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

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STUDIES ON THE MECHANISM OF MURINE CYTOMEGALOVIRUS INDUCED IMMUNOSUPPRESSION by PETER FREDERICK MUIR WHYTE B.Sc,  (Microbiology), U n i v e r s i t y of B r i t i s h Columbia  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY  We accept  this thesis as conforming  to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA Augus t  1983  (Copyright, Peter F.M. Whyte  f  3933  In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of  Pathology  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  Aug. 19, 1983  ii  ABSTRACT  The  present study was undertaken to elucidate the mechanism of  murine cytomegalovirus-induced immunosuppression.  Previous studies  had established the immunosuppressive e f f e c t of murine cytomegalovirus (MCMV) i n v i v o , and i n v i t r o , and had characterized a wide range of immunological function which were impaired  during  MCMV i n f e c t i o n .  Canditioned  medium, prepared from the tissue culture medium o f  cultured spleen c e l l s i n f e c t e d with MCMV, contained  a soluble  immunosuppressive factor which suppressed spleen c e l l response to stimulation by Con A. This v i r u s induced immunosuppressive f a c t o r (VISF) was not present i n conditioned media prepared from c o n t r o l cultures or from cultures o f spleen c e l l s inoculated with various other viruses or latex beads.  VISF was produced within 24 hours  a f t e r i n f e c t i o n o f the c e l l s , and was not dependent upon the a d d i t i o n o f exogenous f e t a l bovine serum to the culture medium.  Attempts to extract VISF by l i p i d e x t r a c t i o n techniques wer unsuccessful: no s i g n i f i c a n t difference was observed between the c a p a c i t i e s o f c o n t r o l and MCMV conditioned media-extracted l i p i d s to suppress Con A stimulation o f spleen c e l l s .  Also, the production o f  iii VISF could not be abrogated by the a d d i t i o n of indomethacin, an i n h i b i t o r o f prostaglandin synthesis, to the spleen c e l l c u l t u r e s . From these s t u d i e s , i t was concluded that VISF was not a prostaglandin and was probably not any other type o f l i p i d molecule.  It was found that VISF could be concentrated i n the r e s i d u a l volume of an u l t r a f i l t r a t i o n c e l l using an Amicon YM2 membrane f i l t e r (molecular exclusion l i m i t o f 1000 d a l t o n s ) , but VISF was not retained when an Amicon YMLO membrane f i l t e r (molecular exclusion l i m i t of 10,000 daltons) was used.  This indicated a molecular  weight range f o r VISF o f 1000-10,000 daltons.  Gel f i l t r a t i o n  chromatography of concentrated samples o f conditioned media on a Sephadex G-25 matrix revealed a low molecular weight f r a c t i o n (MW 1400 daltons) of immunosuppressive a c t i v i t y present i n the MCMV i n f e c t e d sample, but not i n the c o n t r o l sample or i n samples from spleen c e l l s i n f e c t e d with other viruses or p a r t i c l e s .  When MCMV  and c o n t r o l samples were digested with the p r o t e o l y t i c enzyme, proteinase K, p r i o r to f r a c t i o n a t i o n , t h i s immunosuppressive a c x t i v i t y was s i g n i f i c a n t l y diminished, but no s i g n i f i c a n t change occurred i n the corresponding c o n t r o l sample f r a c t i o n .  This  suggested that VISF was a peptide or was, a t l e a s t p a r t l y , peptide i n nature.  To c o r r e l a t e the above findings with immunosuppression i n v i v o , studies were performed on mice i n f e c t e d with a s u b l e t h a l dose o f MCMV.  Virus-free serum c o l l e c t e d four days a f t e r i n f e c t i o n of the  mice was found to contain immunosuppressive a c t i v i t y r e l a t i v e to c o n t r o l serum from uninfected mice.  