Zimmerman et al., 1992). This transmigration  has been shown to result in the loss of L-selectin (Kishimoto  et al., 1989; Julita  from the surface of the PMN  et al., 1989). However, if the conditions  adhesion  and emigration  Whether  this latter event results in a loss of PMN L-selectin is unknown.  This hypothesis  are inadequate,  is attractive  PMN may return  considering:  to the endothelium  circulation.  for firm  to the circulation.  1) Shedding of L-selectin could provide a rapid means for the regulation adhesion  event  Receptor-ligand  with the subsequent binding would initiate  means of receptor modulation.  of leukocyte  release of PMN back into the receptor shedding and serve as a  This concept is attractive  considering the fact that just  a small fraction of PMN delivered to a focus of inflammation  eventually  emigrate  (Coxson et al., 1990; Doerschuk et al., 1994). 2) The interaction  of leukocytes  result in L-selectin  shedding.  “roll” on unactivated endothelium  with “normal”  Using intravital  endotheium,  are activated  unactivated  endothelium  microscopy, leukocytes  which becomes  (Lawrence, and Springer,  may also tumble and  more pronounced  when the  1991; Smith et aL, 1991). In the  pulmonary capillary bed, leukocytes make intimate contact with the endothelium have to deform to negotiate result in PMN L-selectin  their passage through the lung. Both of these events may being lost. The demonstration  slectin in plasma under normal conditions shed L-selectin in the circulation 3) The variable  suggests  (Schleiffenbaum  of high levels of free L  that leukocytes  high, intermediate  PMN released in the circulation  continuously  et al., 1992).  expression of L-selectin on PMN in the circulation  of cells expressing  and  with populations  and low levels of L-selectin contrasts  with the  from the bone marrow which expresses high levels  96 of Lse1ectin (as dicussed in previous chapter). This suggests  that L-selectin is lost  from the surface of PMN during their lifespan in the circulation.  The second part of this thesis explores the fate of L-selectin on PMN during their lifespan in the circulation.  97  WORKING HYPOTHESIS Our working hypothesis 1) Circulating  PMN  intravascular  is that: shed  life resulting  L-selectin  surface  in “older” PMN expressing  2) Low L-selectin expression from the intravascular  from their  during  their  normal  lower levels of L-selectin.  on “older” PMN is a signal for removal of these cells  pool.  As L-selectin is an activation  sensitive  cell surface receptor, this hypothesis  be tested using PMN labelled and transfused  to recipients  had to  with the least possible  manipulation.  SPECIFIC  AIMS  This hypothesis  was examined by pursuing  1) To develop a method of labelling need for in vitro purification potentially  and subsequent  labelling  the  of PMN, all steps that may  in vivo with the thymidine  5’bromo-2’-  analogue  (BrdU).  deoxyuridine  2) To establish  the best method for transferring  model and to determine  circulation  PMN in vivo that decreases or eliminates  activate PMN. To accomplish this goal an animal model was developed  in which PMN were labelled  animal  the following specific aims:  the clearance  as well as their functional  3) To transfuse  labelled PMN to recipients of these labelled  in our  PMN from the  capabilities.  labelled PMN to recipient animals  N and to determine  the expression  of L-selectin on these labelled PMN over time in the circulation. 4) To determine  whether the lack of L-selectin expression on circulating  as a signal for the removal of these cells from the intravascular  pool.  PMN serves  98 Model of in vivo PMN labelling: The following  specific  aims will be addressed  1) To develop a method of labelling need for in vitro purification potentially  in this section:  PMN in vivo that decreases or eliminates  and subsequent  labelling  the  of PMN, all steps that may  activate PMN. To accomplish this goal an animal model was developed  in which PMN were labelled in vivo with 5’bromo-2’-deoxyuridine  2) In this model, to establish  the best method for transferring  (BrdU).  labelled  PMN to  recipients and to determine the clearance of these labelled PMN from the circulation as well as their functional  capabilities.  Previous studies from this (Doerschuk et al., 1987a; Muir et al., 1984) and several other laboratories  (Segal et al., 1976) have reported on the in vivo behavior of PMN  labelled  isotopes  with  diisopropylfluorophosphate  such  as  chromium  Cr),indium 5 ( 1  (“ 1 1 n),  P). These isotope studies require the PMN to be 32 (DP  isolated from whole blood, labelled with an isotope in vitro and transfered recipients.  or  These procedures  make the experimental  protocol both lengthy  expensive (Haslett et al., 1985; McAfee et a!., 1984) with subsequent and disposal of radioactive  waste.  back into  There is also the possibility  and  decontamination  that the purification  and labelling procedures will activate the PMN and cause them to behave abnormally (Haslett synthesis  et al., 1985). The classical  method  for detecting  in vivo is by their uptake of the thymidine  TdR), identified incorporation  using  autoradiography  analogues  (Denekamp  of 3 [ H ]-TdR allows in vivo labelling  cells engaged  and  in DNA  ([ H 3 1-thymidine, 3 [ H 1Kaliman,  1973). The  of cells but provides few if any  99 advantages Kallman,  over the in vitro labelling with gamma emitting isotopes (Denekamp and 1973; Hamilton  and Dobbin,  1983). However,  BrdU, which was labelled with the radioisotope  the thymidine  analogue,  bromine, in early studies (Hakala, 82  1958; Eidnoff et aL, 1959), has now been modified for immunocytochemical  detection  using a monoclonal antibody with high specificity against this halogenated  analogue  (De Fazio et al., 1987b; De Fazio et a!., 1988; Dolbeare et aL, 1985; Gratzner, The immunocytochemical to 3 [ H ]-thymidine  visualization  autoradiography  monitor cell proliferation al., 1988; Plickert  of BrdU appears to be a powerful alternative  and this technique has been used increasingly  and migration  during normal development  et al., 1988), DNA replication  studies involving  growth kinetics  1982).  in solid tumors  (Gratzner, (Denekamp  (Dombrowicz et  1982), particularly  and Kallman,  Goodson et al., 1991; Soriano and Del Rio, 1991), and cytofluorometric  to  in  1973;  analysis of the  cell cycle (De Fazio et al., 1987b; De Fazio et al., 1988; Dolbeare et al., 1983). The  immunocytochemical autoradiography between  staining  is  performed  within  hours  compared  with  which take days and often weeks. The normal PMN turnover  is  100 and 200 billion cells a day (Dancey et al., 1976; Walker and Willemze,  1980) and labelling  these rapidly dividing cells in the bone marrow with BrdU may  prove to be ideal.  By using BrdU as a non-isotopic method to label PMN in vivo, the behavior of these cells can then be studied by transfusing This method  may eliminates  whole blood to serum compatible recipients.  the need for in vitro purification  labelling of PMN, all steps that may potentially us to study the behavior of an activation  and subsequent  activate PMN. It will further allows  sensitive surface molecule such as L-selectin  100 over time in the circulation.  Furthermore,  of isotopes and isotope contaminated  it may also reduces the cost of disposal  specimens and animals.  Described here is a method for labeffing and detecting the proliferating in the bone marrow by administering  BrdU to donor animals.  labelled cells was then studied in serum- compatible been transferred  pool of PMN  The behavior of these  recipients  after the cells had  in either whole blood, leukocyte rich plasma, or purified PMN.  101 METHODS Animals: Twenty female New Zealand white rabbits were used in this study.  Three (4 ±  0.3 kg, mean ± SD) were used as donors and 17 (2.6 ± 0.2 kg) as recipients. study was approved by the Animal Experimentation British  Committee  The  of the University  of  Columbia.  Labelling  DNA of rabbit leukocytes  with BrdU:  Donor rabbits were given BrdU (Sigma Chemical Co., St. Louis, MO) at a dose of 25 mg/kg daily for 7 days. The BrdU was infused slowly through the marginal at a concentration  of 5 mg/mi in normal saline over a period of 15 minutes.  obtained daily from the central ear artery of these BrdU-treated as follows: ethylene  lml was collected in standard  diamine tetra-acetic  counts determined  ear vein  Vacutainer  Blood  rabbits was analyzed  tubes containing  acid (Becton Dickinson, Rutherford,  potassium  NJ) for blood cell  on a model SS8O Coulter Counter (Coulter Electronics, Hialeah,  FA) and for differential  white cell counts on Wright’s stained blood smears; and 2m1  was collected in acid-citrate-dextrose plasma (LRP) as previously  described.  (ACD) for the preparation The resulting  of leukocyte  rich  LRP was cytospun at 180 x g  for 4 minutes to obtain a monolayer of cells on pre-coated slides (Fisher Scientific Co., Pittsburgh,  PA), air dried, and stained  PMN in each specimen.  After labelling  to determine  the number of BrdU labelled  efficiency of PMN was evaluated,  rabbits were used as a source of labelled PMN in all subsequent  experiments.  these  102 PMN purification: PMN were purified laboratory  from 25 ml of donor blood as previously  (Doerschuk et aL, 1987a; Doerschuk and English,  obtained from 25 ml of donor blood was centrifuged and hypotonic  lysis of the residual  described 1991).  from our  Briefly, LRP  and suspend in 1 ml PMN buffer  red blood cells in the LRP was achieved  dilution in 11 ml sterile water. After 18 seconds, 1 lml 2X PBS (27 mM Na 2 HPO mM 4 PO and 2.74 M NaC1) and 10 ml PMN buffer were added. 2 KH , separated  from the mononuclear  cells by centrifugation  PMN were 95 % to 98 % pure with a viability  ,  132  PMN were  in Histopaque  Chemical Co.) with a density of 1.077 g/ml at 150 x g for 13 minutes.  by  (Sigma  The isolated  of 97% as assessed by trypan  blue  exclusion.  Transfer  of BrdU labelled  leukocytes  BrdU labelled leukocytes were transferred  to recipient  rabbits:  from donor to recipient rabbits as either  25 ml whole blood, LRP prepared  from 25 ml blood, or PMN purified  donor blood. Cells were transfused  into the marginal  recipient over a 5 mm  from 25 ml  ear vein of serum-compatible  period. The total number of leukocytes,  PMN, BrdU labelled  PMN, and the amount of BrdU labelled DNA in the 3 preparations  was determined  from fractions of each donor sample. Eight recipients received whole blood, 3 received LRP, and 6 recieved purified circulating  blood was assessed  PMN. The behavior from  blood samples  of BrdU labelled obtained  PMN in the  from the central  ear  artery of all recipients before the BrdU labelled PMN were infused (baseline) at 5, 30 mm, and at 1, 3, 6, and 24 hours. blood cell and differential  The blood samples were used to determine  cell counts, the percentage  of BrdU labelled  white  PMN in  103 cytospun preparations  Assessing  of LRP, and the fraction of BrdU labelled DNA.  the number  The percentage  determined  of BrdU labelled  of BrdU  labelled  than  PMN in the circulation  total number  (American  was  of 200 PMN on each cytospin slide on randomly  a 1000 PMN were counted. of each recipient  of labelled PMN originally  blood volume  blood of recipients  5 % of PMN were labelled, 500 PMN were counted, if less  1 % of PMN were labelled  labelled  PMN in the peripheral  by counting a minimum  selected fields. If less than  PMN in the circulation:  The number  was expressed  of BrdU  as a fraction of the  infused and was corrected for the calculated  Society, 1965) of the recipient in the following  Physiology  manner:  BV x %PMNrB.J  BrdU  Fraction PMN. Cirt  where  Fraction  circulation  PMNU represents  =  BrdU ed  the  as a fraction of the total number  the calculated  number  of BrdU  of BrdU labelled  number of PMN (1 x 106) in the circulation  the fraction of leukocytes  labelled  PMN  PMN infused, PMN  (total white cell count times  that are PMN) times the calculated  blood volume(BV),  %PMN the fraction of BrdU labelled PMN in a cytospin of peripheral the recipient, (PMN count/mi  and PMN  in the  blood in  the number (1 x 106) of BrdU labelled PMN infused  x ml of fluid infused x %BrdU labelled  PMN).  In  three rabbits who  received whole blood more frequent blood samples were taken: at baseline, 2.5, 5, 7.5, 10, 15, 20, 30, and 45 minutes  and  1, 2, 3, 4, 5 and 6 hours after infusion.  Twenty-  104 four hours after infusion of the labelled cells, recipients intramuscular  injections  Philadelphia,  of 100 to 150 mg/kg of ketamine  PA) and 5 to 8 mg/kg acepromazine  of lung, liver, spleen,  immunohistochemical  bone marrow,  hydrochloride  with  (Ayerst,  maleate (Ayerst) and sacrificed in  a supine position by an injection of 2 to 3 mI/kg of saturated Specimens  were anaesthetized  KC1 into the aortic root.  and gut were removed  for the  detection of BrdU labelled cells and for the analysis of BrdU  labelled DNA.  Tmmunocyto-  and histochemical  detection  of BrdU labelled  cells:  The lung, spleen, liver, and gut specimens were fixed in 10 % buffered formalin for 2 to 4 hours while the bone marrow specimens was fixed with B5 fixative.  Randomly  selected blocks from the fixed tissues were embedded in paraffin.  Three m tissue  sections placed on slides coated with 3-aminopropyl-triethoxysilane  (Sigma Chemical  Co.) were baked for 16 hours at 37°C. The paraffin was removed in two 10 minute washes of xylene. For sections of bone marrow the second xylene wash contained 4 % iodine in order to remove mercury remaining rehydrated  from the B5 fixative.  Sections were  in graded ethanol from 100% to 70 %, rinsed twice with distilled water,  and digested at 37°C for 10 minutes in a 0.4% pepsin (Sigma) solution acidified to pH  2.5. All cytospun preparations  of LRP were fixed in methanol for 10 minutes.  in both the tissue sections and cytospun specimens was denatured for 1 hour.  This was followed by neutralization  consecutively  BrdU labelled  in 2 N HC1 at 37°C  in three washes of 0.1 M borate  buffer, pH 8.5, each for 10 minutes. The APAAP technique used to detect  DNA  DNA in cells. Briefly,  (Cordell et al., 1984) was  specimens  were incubated  in 5 % rabbit serum for 15 minutes, then in 0.5 g/ml mouse anti-BrdU  105 antibody (Boehringer-Mannheim,  Mannheim,  Germany) prepared with 1 % BSA in 50  mM Tris Cl, 150 mM NaC1, pH 7.6 (TBS), at room temperature chamber  for 1 hour.  Non-immune  mouse IgG at 0.5g/ml was used as a negative  control in addition to a negative  control where the primary  Incubation  of rabbit  Copenhagen,  in a 1/20 dilution  (DAKO Laboratories).  alkaline  phosphatase  IgG (DAKO Laboratories,  anti-alkaline  phosphatase  The alkaline  phosphatase  sodium nitrite,  complex  was developed for 20  in 100 ml TBS at pH 8.7, after the addition of a mixture 0.2 ml of 5 % fuschin (Merck, Rahway,  naphthol-AS-BI-phosphate  (Sigma  N,N-dimethylformamide.  Endogenous  of 17.5 mg levamisole  preparations  was omitted.  Slides were washed in 0.1 % Tween 20 in TBS for 10 minutes  following each antibody application.  addition  anti-mouse  antibody  Denmark) for 45 minutes was followed by 45 minutes in a 1/50 dilution  of a mouse monoclonal  minutes  in a humidified  were counterstained  Chemical alkaline  NJ) in 2M HC1 and 50 mg  Co.)  dissolved  phosphatase  (Sigma Chemical  in  0.6  Co.) to the colour reaction.  with Mayer’s-hematoxylin  ml  was blocked by the The  for 60 seconds, mounted  in an aqueous medium, and analyzed on a Zeiss Model flR Universal microscope (Oberkochen,  of 0.5 ml of 4%  Research light  Germany).  DNA Analysis: Samples  of individual  organs were frozen in liquid nitrogen  and stored at -70°C.  Nuclei were isolated from whole blood, LRP, and PMN according to the method of Buffone and Darlington  (Buffone and Darlington,  1985) and DNA was isolated from  these nuclei as well as from the lung, liver, spleen, skin, bone marrow, and ileum following procedures described previously  (Strauss,  1990).  Ten g of DNA isolated  106  from blood, LRP, or PMN and 20g of DNA isolated from the above mentioned organs were electrophoresed  through 0.7% agarose gels. The DNA was transferred  gels  filters  to  Hybond-N  manufcturer’s ultraviolet  instructions.  After  were washed  photographed,  Arlington  cross-linking  light at 254 nm for 4 minutes,  mouse anti-BrdU antibodies filters  (Amersham,  Heights,  IL) according  the DNA to the ifiters  to with  BrdU labelled DNA was detected using  following the APAAP method as described above. These  for 5 minutes  in 10 mM Tris Cl, 1 mM EDTA, pH 8.0,  and analyzed on a Ultroscan XL densitometer  software (Phamacia,  from the  using the Gelscan XL  Uppsala, Sweden).  Morphometry: A stereologic analysis to determine  the distribution  of BrdU positive PMN  (PMNB1J)  in the lung, spleen, liver, gut and bone marrow tissue was performed on paraffin embedded tissue sections that had been immunologically BrdU. A point counting technique  (Cruz-Orive and Weibel, 1981) was applied using  a Nikon microscope with a camera-lucida of view generated means  stained for the presence of  attachment,  which allowed random fields  by a computer program to be analyzed at 400X magnification  of a point counting  grid of 400 points  that  was superimposed  by  onto the  microscope image. A total of 20 fields were evaluated per organ. The volume fraction (V) of BrdU labelled PMN was calculated  VV&W)  =  from the equation:  E poinLs )fl PMN&dU  E  on the grid  Then, the total number of PMNBrdU in each organ was calculated  from the equation:  107  organ volume x Number of PMN&dU  =  (PMN  140 m 3  where PMNBrdU represents  the number of BrdU labelled PMN and 140 m 3 represents  the assumed volume of a PMN (Doerschuk et aL, 1993). The calculated of  pjBrdU  in each organ was expressed  as a percentage  total number  of the total number  of  PMNU infused into each recipient rabbit.  Activation  parameters  on BrdU labelled  PMN:  a) Expression of L-selectin and CD18 on BrdU labelled PMN Immunofluorescence expression Butcher)  staining  of L-selectin (Monoclonal antibody DREG 200, kind donation of Dr. E.C. and CD18 (Monoclonal antibody  using flow cytometry. sensitive Julita  of labelled and control PMN was done to determine the  parameters  Changes  60.3, kind donation  in the expression  of cell activation  (Kishimoto  of Dr. J.M. Harlan)  of these molecules on PMN are et al., 1989; Julita  et aL, 1989). A whole blood method for preparing  specimens  et al., 1991;  was used.  The  cells were prepared from EDTA blood for analysis using a commercially available kit (Coulter Clone®, Coulter Electronics, incubated  Florida).  ILl of EDTA blood was 100  with 1 200 . d PBS buffer and 0.4g MoAb DREG 200 or 60.3 for 10 mm  room temperature using non-immune  in the dark.  at  For each blood sample, a negative control was done  mouse IgG (Sigma Chem Co, St Louis, MO). After washing the  cells with PBS buffer, FITC goat anti-mouse incubated  Briefly,  for 10 mm. After washing  0.24g was added to each specimen and  the cells twice, the red blood cells were lysed  108 (Immuno-lyse,  Coulter  paraformaldehyde  specimens  Clone®).  and stored  The remaining at 4°C.  leukocytes  Flow cytometry  were fixed with  was performed  within 24 hours (Model; Profile EPIC II, Coulter Electronics,  Analysis gates for the PMN were established scatter profiles. presented  using the distinctive  A total of 3000 gated cells were evaluated  1%  on the Florida).  forward and side  per specimen and are  as either percent positive cells or mean fluorescence intensity  (log).  b) Superoxide production of BrdU labelled PMN: The possibility determining  procedure was evaluated  by  PMN superoxide anion production with N-formyl-L-methionyl-L-leucyl-L  phenylalanine  (FMLP) and phorbol 12-myristate-13-acetate  Mo) stimulation dismutase  that PMN are primed by the labelling  on purified  (SOD) inhibitable  labelled  appropriate  stimulant,  concentrations  control  PMN, using  reduction of ferricytochrome  et al (1984). We preincubated 23g/ml SOD. To initiate  and  (PMA) (Sigma, St Louis,  2x10 / 6 ml PMN for 5 mm  the  superoxide  C as described by Markert at 37°C with and without  the reaction we added ferricytochrome  C (80MM), and the  either PMA (2nM) or FMLP (10M). All reagents  were final  in a final volume of 750l and were incubated at 37°C for 30 mm. The  reaction was ended by adding 400jl isotonic Ranks buffered saline (HESS), pH 7.4, to the mixture and centrifuging in absorbance  it for 3 mm  at 550mm in a Perkin  converted the results to nanomoles  at 2000 x g. We measured  Elmer Lamda2  the increase  UV/VIS Spectrometer  and  of reduced cytochrome C by using the extinction  coefficient 18.5 mM’ .cm . Values of superoxide produce by a final PMN concentration 1 1 x 106 cells over 30 mm samples  with and without  were determined  by calculating  SOD. Each determination  the difference between  was done in duplicate  and  109 expressed as nanomoles  c) Migration  /0 2 3OminIlxlO  of BrdU labelled  A focal pneumonia  6  PMN.  cells into inflammatory  foci  was produced as has been previously described (Doerschuk et aL,  1990) to test the ability of labelled PMN to migrate into a focus of infection. Briefly, 5 x iO S. pneumoniae  organisms  5 % colloidal carbon.  