Fractionation of the serum by  Sephadex G-25 column chromatography demonstrated several f r a c t i o n s i n which the i n f e c t e d mouse serum was more suppressive than the f r a c t i o n which contained the immunosuppressive a c t i v i t y from the MCMV conditioned medium sample, however the pattern was complicated by the number of other immunosuppressive substances present i n both sera.  : >v  TABLE OF CONTENTS  Chapter I.  Page Introduction I. II. III.  H i s t o r i c a l aspects o f cytomegalovirus  1  Nomenclature  2  Physical  V.  VI.  VII.  properties  o f the v i r u s  3  a.)  Morphology  3  b.)  DNA  3  c.) IV.  1  Proteins  4  Replication  4  a.)  4  Host  Range  1.  Human c y t o m e g a l o v i r u s  4  2.  Murine  5  cytomeglaovirus  b.)  Penetration  o f the c e l l  5  c.)  Replicative  cycle  5  Pathogenesis  7  a.)  Human c y t o m e g a l o v i r u s  7  b.)  Murine  8  cytomegalovirus  Immunological  Interactions  11  a.)  Human c y t o m e g a l o v i r u s  11  b.)  Murine  12  Purpose  cyotmegalovirus  o f the s t u d y  15  v  II.  M a t e r i a l s and I.  II.  Methods  16  Methods  16  1.  Mice  16  2.  Cells  16  3.  Viruses  17  4.  Tissue  5.  F e t a l b o v i n e serum  18  6.  Solutions  18  7.  Chemicals  20  c u l t u r e medium  17  Methods  21  1.  Mouse embryo c e l l  cultures  2.  BHK-21 c e l l c u l t u r e s  22  3.  Spleen c e l l c u l t u r e s  22  4.  P r o p a g a t i o n o f murine c y t o m e g a l o v i r u s  23  5.  Propagation of Sindbis v i r u s  23  6.  Plaque a s s a y o f v i r u s  24  7.  C o l l e c t i o n o f mouse serum  25  8.  Preparation  9.  Mitogen s t i m u l a t i o n t e s t  of conditioned  21  medium  of conditioned  25 26  10.  Ultrafiltration  media  11.  E x t r a c t i o n of l i p i d s  12.  Sephadex G-25  13.  Assay o f Sephadex G-25  14.  Proteinase  K digestion  30  15.  Polyacrylamide g e l electrophoresis  30  from c o n d i t i o n e d  27 media  f r a c t i o n a t i o n of conditioned fractions  28 media  29 29  i .  vii  III.  16.  Staining  17.  Liquid s c i n t i l l a t i o n  Results I:  o f gels  31 counting o f r a d i o a c t i v i t y  Evidence f o r the presence o f a  soluble  suppressor f a c t o r . I.  33  Immunosuppressive e f f e c t o f serum from MCMV i n f e c t e d mice  II.  III. IV.  33  In v i t r o p r o d u c t i o n o f a v i r u s  induced  suppressor  factor  36  S p e c i f i c i t y o f the v i r u s induced suppressor f a c t o r  43  F a c t o r s a f f e c t i n g the p r o d u c t i o n o f t h e v i r u s induced suppressor f a c t o r  IV.  Results  I I : Characterization  44  o f the v i r u s  induced  suppressor f a c t o r . I.  53  E f f e c t o f i n d o m e t h a c i n on t h e p r o d u c t i o n o f t h e v i r u s induced suppressor f a c t o r  II.  C o n c e n t r a t i o n o f the v i r u s  53  induced suppressor  factor  by u l t r a f i l t r a t i o n III.  Fractionation  55  of conditioned  medium by g e l  f i l t r a t i o n chromatography IV.  Fractionation  59  o f mouse serum by g e l f i l t r a t i o n  chromatography V.  32  Fractionation  64 of Sindbis  v i r u s and LB8  media by g e l f i l t r a t i o n chromatography  conditioned 69  VI.  Proteinase K digestion of the v i r u s induced suppressor factor  VII.  Electrophoresis of mouse serum and conditioned medium  Discussion  Bibliography  . • ix LIST OF TABLES  —  Page o  I.  Effect of conditioned media on the uptake of  H-dThd  by unstimulated and Con A stimulated spleen c e l l s .  II.  E f f e c t of the p u r i f i c a t i o n of conditioned media on the suppression of Con A stimulation of spleen c e l l s .  III.  42  Effect of l i p i d s from conditioned media on the stimulation of spleen c e l l s by Con A.  IV.  40  58  Effect of u l t r a f i l t r a t i o n through a YM2 membrane f i l t e r on the a b i l i t y of conditioned media to suppress Con A stimulation of spleen c e l l s .  