This mixture  anaesthetized  were dissolved in 0.5 ml sterile saline containing was instilled  into the left lower lobe of two  rabbits through a paediatric feeding tube inserted between the tracheal  rings and positioned under fluoroscopy. An equal volume of sterile saline with 5 % colloidal carbon was instilled  into the right lower lobe to serve as a control.  Two 1  cm x 1 cm foam sponges, one soaked by immersion in the solution of S. pneumoniae and the other in the control solution, were then inserted  subcutaneously  sides of the linea alba.  anaesthetized  The animals  were maintained  position for 3 hours by the administration  of additional  ketamine  on opposite in the prone  HC1 as required.  At this point, 25 ml whole blood in ACD from a BrdU labelled donor was transfused intravenously pneumonic  as above and the rabbits were sacrificed and control  surrounding  lung areas  marked  STATISTICAL  of  detection of BrdU  of BrdU labelled DNA.  METHODS  All values are expressed as mean± standard  disappearance  Specimens  by the colloidal carbon and of skin  the sponges were excised for immunohistochemical  labelled cells and for analysis  To evaluate  1 hour later.  the differences rate  error when except otherwise mentioned.  in the mode of transferring  of labelled  PMN in each  recipient  labelled  leukocytes,  was estimated  the  as the  110 relationship within  between the log of the fraction of PMNBrdU and time. The family of lines  each group were then compared  (REML) method described by Feldman compared using a chi-square  statistic  were considered to be significant estimated  using the restricted (Feldman,  maximum  likelihood  1988). The estimates  were then  and differences in slopes, intercepts  when a probability  slope, the half-life was calculated  and lines  than 0.05 was found. Using the  for each group (Sachs, 1982).  111 RESULTS  Labelling  of PMN with BrdU:  Figure 18 shows that a daily intravenous increased  in the percentage  labelling  dose of 25 mg/kg of BrdU produced a rapid  of BrdU labelled  PMN. The immunocytochemical  of PMN was variable and all cells with visible stain were deemed positive  (figure 19a). The increased in BrdU labelled PMN occurred in a linear fashion from day 1 to day 5 and gradually labelled.  Immunoblot  levelled out by day 7 when 80±2.3% of the PMN were  analysis  of the DNA extracted  from blood leukocyte samples  showed that the amount of BrdU labelled DNA increased in a similar fashion (data not shown).  Washout  of BrdU labelled  PMN in recipient  blood  Table VI shows that the number of PMN transferred PMN was  approximately  one-half of the number  as either LRP or as purified  of PMN in the same volume of  whole blood. After the infusion of donor whole blood into recipients, BrdU positive PMN in the circulation, minutes  rose in an irregular  fashion to a peak at 60  and then decreased rapidly over the next 24 hour period (figure 20a). When  LRP (figure 20b) or purified PMN (figure 20c) was infused, labelled  the number of  PMN in the recipient’s  the number  of BrdU  blood was lower at all the time points up to 60  minutes.  Calculation  of the half-life of BrdU labelled  The time required circulating  to achieve the maximal  blood of the recipients  20a). This time,  PMN  number  of BrdU labelled  PMN in the  who received whole blood was 60 minutes  (figure  was applied to the rate of decay equation to calculate the half-  II2  100  T z  60  I  20  I  T J  0  0  1  2  4  5  6  Time (days)  Figure 18: Time course of BrdU incorporation into PMN. Blood samples were drawn daily from donor rabbits infused with 25 mg/kg/day of BrdU for 7 days. The fraction of BrdU positive PMN on cytospins prepared from LRP in donor blood is plotted against the days elapsed after the start of BrdU treatment. Day 0 represents the time before the first injection. Each point represents the mean of three experiments ±SE.  7  “3  Table VT COMPARISON OF THE NUMBER OF WBC, PMN AND BrdU LABELLED PMN RECOVERED IN THREE DIFFERENT PREPARATIONS OF DONOR BLOOD  )* 6 Number of leukocytes (x10  Type preparation WBC  PMN  BrdU labelled PMN  Whole blood(8)t  257.7±8.8  116.3±8.9  85.9±22.1  LRP (3)  132.3±1.8  52.5±3.8  41.6±3.9  60.0±5.0  46.2±3.1  Purified PMN (6)  *lkt  -  obtained from 25m1 donor blood.  tnumber of samples analyzed. *average±SE.  1/4  I .:,.  Figure 19: Immunohistochemical detection of BrdU labelled cells. Panel (a) demonstrates a cytospin prepared from LRP of the donor blood after 7 days of BrdU 25gfkg/day treatment. BrdU labelled PMN in the spleen (panel b) 24 hours after infusion of labelled PMN into recipient, the control lung (panel c) and Streptococcus pneumoniae infected lung (panel d) of recipients. BrdU labelled cells were transfused as whole blood 3 hours after the instillation of either Streptococcus pneumoniae or a vehicle into the lung with colloidal carbon as marker. Bar =1Om.  100  a  80  J  03  C  z  II  60  40  20  0  I  0  2,40  120  360 1440  1450  Time (mm) 100  b 80  J  03 03  C  60  L  z  ;  .3  20  0  ‘i 120  = 240  360 1440  1450  360 1440  1450  Time (miii) 100  C 80 0? 0?  bO  60  C  40  20 .1  0  0  I  0  120  240  Time (miii)  Figure 20: BrdU labelled PMN in recipient’s circulation as a fraction of the number of BrdU labelled PMN infused after the infusion of whole blood (19a, n=8), LRP (19b, open circles n=3) or purified PMN (19c, op.en circles n=3). Each data point represents the mean with the SE. BrdU-PMN(% of injected) represents the number of BrdU labelled PMN in the circulation as a fraction of the total number of BrdU labelled PMN infused (see text).  116 life of PMN in the circulation:  N  where k represents  NTe  -b  the positive rate of loss of BrdU labelled PMN (slope),  t the time after  circulation  =  e = 2.7 1828, N the number of BrdU labelled PMN in the  at time t and NT,,,X the number of BrdU labelled PMN in the circulation  at time Tm. Since the half-life can be estimated equation  as the time at which N  =  NTX  for the half-life, , 112 becomes t t 112 = ln 2/k. The constant,  using the restricted  maximum  confidence interval  (Sachs, 1982) was obtained  k, was calculated  likelihood method described by Feldman (1988). The  bounds of the 95 % confidence calculation,  /2, the rate-decay  interval  by deriving  for the slope, k.  the lower and upper Using this method  the half-life of the BrdU labelled PMN in the circulation  of recipient  rabbits using the whole blood infusion method was found to be 270 minutes 95 % confidence interval  Distribution  of 248 and 296 minutes  of BrdU labelled  of  with a  (table VII).  PMN in recipient  animals  Twenty-four hours after the recipients received the BrdU labelled cells, 55±11 % and 58±9% of the original respectively,  labelled  PMN infused  as either  whole blood or LRP,  were found in the spleen (Table VII, figure 19b). After the infusion of  purified PMN, only 13±10% of the injected cells were recovered in the spleen. Very few BrdU labelled PMN were observed in sections of the liver, lung and bone  117 Table VII  HALF-LIVES OF BRDU LABELLED PMN INFUSED AS EITHER WHOLE BLOOD, LEUKOCYTE RICH PLASMA OR PURIFIED PMN  Type preparation  slope  halflife*  Whole Blood  -0.0011132  270.4  248-296  LRP  -0.0011841  254.2  184-408  Purified PMN  -0.0024649  122.1  90-186  *  95% CI  Half-life and confidence intervals (CI) are given in minutes  calculated from the equation Nt=Naxed (see text)  118 marrow. In the lung and bone marrow the occasional microvessels.  labelled PMN was seen in  The point counting technique based on a grid density of 400 points was,  however, not sensitive  enough to quantitate  the number of labelled PMN in these  organs. The majority of BrdU labelled PMN seen in the liver were extravascular. analysis of DNA extracted or LRP showed significantly  that  The  from these organs (figure 21) after infusion of whole blood  the amount  of BrdU  higher than that found elsewhere.  labelled  DNA in the  spleen  was  In addition, Southern analysis of the  labelled DNA found in the spleen formed a ladder pattern typical of DNA from cells undergoing  programmed  cell death or apoptosis (figure 22). This ladder pattern was  not present in the DNA prior to infusion nor was it detected on the ethidium bromide stained  gels of the total DNA extracted  from the recipient  spleens.  BrdU labelled  DNA was also found in the liver, bone marrow, gut and lung after whole blood or LRP infusion (figure 20). When adequate amounts of labelled DNA were present, as was the case for the lung, the same ladder pattern (figure 22). equivalent Activation  After the infusion amounts  of purified  found in the spleen was present  PMN, BrdU was not detectable  of DNA from these same organs, including  parameters  L-selectin expression similar as measured  of BrdU labelled  on BrdU labelled  in  the spleen.  PMN  (82±8.5% labelled)  with indirect immunofluorescence  and control PMN was  using flow cytometry  (MFI  11.2 ± 2.8 versus 10.8 ± 3.1, BrdU labelled versus control PMN, n = 3). Similarly, CD 18 expression  was not different  PMN to generate  in BrdU labelled PMN. The ability  superoxide with FMLP stimulation  of BrdU labelled  was the same as control PMN  (7.1±3 versus 6.8±2.7nmolO 2/30 mm, BrdU versus controls, n=3). Likewise, PMA stimulation,  BrdU labelled  and control PMN superoxide production was  with  siq  .—  —4  -4  C)  .—  I  0  Liver  Bone marrow  Spleen  Gut  Lung  Organ  Figure 21: Semi-quantitation of BrdU labelled DNA in recipient organs 24 h after the transfusion of whole blood (solid bars, n=5) and LRP (hatched bars, n=3). The Southern blots were analyzed by densitometry and bars represent the average intensity of immunological staining of DNA ± SE. Results were corrected for the number of BrdU labelled PMN infused.  I2O  40 4 0  4  ,  Figure 22: Immunological detection of BrdU labelled in DNA extracted from organs of one of the recipient rabbits after the transfusion of whole blood from a donor rabbit. A Southern blot of 20 g of DNA from each organ was stained for BrdU as was 1Oig of DNA from donor peripheral blood used prior to transfer. Note the ladder pattern in the lung and the spleen lanes, indicating PMN apoptosis in these organs. Lanes are as indicated.  121 similar.  These findings  indicate  that the labelling  procedure  did not activate  or  prime the labelled PMN. White cell, PMN and platelet counts did not change over the 24 hour study  period  with  any of the transfusion  modes, indicating  transfusion  per se did not affect the behavior of infused PMN.  Migration  of BrdU labelled  PMN into inflammatory  Four hours after the instillation pneumonia,  of bacteria  that  the  foci  into the left lower lobe to induce  there were more BrdU labelled PMN in the pneumonic  area and BrdU  labelled PMN had migrated into the alveolar spaces (Figure 19c and 19d). The tissue surrounding more  the subcutaneous  intravascular  surrounding  and  sponge saturated  extravascular  the control sponge.  BrdU  with S. pneumoniae labelled  PMN  also contained  than  the  tissue  122 DISCUSSION Labelling PMN with radioisotopes in vivo has been well described  ex vivo to study their turnover rates and behavior (Denekamp  and Kaliman,  1973; Doerschuk et aL,  1987a; Muir et aL, 1984; Segal et at., 1976). These procedures disadvantages  and pitfalls,  have numerous  with one of the major concerns being the activation  PMN during cell purification  of  and labelling procedures (Haslett et aL, 1985; Hamilton  and Dobbin, 1983; McAfee et aL, 1984). Whether isotope labelled PMN represent the in vivo behavior of circulating have demonstrated  PMN has been questioned.  In the present study we  that BrdU can be used to label leukocytes in vivo and that these  labelled leukocytes can be successfully tranferred to recipient animals with minimum manipulation.  In a previous study de Fazio et al (1987a) measured  serum levels of BrdU in a mouse  exposed to a dose of BrdU similar to what we have used. Steady state levels were well in excess of circulating detected  thymidine  with their dosing regime over 7 days. They also demonstrated  percentage  of cells labelled  after a 30 mm  obtained for the S-phase percentage flow cytometry,  indicating  pulse of BrdU was similar  that the to values  of normal mouse bone marrow as determined  satisfactory  Following 7 days of intravenous circulating  levels and no bone marrow toxicity could be  labelling  administration  by  efficiency with this dose of BrdU. of BrdU, approximately  PMN were labelled with BrdU. The intermittent  80 % of the  daily dose of BrdU we  used in our model may explain the ±20% BrdU negative PMN in the circulation after a 7 day labelling  period.  123 The first BrdU labelled PMN were detected in the circulation  of the donors after 24  hours with a small increase after 48 hours and a sharp rise to 28 ± 10% positive PMN within 72 hours (figure 17). This represents in the last myelocyte generation circulation.  the minimum  cycle until the mature  Using H 3 TCIR the minimum  transit  time from DNA synthesis PMN are released  in the  time is 96-144 hours in humans  (Fliender et at., 1964) and 48-72 hours in dogs (Maloney and Patt, 1968). Our study suggests that the myelocyte blood transit time in rabbits is between 24-48 hours. In this study using a whole blood transfusion labelled PMN in the circulation  method the calculated half-life of the BrdU  was 270 minutes  or 4.5 hours (95 % CI 248-296  minutes). This half-life is shorter than the 6.5 hours calculated  in a previous study  when PMN in rabbits were labelled with 51 Cr (Doerschuk et at., 1987a) although in that study purified PMN were used and the half-life was calculated experiment.  from a single  It is also shorter (between 6 and 10 hours) if compared to studies where  isotopes such as DP P, 1 32 ‘ I n, 3 Cr were used in humans [ H ITdR and 51  (Athens et at.,  1961; Filender et at., 1964; Segal et al., 1976).  Transfusion of whole blood was the most convenient and effective way of transferring these labelled PMN to recipient  animals.  Using the same volume of blood, PMN  purification resulted in a calculated loss of approximately  50 % of the PMN due to the  purification procedure. Accelerated removal of these PMN from the circulation early following  infusion  suggests  they  underwent  changes  during  the  purification  procedure. Similar results were obtained when leukocyte rich plasma was infused. The most likely explanation results in sequestration  for these observations  is that the processing of PMN  of these cells and faster removal from the circulation  pool  124 than PMN in whole blood (Haslett et al., 1985). Transfusing  whole blood from the  donor to the recipient as described in this study avoids most of the problems that are encountered  when labelling  The in vivo labelling determined  cells in vitro with isotopes.  of PMN with BrdU did not activate  by changes  in the expression  of two activation  the PMN, as was sensitive  surface cell  adhesion molecules, L-selectin and CD 18, or prime the PMN, as was determined their ability to produce superoxide. The functional capabilities transfused  into recipients were further evaluated  of inflammation.  We have demonstrated  pnewnoniae generated inflammatory lung  into  subcutaneous  the  alveolar tissue.  space  out of systemic  This finding qualitatively  PMN suggests  in foci of inflammation  evaluation  that the labelled  similar to the unlabelled  by their ability to migrate into foci  that BrdU labelled PMN accumulated  labelled PMN to migrate across the endothelium  labelled  of BrdU labelled PMN  in S.  foci and migrated out of the microvessels of the  and  space in the lung. This preliminary  by  cells. The linearity  microvessels  assesses  into  the ability  into the interstitium of the functional  infected  of the BrdU  and/or alveolar  capabilities  of BrdU  PMN appeared  to behave in a fashion  of recruitment  of BrdU labelled PMN  should, however, be addressed  in a quantitative  study with  a variety of stimuli in a dose-related manner, which is outside the scope of this thesis.  It has been suggested  that effete PMN leave the circulation  according to their age, do not re-enter the circulation  randomly  rather than  (Fliedner et al., 1964a) and  meet their fate in situ (Hurley, 1983). The normal site for PMN emigration  from the  125  circulation  and death is unclear (Bainton,  1988) but our data show that over half of  the BrdU labelled PMN infused as whole blood or LRP were found in the spleen 24 hours after infusion.  This figure contrasts  with the 13±10% of BrdU labelled PMN  recovered in the spleens of the recipients who received purified PMN. This low value for removal of purified PMN by the spleen is similar to data obtained from a study that used 51 Cr labelled PMN in rabbits (Doersehuk et aL, 1987a). We attribute difference to PMN activation  this  during the isolation procedure, which results in rapid  removal of labelled cells by several organs, especially the liver. Previous studies with Cr and 111 51 1n labelled PMN showed a substantial  removal in the liver (Doerschuk et  aL, 1987a; Muir et al., 1984). We suspect that the dispersion  of the BrdU labelled  PMN in an organ the size of the liver (100 times larger than the spleen in rabbits)  makes it difficult to detect this shift of sequestration liver using our morphometric  of PMN from the spleen to the  method. It is also possible that a fraction of the PMN  removed into the tissues during the time frame of our experiment  have already been  degraded  into the tissues  and processed. The fate of PMN after they migrate  unknown but they probably live for only 1 to 2 days (Bainton,  The immunodetection  labelled WBC undergoing  disappearance  1988).  methods used in conjunction with the Southern analysis of the  DNA in this study showed the typical fragmented  The finding  is  programmed  that apoptosis  ladder pattern indicative of BrdU  cell death (apoptosis) in the spleen and lung.  of WBC occurs in the spleen is consistent  with the  and death of PMN in that organ. It is unlikely that apoptosis of other  leukocytes contributes  significantly  to the apoptosis pattern observed in the spleen.  Lymphocytes are known to have a long half-life and the fraction of other granulocytes  126 in donor blood (<5%) is small. The observation lung is consistent  with reports (Hogg, 1987) showing that there is a population  PMN which have very long transit proportion  that apoptosis also occurred in the  of the PMN marginated  times in the lung.  of  As the lung contains a large  pool, it is conceivable  that some PMN remain  there long enough to undergo apoptosis.  This technique  of labelling  cells to recipients  donor leukocytes  circulation.  the labelled  in whole blood, provides a novel way to study PMN behavior in  vivo that is fast and simple. determine  in vivo and transferring  the kinetics  This method was used in the subsequent  of L-selectin  on PMN during  their  time  spent  study to in the  127 Changes  in L-selectin  expression  on PMN  during  their  lifespan  in the  circulation The following  specific  1) To transfused  aims wifi be address  BrdU labelled  in this section:  PMN to recipient  animals  and detennine  the  expression of L-selectin on these labelled PMN over time in the circulation. 2) To determine  whether the lack of L-selectin expression on circulating  as a signal for removal of these cells from the intravascular  PMN serve  pool of PMN.  Introduction Studies  on the behavior of PMN in the systemic circulation  migration  of PMN out of the vasculature  endothelium mediated  of post capillary  by the selectins  have shown that the  is preceded by the rolling of PMN on the  venules (Ley et al., 1991b; Von Andrian et al., 1991). (Lasky,  1992a). The leukocyte  associated  selectin,  L  selectin, has been shown in vivo to mediate rolling of rabbit, rat and human PMN in mesenteric  venules  phenomenon  (Von Andrian  et al., 1991; Ley et al., 199 ib). This rolling  allows PMN to marginate  in post-capillary  or collecting venules, is more  pronounce on inflamed venules which allows the PMN to slow down and stop under shear force (Lawrence, and Springer, essential  feature of PMN function in the presence of shear force both in vivo and in  vitro and may be facilitated on the tips of microvillus  by the conspicuous spatial distribution  of the L-selectin  projections on the PMN cell surface (Picker et al., 1991).  Rolling along the endothelium circulation  1991; Smith et al., 1991). Roffing of PMN is an  or by firm adherence  is followed by either a return in preparation  for migration  space. This latter event is mediated by the activation-induced  of the PMN to the out of the vascular  increases in avidity of  128 the integrins for their immunoglobulin-like  counter receptors, intercellular  adhesion  molecule (ICAM-l and ICAM-2) and vascular cell adhesion molecule (VCAM-l) on the endothelial  cell surface (Butcher,  1991; Larson and Springer,  1990; Zimmerman  et  al., 1992).  