V.  61  Effect of proteinase K on the f r a c t i o n a t i o n of the v i r u s induced suppressor factor by Sephadex G-25 chromatography. 75  ; .X  LIST OF FIGURES  Figure 1.  Page E f f e c t o f normal and i n f e c t e d mouse serum on Con A s t i m u l a t i o n o f spleen c e l l s .  2.  Suppression of Con A s t i m u l a t i o n of spleen c e l l s by murine cytomegalovirus c o n d i t i o n e d medium.  3.  50  E f f e c t o f f e t a l bovine serum on the production of the v i r u s induced suppressor f a c t o r .  6.  46  K i n e t i c s o f production o f the v i r u s induced suppressor factor.  5.  39  E f f e c t o f v a r i o u s c o n d i t i o n e d media on Con A s t i m u l a t i o n of spleen c e l l s .  4.  35  52  E f f e c t o f indomethacin on the production of the v i r u s induced suppressor f a c t o r .  56  x  7.  E f f e c t o f the c o n c e n t r a t i o n o f the v i r u s suppressor  8.  factor  Sephadex G-25  9.  Sephadex G-25 cytomegalovirus  10.  Sephadex G-25  Sephadex G-25 control  of control  fractionation  66  o f normal and murine  i n f e c t e d mouse serum.  fractionation  o f S i n d b i s and  68  LB8 72  1  fractionation  63  and murine  c o n d i t i o n e d media.  c o n d i t i o n e d media.  11.  on Con A s t i m u l a t i o n o f s p l e e c c e l l s .  fractionation  cytomegalovirus  induced  of proteinase K digested  and murine c y t o m e g a l o v i r u s  c o n d i t i o n e d media.  74  i  ABBREVIATIONS  N,N'-bis-methylene acrylamide concanavalin A deoxythymidine f e t a l bovine serum gravitational  force  tritium Hanks' balanced s a l t  solution  human cytomegalovirus immunoglobulin infected mouse serum modified Eagle's medium murine cytomegalovirus m u l t i p l i c i t y of i n f e c t i o n normal mouse serum phosphate buffered s a l i n e sodium dodecyl s u l f a t e v i r u s induced suppressor factor  xiii ACKNOWLEDGEMENTS  I would l i k e to extend my most sincere thanks to my supervisor, Dr. J.B. Hudson, for allowing me this opportunity  to study and carry out this project  i n his laboratory, and for his guidance throughout the course of this study. My gratitude i s a l s o extended to the members of my supervisory committee, Dr. C.E. Slonecker, Dr. W.H. Chase, Dr. S. Sacks and Dr. R.H. Pearce, for t h e i r guidance and h e l p f u l suggestions. and  I would a l s o l i k e to thank Ms. Cathy Moore  Ms. Elizabeth Graham for t h e i r t e c h n i c a l assistance, and Mr. Douglas  Walker f o r h i s h e l p f u l discussions.  1  CHAPTER I  INTRODUCTION  I.  HISTORICAL ASPECTS OF  CYTOMEGALOVIRUS  In 1926, the d i s c o v e r y o f a f i l t e r a b l e , h e a t l a b i l e capable of transmitting  ' c y t o m e g a l i a ' i n guinea  factor  p i g s brought  an end  to a twenty-two y e a r l o n g debate c o n c e r n i n g the n a t u r e and e t i o l o g y o f the c y t o m e g a l i c c e l l s (22,74). cellular  The e a r l i e r  observed  i n various tissues  t h e o r y o f a p r o t o z o a n n a t u r e o f the l a r g e  b o d i e s had been g r a d u a l l y  g i v i n g way t o the t h e o r y o f a  v i r a l e t i o l o g y , r e s u l t i n g i n swollen host c e l l s . lesions included  observed  i n the s a l i v a r y g l a n d s  of several  f a c t o r p r e s e n t i n the s a l i v a r y g l a n d s 1954 t h a t  i s o l a t e d and propagated isolates  were  d e s c r i b e d by  by members o f the herpes  McCordock and Smith d i s c o v e r e d a s i m i l a r  was n o t u n t i l  animals  Today, the m a j o r i t y o f Cowdry's Type A  i n c l u s i o n s a r e r e c o g n i z e d as b e i n g caused family.  The c y t o m e g a l i c  i n the o r i g i n a l Type A n u c l e a r i n c l u s i o n s  Cowdry i n 1934 ( 2 3 ) .  of infants  transmissible  o f mice i n 1936, however i t  the s a l i v a r y g l a n d v i r u s i n tissue culture  (122).  