The only well described enzymatic  of regulation  of L-selectin  cleavage of the receptor through biochemical  well understood bearing  mechanism  (Kishimoto  on PMN is the  processes that are still not  et al., 1989; Tedder, 1991). Stimulation  cells with activation-inducing  agents  of the receptor-  or phorbol esters results  period of enhanced avidity of L-selectin for its ligand on endothelial immediate  receptor  shedding  (Kishimoto  into inflamed  tissues.  cells followed by  et al., 1989; Porteu and Nathan,  Spertim et al., 1991b). This shedding of L-selectin may be essential migrate  in a short  The hypothesis  that  1990;  to allow PMN to  receptor-ligand  interaction,  involved in the rolling of PMN, results in the removal of L-selectin from the surface of PMN is attractive  for several reasons: 1) the shedding of L-selectin provides a rapid  means for the de-adhesion the signal  of the PMN allowing it to return to the circulation  for firm adhesion  is insufficient;  supematant  of cultured lymphocytes  recirculating  lymphocytes  to peripheral demonstrated  lymph  2) L-selectin  (Lanier et al., 1989; Spertim  (Gallatin  et al., 1983a);  free in the plasma (Schleiffenbaum  selectin may be constitutively  lost from circulating  the fate of L-selectin on circulating ability to synthesize  can be found in the et al., 1991b); 3)  shed L-selectin as they leave the vascular  nodes  when  4) and  space to home  L-selectin  can be  et al., 1992), suggesting  that L  leukocytes. Little is known about  PMN, which do not recirculate  and lack the  L-selectin after leaving the bone marrow (Spertini et al., 199 ib).  129 The variable  expression  of L-selectin on circulating  PMN in contrast  to the high  expression on PMN released from the bone marrow further suggests that PMN lose L-selectin in the intravascular  space. Doershuck et al (1994) have demonstrated  just a small number of PMN delivered to an area of pneumonia into the airspace, suggesting endothelium  eventually  that the majority of PMN that interact  in an inflammatory  that  emigrate  with activated  focus do not emigrate but return to the circulation.  We conducted this study to test the hypothesis that PMN continuously  shed L-selectin  during their stay in the circulation,  resulting  of L-selectin. We further questioned  whether the lack of L-selectin on the surface of  in older PMN expressing  PMN signals their removal from the circulation.  lower levels  The study used the technique  tracing donor PMN labelled with BrdU in recipient animals  of  (as has been described  above), and allowed us to measure the expression  of L-selectin on labelled PMN in  relation to the time PMN spent in the circulating  blood.  130 METHODS: Animals:  Twenty female New Zealand white rabbits were used in this study. Five (3.8 ± 0.3 kg, mean ± SD) were used as donors and 15 (2.3 ±0.3 kg) as recipients. approved by the Animal  Experimentation  Committee  The study was  of the University  of British  Columbia.  Cell preparation: BrdU labelling of donor PMN:  The DNA of rabbit leukocytes  was labelled with BrdU, a thymidine  analogue,  as  previously described (Bicknell et aL, 1994). Briefly, 5 donor rabbits were given BrdU (Sigma Chemical Co., St. Louis, MO) at a dose of 25 mg/kg daily for 7 days. was collected from the central ear artery for blood cell counts, differential  Blood  white cell  counts and to prepare LRP which was cytospun onto 3-aminopropyl-tri-ethoxysilane coated slides.  The cytospin specimens were air dried and stained using the APAAP  method (Cordell et al., 1984) to determine the fraction of BrdU labelled PMN in each specimen (Bicknell et al., 1994). PMN punfication:  The PMN were purified from donor rabbit blood as has been previously  described  (Doerschuk et al., 198Th). Briefly, LRP obtained from each 75 ml of donor blood was centrifuged and resuspended  in 1 ml PMN buffer. Hypotomc lysis of the residual red  blood cells in the LRP was achieved by dilution with 11 ml sterile water. seconds, 11 ml 2X PBS (2X PBS is 27 mM , 4 H 2 Na PO NaC1) and  10 ml PMN buffer  were added.  After 18  132 mM KH 4 P0 and 2.74 M 2 ,  PMN were separated  from the  131 mononuclear  cells by centrifugation  in Histopaque  density of 1.077 g/ml at 150 x g for 13 minutes. pure with a viability  (Sigma Chemical  The isolated PMN were 96% to 98%  of 96 % as assessed by trypan blue exclusion.  Removal of L-selectin by chymot,ypsin  treatment of PMN  Selective removal of L-selectin from PMN by chymotrypsin behavior of a population of predominantly To remove L-selectin  allowed us to study the  L-selectin negative PMN in the circulation.  from the surface of PMN we used the method described by  Jutila et al (Jutila et a!., 1991) who demonstrated from leukocyte  surfaces without  activating  that chymotrypsin  and PMN were purified as previously  (control) or chymotrypsin cells/lU incubated  and were incubated  in  stimulus  Donor blood (75 ml) was  described. The purified PMN were in either denatured  chymotrypsin  type IV (Sigma, St. Louis, MO) (diluted lU/mi for 3 x 106  for 10 minutes  incubated with heat denatured of 5, 15, 30 and 60 mm  by changes  to a chemotactic  and changes in cell morphology measured by flow cytometry.  divided into two specimens  cleave L-selectin  the PMN, as measured  CD1 lb/CD 18, PMN random adhesion to plastic, migration  obtained  Co.) with a  at 37°C).  chymotrypsin.  heat denatured  PMN infused in control rabbits were In preliminary  chymotrypsin  experiments  the ability  to cleave a substrate  N-Benzoyl  L-tyrosine ethyl ester was tested using a method described by Hummel et al (1959). The  ability  of  chymotrypsin  spectrophotometrically Spectrometer denatured abolished  (Perkin  with  to  the  cleaves  use  of a Perkin  Elmer & Co, Uberlinger,  chymotrypsin  the ability  and this denaturing  the  substrate Elmer  Germany).  of the enzyme  was Lamda  determined 2 UV/VIS  With 30 mm  of heat  to cleave the substrate  time was used in all subsequent  experiments.  was The  132 efficiency of L-selectin removal by chymotrypsin by two methods: a) immunocytochemically  from the PMN surface was evaluated  by using the APAAP method to stain for  the presence of L-selectin on isolated PMN cytospun on coated slides before and after the chymotrypsin analysis  treatment  and b) immunofluorescence  of PMN before and after the chymotrypsin  In vitro effect of chymotrypsin  treatment  treatment  to verify that the chymotrypsin  treatment  on PMN activation  parameters  one with denatured  was tested  did not influence the behavior of PMN in  Isolated PMN were divided into 3 samples,  chymotrypsin,  (see below).  on PMN  The effect of the chymotrypsin  the circulation.  staining and flow cytometric  chymotrypsin  one was treated  and the remaining  with  one incubated  with buffer only before analysis. a) The expression selectin  (MoAb DREG 200, kind donation  determined  with immunofluorescent  b) The viability trypan  of CD18 (MoAb 60.3, kind donation  chymotrypsin  method  by Dr. E.C. Butcher)  on PMN were  flow cytometric analysis.  of isolated PMN treated  blue exclusion  by Dr. J.M. Harlan) and L  which  with chymotrypsin found no change  was also tested by the from values  before the  treatment.  Experimental  protocols:  The leukocytes of donor rabbits were labelled in vivo with BrdU and these leukocytes were transfused  as either  whole blood or purified  recipients.  These BrdU labelled  recipients  over a 24 hour period.  PMN to serum  compatible  PMN were then followed in the circulation  of  133 a) Twenty-five obtained  ml of donor whole blood (ACD as anticoagulant)  from 25m1 of whole blood (n=5) was transfused  recipients.  The disappearance  of BrdU labelled  monitored over a 24 hour period by obtaining  or purified  to serum  PMN  compatible  PMN from the circulation  was  blood specimens from the central ear  artery at baseline (before infusion), at 2.5, 5, 7.5, 10, 15, 20, 30, 45, 60 mm  and then  hourly until 6 hours after the infusion. A late specimen was collected after 24 hours. The white cell counts were determined Electronics,  Florida) and differential  on a Coulter Counter (Model SS8O, Coulter white cell counts on Wright’s  stained  blood  smears. Leukocyte rich plasma was prepared from each specimen and cytospins were made as described earlier.  b) To determine intravascular  the effect of removing  from the PMN surface on their  behavior, purified PMN from 75m1 of donor blood were diluted in 20m1  PMN-buffer. The specimens incubated  L-selectin  were divided into two equal aliqouts  with either denatured  chymotrypsin  which were then  (control) or chymotrypsin  type IV as  described earlier. The PMN treated in this way were then infused into the recipients and the disappearance  of BrdU labelled PMN from the circulation  over a 24 hour period by obtaining baseline, immediately  blood specimens  was monitored  from the central ear artery at  after infusion of the PMN, 5, 10, 30, and 60 mm, and 3, 6 and  24 hours later. White cell counts were obtained using a Coulter Counter (Model SS8O, Coulter Electronics, Florida) and differential  white cell counts using Wright’s stained  blood smears. Leukocyte rich plasma was prepared from each specimen and cytospins were made as described earlier.  134 Evaluating  BrdU labelled  Double Immunoenzymatic  PMN in recipients  staining of leukocytes  Leukocytes on cytospins prepared from peripheral blood were stained for the presence of surface technique.  L-selectin  and  nuclear  The APAAP technique  BrdU  using  a double  alkaline  phosphatase  (Cordefi et al., 1984) was used for both antigens.  Surface L-selectin was first labelled followed by nuclear BrdU labelling. slides were fixed in acetone for 10 mm, incubated minutes before the application 200 (5g/ml) for 60 mm  Briefly,  with 5 % rabbit serum for 15  of the monoclonal antibody against L-selectin, DREG-  in a humidity  chamber at room temperature.  Non-immune  mouse IgG (5g/ml) and the omitting of the primary antibody was used as negative controls. As a linking antibody, a 1/20 dilution of rabbit anti-mouse IgG (Dako Z259) was applied conjugated  for 45 minutes,  followed by the anti-mouse  alkaline  complex (Dako D651) in a 1/50 dilution for 45 minutes.  phosphatase All antibodies  were prepared in 50mM TrisCi, 150 mM NaCl, pH7.6 (TBS) with 1 % BSA and slides were washed alkaline  in TBS twice for 10 mm  phosphatase  was developed  HistoMark Red® (Kirkegaard  Cell membranes  by using  each antibody a commercially  and Perry, Gaithersburg,  the dark. After a 10 minutes minutes.  between  The  available  kit,  Maryland) for 10 minutes in  wash, slides were fixed 1 % paraformaldehyde  were further permeablized  which time DNA was denatured The 2N HCI was neutralized  application.  by incubating  by methanol  for 10 mm  for 10  after  slides in 2N HCL at 37°C for 60 mm.  by washing the slides 3 times with 0. 1M borate buffer  (BDH), pH 8.5. This was followed by the second APAAP procedure where mouse anti BrdU (Boeringher-Mannheim, primary  antibody.  Mannheim,  Germany)  0. 1g/ml was used as the  With this procedure all slides were washed between antibody  135 applications  with 0.1 % Tween 20 in TBS (pH 7.6) for 10 minutes.  phosphatase  was developed with HistoMark Blue for 10 minutes in the dark. Slides  were washed for 30 minutes  in distilled  water, mounted  The alkaline  in an aqueous  medium  (Gelvatol®) and analyzed on a Zeiss Universal Research light microscope (Model 1W, West Germany). The influence of the double labelling procedure on the presence of surface L-selectin and nuclear  BrdU expression  number of positive PMN for each antigen  was evaluated  by comparing  with paired slides stained  the  for a single  antigen using the APAAP method as described above.  Evaluating  BrdU labelled PMN  The slides were coded and evaluated without knowledge of their origin. BrdU labelled PMN were evaluated  on computer generated  randomly selecting fields counting  100  PMN per slide. BrdU labelled PMN were identified by a deep blue staining of the cell nucleus  (figure 23). Cells were categorized  as either BrdU positive (blue nucleus),  BrdU and L-selectin positive (double labelled), L-selectin positive (red cell surface), or negative  for both labels. If less than 10 % of the PMN were BrdU labelled, 200  PMN were counted, if less than 5 %, 500 PMN were counted, and if less than 1 %, 1000 PMN were counted. The total number BrdU labelled PMN evaluated  in each  slide averaged 102± 19 with a range of 50 to 156. The number of BrdU labelled PMN present in the circulation  of the recipients  at each time point was expressed as a  fraction of the total number of labelled PMN infused corrected for the calculated blood volume (American Physiology Societ, 1965) in the following manner:  136  Fraction PMNBrdU Circ  =  PMNCWC  X  BV x %PMNe:em BrdU  PMNed  where the fraction PMN circulation  represents  the number of BrdU labelled PMN in the  as a fraction of the total number of BrdU labelled PMN infused, PMN CfrC  the calculated  number of PMN (1 x 106) in the circulation  (total white cell count times  the fraction of leukocytes that are PMN), BV the calculated blood volume, %PMNT the fraction of BrdU labelled PMN in a cytospin of peripheral and PMN  blood in the recipient,  the number(1 x 106) of BrdU labelled PMN infused (PMN count/mi x  ml of fluid infused x %BrdU labelled PMN). The BrdIJ labelled PMN were further stratified  as either stained for BrdU alone or  double stained for BrdU and L-selectin, and these fractions were calculated separately using the equation  Calculation  of the half-life of BrdU labelled PMN  The time required circulating  above.  to achieve the maximal  blood of the recipients  the rate of decay equation  of BrdU labelled PMN in the  who received purified PMN, Tmax, was applied to  to calculate  described in the previous section.  number  the half-life of PMN in the circulation  as  ‘37  a  0  ‘4  .-  .  II b  4 Figure 23: Immunocytochemical detection of BrdU label led cells. Panel (a) demonstrates a cytospin prepared from LRP of the donor blood after 7 days of BrdU 25gfkg/day treatments. Cells were stained for the prese nce of BrdU using the APAAP method, with BrdU labelled cells staining red (see text). Panel (b) demonstrates double immunolabelling of PMN on cytosp ins for both nuclear BrdU (blue) and surface L-selectin (red) using a double alkaline phos phatase technique (see text). Bar=lOMm.  138  Statistical  analysis:  Statistical Inc., Evanston,  analysis was performed using SYSTAT® Version 5.1 software (Systat, IL) (Wilkinson,  A one-way analysis  1990).  of variance  was used to evaluate  the changes  in L-selectin  expression on PMN in the circulation  over time, and a two-way analysis  was used to compare the transfusion  of whole blood with isolated PMN over time.  To evaluate labelled estimated  the differences in the behavior of chymotrypsin-treated  PMN the disappearance as the relationship  rate  maximum  likelihood  (REML) method  were then compared using a chi-square statistic  intercepts  and lines were considered significant  aL, 1990).  pBrdU  was  and time.  animals within each group were then compared  estimates  The estimated  or control BrdU  PMN in each recipient  between the log of the fraction of  The family of lines for the individual using the restricted  of labelled  of variance  (Feldman,  1988). The  and differences in slopes,  when the p-value was less than 0.05.  slope was then used to calculate the half-life for each group (Bryan et  139 RESULTS In vivo labeffing  of PMN with BrdU  Daily intravenous  injections  percentage  of 25 mg/kg of BrdU produced a rapid increase in the  of BrdU labelled of the PMN in donor rabbits with 82±4% PMN labelled  by 7 days. AU cells with visible nuclear BrdU stain were deemed positive (figure 23a). In double labelled slides, the surface L-selectin was stained red and the nuclear BrdU blue with overlapping areas stained purple (figure 23b). Expression of both L-selectin and BrdU was similar in paired single and double stained  L-selectin  changes  on PMN during their intravascular  slides.  lifespan  The fraction of L-selectin negative BrdU labelled PMN increased with time spent in the circulation  whether or not the PMN were infused as whole blood (p <0.001) or as  purified  PMN (p <0.0001) (figure 24). The fraction  negative  PMN infused was higher for the purified PMN (14.2±1.9%) than for the  whole blood (7.5±2%), resulting  of BrdU labelled  in a higher fraction of L-selectin  labelled PMN in the recipient circulation  L-selectin  negative  BrdU  directly after purified PMN were infused  (23±5.2%) than after the infusion of whole blood (11±2.6%). This difference was attributed  to a small loss of L-selectin from PMN during the purification  which was confirmed  with flow cytometric  (figure 25a and b). In both transfusion PMN occurs within  the first 30 mm  analysis  of these populations  process, of PMN  modes, a rapid increase in L-selectin negative after infusion  followed by a more gradual  increase over the rest of the study period. After 24 hours, with the infusion of either whole blood or purified PMN, nearly all the PMN were negative for L-selectin (figure 24).  /4’O  z 100-  I.  80C  T  60-  ,o-..  -  -.  -  -  /  0  80  160  240  320  I  400400 1440  Time (mm) •  Whole Blood  -  -0  -  Purified PMN  Figure 24: L-selectin changes on PMN during time spent in the circulation when BrdU labelled PMN were infused as whole blood (closed circles, n=5) and purified PMN (open circles, n=5). Values are the mean±SE. PMN on cytospins made of leukocyte rich plasma were double immunolabelled for BrdU and L-selectin (see text). The fraction of L-selectin negative BrdU labelled PMN (y-axis) increased significantly over time in both groups (p <0.001) with nearly all the BrdU labelled PMN being negative for L-selectin after 24 hours.  14!  a  b  c  1  1  4-’  D 0 C.)  a) 0  d  I  1  e  f  I_  1.  1  •  Mean Fluorescence Intensity Figure 25: The effect of chymotrypsin (lU/mi for 3x10 6 PMN incubated for 15 miii) on the expression of L-selectin (a, b and c) and CD18 Cd, e and f) as measured with flow cytometry. Panels a and d represent baseline expression of PMN L-selectin and CD18 respectively determined on whole blood. Panels b and e represent similar expression after PMN purification (note the small loss in L-selectin with PMN purification). Panels c and f represent PMN L-selectin and CD18 expression after chymotrypsin treatment respectively. Note the significant loss of L-selectin from the PMN with chymotrypsin treatment but no changes in the expression of CD18. In panels the events left of cursor 1 represent non-specific labelling. The x-axis represents events and the y-axis the mean fluorescence intensity plotted on a logscale.  142 In vitro effect of chymotrypsm  on PMN  Cleavage of L-selectin from PMN by chymotrypsin  There was no difference between the expression of L-selectin on PMN incubated denatured  chymotrypsin  or buffer alone, therefore all subsequent  PMN incubated in denatured and 25c demonstrated following  chymotrypsin  immunocytochemistry  treatment.  This  loss  by grading the expression  cytospins prepared from leukocyte rich plasma,  present was  on PMN was lost confirmed  with  of L-selectin on PMN present in where PMN highly positive for L  selectin decreased from 63 ± 6.8 % to 4.6 ± 3 % with chymotrypsin  Effect of chymotrypsin  results are from  before analysis and infusion. Figure 25b  that 78±8.2% of the L-selectin  chymotrypsin  in  treatment.  on PMN activation parameters  The viability of PMN, accessed by the trypan blue exclusion, was the same before and after chymotrypsin  treatment  and the treatment  did not change the expression  of  CD18 (figure 25d and 25e) as measure by flow cytometry.  The clearance  of chymotrypsin  treated PMN from the circulation  The fraction of BrdU labelled PMN present in the circulation  of recipients  directly  after infusion was the same in both the control (48±12.6%) and the chymotrypsin treated  (46±9.6%) groups.  Although  the number  of chymotrypsin  decreased in the first hour following infusion, they returned hours and then disappeared  from the circulation  group (figure 26). This similarity  treated  to the circulation  PMN  by 3  at the same rate as in the control  was reflected in the overall half-lives  (262 versus  296 minutes) of both groups (table Vifi). In both groups L-selectin positive PMN were  100 C)  0  80  .4-  0  60  z  0  80  160  240  320  4001400  1500  Time (mm) 0  Control  —•  Chymotrypsin  Figure 26: BrdU labelled PMN in the circulation of recipients after the infusion of purified PMN treated with either denatured chymotrypsin (open circles, n=5) or chymotrypsin (closed circles, n=5). PMN were incubated with chymotrypsinldenatured chymotrypsin (1U/ml for 3 x 106 PMN) for 15 mm. Values are mean±SE of the number of BrdU labelled PMN in the circulation as a fraction of the number of BrdU labelled PMN infused.  