o f mice was Shortly  after,  o f the hunam s a l i v a r y g l a n d v i r u s were made (112,123,135).  By 1960, the s a l i v a r y g l a n d v i r u s had been demonstrated t o produce i t s characteristic lesions  i n tissues  o t h e r than the s a l i v a r y  2  g l a n d s , and t h e r e f o r e was renamed c y t o m e g a l o v i r u s , a f t e r the m i c r o s c o p i c morphology o f the l e s i o n s i t caused  II.  (135) .  Nomenclature  The  development o f the n e g a t i v e s t a i n i n g procedure  microscopy  for electron  i n 1959 made p o s s i b l e the e x a m i n a t i o n o f the s i z e and  morphology o f v i r u s e s , a l l o w i n g g r o u p i n g o f v i r u s e s by these criteria  (12).  S h o r t l y a f t e r t h i s development, C.H. Andrewes  proposed a r e v i s e d scheme f o r the taxonomic  grouping o f v i r u s e s  based upon f o u r c r i t e r i a , one o f which was morphology ( 1 ) . proposed  c l a s s i f i c a t i o n , the c y t o m e g a l o v i r u s e s formed  the NITA ( n u c l e a r i n c l u s i o n Type A) v i r u s c l a s s . the proposed 1962  In h i s  a group  within  Later that year  c r i t e r i a were a d o p t e d by the V i r u s Subcommittee a t the  I n t e r n a t i o n a l Nomenclature Committee m e e t i n g , however the NITA  group was renamed h e r p e s .  P r e s e n t taxonomic  g r o u p i n g s d e s i g n a t e the human c y t o m e g a l o v i r u s  (HCMV) and t h e murine c y t o m e g a l o v i r u s (MCMV) as s e p a r a t e genus o f the H e r p e s v i r i d a e f a m i l y ( 8 9 ) .  3  III.  P h y s i c a l P r o p e r t i e s o f the Virus  a . ) Morphology  Comparison o f herpes v i r u s e s by e l e c t r o n m i c r o s c o p y indistinguishable virion  particles  revealed  f o r many o f the members o f t h e  herpes v i r u s f a m i l y i n c l u d i n g HCMV and MCMV (87,104,121,138,140). Features  o f t h e v i r i o n i n c l u d e an i n n e r n u c l e o p r o t e i n  c o n s i s t i n g o f 162 h e x a g o n a l and p e n t a g o n a l e l o n g a t e d  core capsomers i n a  5:3:2 s y m m e t r i c a l arrangement ( 5 4 ) . Each e l o n g a t e d itself  capsomer i s 9.5 x 12.0-13.5 nm.  The c a p s i d  i s a p p r o x i m a t e l y 100 nm i n d i a m e t e r and i s s u r r o u n d e d by a n  envelope, derived  from t h e n u c l e a r membrane o f t h e i n f e c t e d c e l l ,  m e a s u r i n g 4.0-10.0 nm i n t h i c k n e s s  and b e a r i n g  projections  (87,138,140).  A d i s t i n g u i s h i n g f e a t u r e o f MCMV i s t h e p r e s e n c e o f m u l t i c a p s i d v i r i o n s which a r e o f t e n observed i n a d d i t i o n t o s i n g l e c a p s i d virions (60).  b . ) DNA  The  c y t o m e g a l o v i r u s e s c o n t a i n l a r g e double s t r a n d e d ,  genomes.  l i n e a r , DNA  HCMV has a genome o f 150 x 10^ d a l t o n s w i t h a buoyant  4  3 d e n s i t y o f 1.718 g/cm and a G + C c o n t e n t o f 58% (27,30,58). MCMV c o n t a i n s a genome o f 132 x 10^ d a l t o n s w i t h a buoyant d e n s i t y of  1.718 g/cm  3  and a G + C c o n t e n t o f 59% ( 9 2 ) .  c.) P r o t e i n s  S t u d i e s on HCMV r e v e a l a t l e a s t 35 s t r u c t u r a l p r o t e i n s , i n c l u d i n g a minimum o f 7 g l y c o p r o t e i n s (125) . quite similar;  REPLICATION  a.)  Host Range  1. Human  t o be  33 s t r u c t u r a l p r o t e i n s , i n c l u d i n g a t l e a s t 6  g l y c o p r o t e i n s , have been r e p o r t e d  IV  MCMV appears  (19,71).  Cytomegalovirus  HCMV can be i s o l a t e d from many t i s s u e s a n d body f l u i d s  including  u r i n e , m i l k , b l o o d , semen, s a l i v a r y g l a n d s , k i d n e y s , c e r v i x , and  stools  (24,31,65,77,137).  