100  144 a  U) 0 U)  80  0  60 z a 40  T  o  K  I  ---  0  80  160  240  320  4001400  100 0 0  b  4-  o  80  t  0  60  z a •  40 T S...,  20  ---  .  T  7—  0 0  80  160  240  320  4001400  Time (mm) 0  L-seIectin+  —-•--—  L-seiectin  Figure 27: BrdU labelled PMN in the circulation of recipients after the infusion of purified PMN treated with either denatured chymotrypsin (a, n=5) or chymotrypsin (b, n=5). PMN were incubated with chymotrypsinldenatured chymotrypsin (1U/ml for 6 PMN) for 15 mm. Values are mean±SE of the number of BrdU labelled PMN 3x10 in the circulation expressed as fraction of the number of BrdU labelled PMN infused. The closed circles represent the washout of L-selectin negative PMN and the open circles L-selectin positive PMEN. In both groups L-selectin positive PMN clear from the circulation faster than L-selectin negative PMN. With chymotrypsin treated PMN (b), L-selectin positive PMN cleared from the circulation significantly faster than L selectin negative PMN (p < 0.05).  145  cleared from the circulation 27b). In the chymotrypsin  faster than L-selectin negative  PMN (figure 27a and  group, L-selectin positive PMN were cleared from the  circulation faster than L-selectin negative PMN. However, the half-lives of L-selectin negative population  PMN in both group did not differ from that of the half-life of the whole of PMN (table VIII).  ‘4’ TABLE Vifi Half-life of PMN treated with and without Chymotrypsin  Cell populations  112 T  Confidence intervals Upper(min)  Lower(min)  Control PMN’; All  262  458  184  L-selectin+  178  311  134  L-selectin-  219  320  135  Chymotrypsin treated PMN; All  296  570  199  L-selectin+  133  238  118  L-selectin-  302  722  180*  #Control PMN were incubated with denatured chymotrypsin *Half..life of the L-selectin negative PMN was significantly longer than the L-selectin positive PMN in the group that was infused with chymotrypsin treated PMN.  147  DISCUSSION The results increases  presented  here show that  the fraction  during their stay in the circulation.  PMN  and changing to negative cells  removal of L-selectin positive PMN from the circulation.  data favour the hypothesis PMN as they marginate expressing  negative  This may be either from L-selectin  positive PMN losing their L-selectin in the circulation or from the preferential  of L-selectin  that L-selectin is progressively  and demarginate  lost from the surface of  in the circulation,  resulting  lower levels of L-selectin. We also demonstrated  from the surface of PMN with chymotrypsin  Our  in older PMN  that cleaving L-selectin  did not result in the accelerated removal  of these PMN from the circulation.  The adhesive interaction sequential  1991)  rolling which is mediated  molecules  that  recognize  fucosylated  constitutively  carbohydrate  (sLex) (Lasky,  1992a;  expressed on nearly all circulating  et aL, 1990a) that interact rolling and margination  with endothelial  on activated  endothelium-associated  endothelium  of the selectin  1991; Lawrence ligands, Springer,  1990b).  family of  and Springer,  especially  structures L-selectin  is  PMN (Griffin et al., 1990; Tedder  cell surface ligands,  of PMN in post-capillary  has been shown to occur or normal unactivated  cells involves multiple  1991). The initial adhesive  by members  (Abbassi et al., 1993; Butcher,  sialylLewisx  containing  enhanced  and endothelial  steps (Butcher, 1991; Lawrence and Springer,  step is leukocyte adhesion  between leukocytes  venules.  resulting  in the  This rolling phenomenon  endothelium  with L-selectin presenting  (Ley et al., 1993) but is sLex to the inducible  selectins, P- and E-selectin (Picker et al., 1991; Spertini et al.,  1991a). The fraction of leukocyte rolling increases  from under 20% on unactivated  148 endothelium  to over 30 % on cytokine activated  endothelium  with a marked decrease  in rolling velocity (Ley et al., 1993; Ley et al., 1991b). Rolling cells become activated when they encounter appropriate endothelial  surface or in the extravascular  both functional allows  activating  firm  adhesion  CD18/ICAM-1 activation  activation  system  or chemotactic compartment.  and up-regulation PMN and  between  stimuli generated PMN activation  of the 2 -3 integrins  results in  (CD 11/CD 18) and  the endothelium  (Arfors et al., 1987; Lawrence  on the  mediated  and Springer,  via the  1991). PMN  has also been shown to result in the shedding of L-selectin as a deadhesive  event. This chain of events emigrate  has been shown in vivo to be essential  into the extravascular  1987; Von Andnan  space toward an inflammatory  for PMN to  focus (Arfors et al.,  et al., 1992).  The principle finding in this study is that PMN L-selectin expression decreases with time spent in the circulation,  which may be either from L-selectin positive PMN  losing their L-selectin in the circulation preferential  and changing  to negative  cells or from the  removal of L-selectin positive PMN from the circulation.  the concept that  PMN shed L-selectin  shedding is the principal mechanism  during  their  Our data favor  intravascular  life because  for PMN to regulate their L-selectin expression  (Spertini et al., 1991b; Tedder, 1991). It is conceivable that PMN could shed their L selectin when they encounter mildly activated vascular beds such as the gums, upper respiratory  tract and bladder where they are inadequately  adherence  and migration  express  lower levels  demonstrated  and that PMN released  of L-selectin.  intravascular  Previous  activation  stimulated  to support firm  from these vascular  studies  beds could  from our laboratory  of PMN with shedding  of L-selectin  have and  149  *  without PMN migration  in the lungs of rabbits who had been exposed to cigarette  smoke and complement  fractions (Doerschuk and Allard, 1989; Kiute et aL, 1993).  However,  this is unlikely  expression  to be the major mechanism  on the PMN in our study because  for the loss of L-selectin  the size of these mildly activated  vascular beds is small in relation to the total microvascular  Spontaneous  shedding of L-selectin in lymphocytes  by Spertini  et al (1991b), but  Furthermore,  high levels of circulating  been demonstrated continuously attractive  whether  hypothesis  PMN behave  similarly  et al (1992), suggesting  while while they in the intravascular  is that  ligand  binding  provides a rapid means for the regulation with the subsequent  has been demonstrated  initiates  receptor  are inadequate  vascular  sites. The stage at which L-selectin  of PMN on “normal” or activated derived activating  leukocytes  space. A more shedding  which  or alternatively  has not been established. endothelium,  factor, or the migration  cells  if the conditions for  firm adhesion and migration  interaction  that  This de-adhesion event  may be necessary to allow the to PMN return to the circulation  leukocyte-endothelial  is unknown.  of leukocyte adhesion to endothelial  release of the cell allowing de-adhesion.  space at inflamed  in culture  free L-selectin (sL-selectin) in plasma have  by Schleiffenbaum  shed L-selectin  surface available.  to migrate out of the is shed during  Hypothetically,  the rolling  the exposure of PMN to endothelium  process through  the endothelium  itself  may all be associated with L-selectin shedding. Shedding may also occur at all above mentioned stages of leukocyte-endothelial show that normal circulating  interaction  in a graded fashion. Our results  PMN decrease their expression of L-selectin over time  and that PMN that remaining  in the circulation  for 24 hours are universally  L  150 selectin negative. the endothelium  Technical  This finding suggests that the normal contact between PMN and may result in a loss of surface L-selectin.  factors could contributing  seen in our model specifically, purification  or transfer procedures.  to the decrease in PMN L-selectin expression  activation  of the PMN by either  the labeffing,  We have shown though that labelling PMN with  BrdU did not activate or prime the cells (previous section). This method of labelling PMN allowed us to study the behavior of an activation such as L-selectin, over time in the circulation.  surface molecule,  sensitive  It is thus unlikely that the labeffing  procedure per se result in PMN activation.  The purification  procedure of PMN resulted in a small loss of PMN L-selectin (figure  25a and b), suggesting procedure. against  that the PMN were mildly activated  However, fact that CD18 expression  significant  cell activation.  human PMN to purification  during the purification  did not change that would argue  This finding is in contrast  to the response  (Kuijpers et al., 1991), where CD18 expression increases  with PMN purification and L-selectin expression remains unchanged. have been shown to behave differently  in the circulation  cells and are removed from the circulation this series of experiments  of  Activated PMN  than normal unactivated  faster (Haslett et al., 1985). However, in  the removal of purified PMN (262 mm  95 % CI 458-184) was  similar to values obtained when PMN were infused as whole blood (270 mm  95 % CI  248-296).  It is unlikely that the transfusion  per se influenced  the PMN behavior as both whole  151 blood and the purified PMN were given to serum-compatible  recipients  and neither  produced changes in total white cell, PMN, band cell and platelet counts or increased CD18 expression conclude  that  significantly  on PMN over the 24 hour study period (data not shown). We the  labelling,  activated  To test the hypothesis  purification  and  transfusing  procedures  did not  the PMN.  that the lack of L-selectin on the surface of PMN is a signal  for their removal from the circulation,  we selectively  removed L-selectin from the  surface of PMN. We have shown that PMN L-selectin expression decreases over time in the circulation population  which implies that L-selectin negative  PMN  represent  of PMN. An older population of PMN should theoretically  an older  have a shorter  half-life than newly released cells from the bone marrow. Removing L-selectin from PMN with chymotrypsin selectin negative into recipients,  (Jutila et al., 1991) greatly increased the population  PMN we transfused an early temporary  was seen, but they reappeared similar  half-lives  of L  (figure 25). After infusing these treated PMN removal of these cells from the circulating  pool  in the circulation between 1 and 3 hours. Additionally,  were calculated  for the control and the chymotrypsin  treated  groups. These findings suggest that the lack of L-selectin on PMN did not result in their permanent The calculated chymotrypsin  removal from the intravascular half-lives  treated  of the L-selectin  compartment.  negative  PMN in the control and the  groups were similar if compared to the whole population  labelled  PMN (table VIII). This longer than  negative  PMN may represent  negative  PMN in the circulation,  a continuous  expected  half-life  of  of the L-selectin  shift of L-selectin positive to L-selectin  which may also account for the shorter half-lives  152 of the L-selectin  positive PMN in both the control and the chymotrypsin  treated  groups.  The daily turnover majority  rate of PMN in humans  is 100 to 200 billion cells, with the  of these PMN being removed by the spleen and liver (Andrewes,  19 lOb;  Andrewes, 1910a; Bicknell et al., 1994). Our results suggest that a lack of L-selectin expression is not the signal responsible circulation  for the removal of these older PMN from the  because removing L-selectin from PMN with chymotrypsin,  “older” PMN, does not result in the accelerated  removal  simulating  of these cells from the  circulation.  Our data show a smaller  fraction of L-selectin positive than negative  first specimen of circulating  PMN in the  blood taken after the infusion of labelled PMN (figure  27a and b). This finding suggests that the L-selectin positive PMN are preferentially removed from the circulating previous studies demonstrating  pooi of blood early after infusion and is consistent preferential  sequestration  in the lungs of rabbits exposed to zymosan activated Quinlan  et al., 1992). Our findings  selectin  positive PMN preferentially  of L-selectin positive PMN  plasma (Graham et aL, 1992;  extend these observations marginate  with  by showing that L  in normal unstimulated  animals.  Moreover, a rapid increase in L-selectin negative PMN was seen within the first 30 mm  after the infusion of labelled whole blood and purified PMN (figure 24), which  may represent preferential these findings return  suggest  margination  preferential  to the circulation  of L-selectin positive PMN. Taken together,  margination  with less L-selectin  of L-selectin resulting  positive  PMN that  in an increasing  pool of  153 circulating  L-selectin negative  Other studies  PMN.  from our laboratory  PMN in humans  have demonstrated  that in normal  circulating  (previous sections) and rabbits (Klute et aL, 1993) the expression of  L-selectin is variable with populations levels of L-selectin,  of PMN expressing high, intermediate  These PMN contrast  with mature  and low  PMN in the bone marrow  where the majority express high levels of L-selectin (Lund-Johansen  and Terstappen,  1993; previous section) resulting in an increase in L-selectin expression on circulating PMN with the bone marrow release of PMN. It has been shown in vitro and in vivo that in the chain of events responsible for leukocyte-endothelial is essential  for PMN emigration  Lund-Johansen,  and Terstappen,  interaction,  to an area of inflammation  L-selectin  (Ley et al., 1991b;  1993; Mulligan et a!., 1991; Mulligan et al., 1994;  Tate and Repine, 1983a), implying that PMN recently released from the bone marrow are equipped  to be preferentially  PMN with less L-selectin  recruited  to foci of inflammation  may be less able to be recruited  levels of L-selectin on neonatal  importance speculate  of a critical  receptor  density  endothelium further  for L-selectin-dependent  that PMN with reduced or absent L-selectin represent  older circulating marginate  PMN deficit in L-selectin  PMN with reduced functional  and emigrate  capabilities,  to areas of inflammation.  older  to these sites. Reduced  PMN (±50% of adult values) are related  reduced ability to adhere under flow conditions to activated et aL, 1991). This neonatal  whereas  to their  (Anderson  emphasizes  the  function.  We  a subpopulation  of  such as their ability to  154  6) SUMMARY AN]) FUTURE DIRECTIONS of PMN to sites of inflammation  The emigration During  years  recent  there  has been an extraordinary  concerning the mechanisms during inflammation  intercellular increase  (Butcher, 1991; McEver, 1992 Springer,  adhesion.  in information  that mediate this leukocyte-endothelial  aL, 1992; Yong, and Khwaya, in this interaction  requires  cell interaction  1990b; Zimmerman  et  1990). The cell adhesion molecules play a pivotal role  during inflammation  in the systemic and pulmonary  However, the role of these molecules in the trafficking  microvessels.  of PMN from the bone marrow  into the blood and changes during their lifespan in the circulation this thesis we have explored the fate of the cell adhesion  are less clear. In  molecule, L-selectin,  on  PMN from the time of their release from the bone marrow into the bone marrow venous sinusoids and the general circulation until their permanent intravascular  pool (figure 28). We have demonstrated  PMN in the bone marrow  hematopoietic  tissue  removal from the  that the mature  express  the highest  segmented levels of L  selectin. Some of this L-selectin is shed when these cells cross the bone marrow-bloodbarrier. During a chemotactic more permeable  and this  stimulus  of the bone marrow the barrier may become  loss of L-selectin  conditions  the expression  expression  on bone marrow PMN, although  of L-selectin  may be less marked.  on circulating  PMN nearly  Under equals  the same level of expression  these the  is never  reached. This also suggests that the PMN expressing high levels of L-selectin in the circulation  at baseline  are newly released  PMN from the bone marrow. We have  further demonstrated  that PMN in the circulation  longer  in the circulation.  they remain  expression of L-selectin on circulating  progressively  This finding  lose L-selectin the  may explain  PMN at baseline, with  the variable  newly released PM  7’  L-selectin during bone marrow release of PM N’s Baseline  Precursor  Bone marrow Mature PMN  Bone Marrow sinusoi  •  ..  zz Circulating blood  * edding • L-seleCtifl  Tissue  156 expressing the highest and older PMN the lowest levels of L-selectin. This lack of L selectin on older PMN in the circulation  has been shown to be not a signal for their  removal from the circulation.  It is known that many chemotactic particularly  factors play a key role in leukocyte homeostasis,  in the setting of inflammation.  These factors also stimulate  of PMN from the bone marrow which functionally chemotaxis regulated  and eventual  neutrophilia  at the cellular level.  leukocytosis  links the initial  as a continuous  transverse  process. In leukemia  may play a key role in this  immature  white  cells are able to  the bone marrow-blood barrier and appear in the blood in contrast to their  normal immature and  for example,  leading to neutrophilic  Adhesion events between the  egressing cells and the bone marrow microenvironment  dynamic  neutropenia,  dynamic process that is self-  The precise mechanism(s)  at the cellular level remain undefined.  the release  counterparts.  promyeloblasts  agranulocytosis,  Even when there is a relative increase in myeloblast  as compared  to differentiated  there is no egress of immature  granulocytes,  as seen with  forms into the blood. The exception  occurs when recombinant  hematopoietic  patients in pharmacological  doses. These factors also alter the adhesive properties of  leukocytes  which may contribute  Similarly,  adhesive  properties  growth  factors  (GM-CSF) are given to  to their ability to egress from the bone marrow. of leukemic  cells differ from normal  immature  leukocytes that may explain their ability to egress from the bone marrow. Both GM CSF stimulated L-selectin  and acute and chronic myeloid leukemic cells express low levels of  (Demetri  and Antman,  adhesion molecule may be partially  1992; Spertini responsible  et al., 1991b). This lack of the for their relocation  from the bone  157 marrow into the circulation. potentials  In aleukemic  leukemia  the blast cells have different  to gain access to the circulation and it is tempting  cells have similar adhesive qualities  as “normal” myeloblasts.  to speculate that these The development  of a  L-selectin knock-out animal may shed further light on the role of L-selectin in the egress of PMN from the bone marrow.  Our finding that intravascular to release PMN expressing Polymorphonuclear important  complement  stimulates  leukocytes  have been implicated  in the pathogenesis  lung destruction  distress syndrome (ARDS), ischaemia-reperfusion  the heart and multi-organ 1983; Tate and Repine, increase in circulating  failure associated  1983a; Tennenberg segmented  stores has been attributed  the bone marrow  high levels of L-selectin may have clinical implications.  clinical conditions that including  respiratory  activation  of several  in emphysema,  adult  injury in the gut and  with septic shock (Hernandez  et al.,  et al., 1987; Senior et aL, 1977). The  and non-segmented  PMN from the bone marrow  to a variety of stimuli (Jagels and Hugli, 1992; Marsh et  al., 1967; Ulich et al., 1989). Chronic cigarette smoking for example, produces a 20%25 % increase in peripheral blood leukocyte counts compared to non-smoking controls which may be related to excess marrow release (Corre et al., 1971; Hunninghake  and  Crystal, 1983; Janoff, 1983). Recent evidence suggests that PMN in the bone marrow pool are mobilized after intravascular  complement activation  in rabbits (Doerschuk  and English, 1991) and sheep (Rosolia et aL, 1992). When PMN are released from the  bone marrow following complement activation (Doerschuk and English, 1991; Rosolia et al., 1992), the PMN expressing preferentially  high levels of L-selectin  have been shown to  sequester in the lungs (Quinlan et al., 1992; Graham et al., 1992). In  158 this study we have shown that newly released PMN express high levels of L-selectin, and it is tempting marginate  to speculate  that  these  and adhere to activated endothelium  newly released  and emigrate into inflammatory  Therefore, they could play a critical role in the pathogenesis injury such as ARDS and multi-organ therapeutic  intervention  PMN preferentially  of PMN-induced  foci.  organ  failure, and in doing so are a prime target for  in conditions of PMN mediated  tissue injury.  Our finding that newly released PMN express high levels and older PMN lower levels of L-selectin, creates the opportunity and trafficking the circulating randomly  to selectively  of these different populations  study the functional  of circulating  capabilities  PMN. 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