The v i r u s appears  liver  t o be c a p a b l e o f  r e p l i c a t i o n and p e r s i s t e n c e i n v i r t u a l l y a l l t i s s u e s a s w e l l as i n s e v e r a l tumours (16,43,59,103). difficult,  P r o p a g a t i o n i n v i t r o i s much more  p r o d u c t i v e i n f e c t i o n o c c u r s i n few c e l l s o t h e r t h a n human  f i b r o b l a s t s and f i b r o b l a s t c e l l  lines  (61,73,105,118).  5  2. Murine  Cytomegalovirus  Many murine t i s s u e s a r e s u s c e p t i b l e v i v o , a l t h o u g h i n many c a s e s the c e l l  t o i n f e c t i o n by MCMV i n  types have n o t been  d e t e r m i n e d , n o r has the e x t e n t o f r e p l i c a t i o n i n those (11,48,61,141).  I n v i t r o i n f e c t i o n o f embryonic  l i n e s , spleen c e l l s virus  cells  fibroblast  or v a r i o u s primary c u l t u r e s a l l y i e l d  cell  infectious  (7,51,70,81).  b. ) P e n e t r a t i o n o f the C e l l  V i r a l e n t r y i n t o the c e l l i s f a c i l i t a t e d independent envelope  attachment  by a  mechanism i n v o l v i n g f u s i o n o f the v i r a l  t o t h e c e l l membrane.  The n u c l e o c a p s i d i s then  i n t e r n a l i z e d i n t o the c y t o p l a s m i n a temperature then m i g r a t e s toward  c. ) Replicative  The  temperature  the n u c l e u s  dependent e v e n t and  (119,131).  Cycle  r e p l i c a t i v e c y c l e f o r cytomegaloviruses i s s i m i l a r t o other  herpes v i r u s e s .  Approximately  r e l e a s e o f the f i r s t  f o u r days a r e r e q u i r e d  progeny v i r u s  (118).  The c y c l e s o f HCMV and  MCMV r e p l i c a t i o n a r e n o t w e l l c h a r a c t e r i z e d b u t appear similar. below.  f o r the  t o be  S e v e r a l d i f f e r e n c e s have been r e p o r t e d and a r e d i s c u s s e d  6  A f t e r p e n e t r a t i o n i n t o the c e l l q u i c k l y toward degraded  t h e n u c l e u s where t h e c a p s i d i s thought  and t h e v i r a l  of  genome i s r e g u l a t e d by a cascade  system  t o t h a t d e s c r i b e d by Honess and Roizman f o r the r e p l i c a t i o n  herpes  simplex v i r u s  (53).  The genome i s d i v i d e d i n t o  e a r l y , e a r l y , and l a t e gene c l a s s e s . of  t o be  DNA e n t e r s the n u c l e u s ( 8 7 ) .  E x p r e s s i o n o f the v i r a l similar  the n u c l e o c a p s i d migrates  immediate  Only t h e immediate e a r l y genes  HCMV have been mapped, and no genes have been mapped i n MCMV  (126) .  A DNA polymerase  has been i d e n t i f i e d and i s p r o b a b l y a  member o f t h e e a r l y gene c l a s s  Although e f f o r t s  to find  (63,96,99).  t h y m i d i n e k i n a s e genes i n t h e v i r a l  genome have f a i l e d , i n c r e a s e s i n c e l l u l a r  thymidine k i n a s e a c t i v i t y  d u r i n g HCMV i n f e c t i o n s have been r e p o r t e d (35,142). contrast to results  o b t a i n e d u s i n g MCMV, which have demonstrated a  decrease i n thymidine kinase a c t i v i t y  (95).  between t h e two v i r u s e s i s t h e i r e f f e c t While  MCMV has l i t t l e  stimulates host c e l l polymerase  activity  This i s i n  o r no e f f e c t  A second  on h o s t c e l l  difference synthesis.  on c e l l u l a r DNA s y n t h e s i s , HCMV  DNA s y n t h e s i s and i n d u c e s DNA dependent RNA (62,127).  Assembly o f t h e n u c l e o c a p s i d o f HCMV t a k e s p l a c e i n t h e n u c l e u s by t h e t h i r d day f o l l o w i n g i n f e c t i o n appear  ( 8 7 ) , a l t h o u g h v i r u s does n o t  i n the s u p e r n a t a n t f o r an a d d i t i o n a l  2-5 days ( 1 3 1 ) .  The  nucleocapsid moves to the cytoplasm  by budding through the nuclear  membrane, a c q u i r i n g an envelope i n the process.  A second coat may  be acquired by budding i n t o lysosomes i n the cytoplasm  V.  (120) .  Pathogenesis  a.) Human Cytomegalovirus  High percentages of seropositive i n d i v i d u a l s i n the population r e f l e c t the prevalence  of HCMV i n s o c i e t y .  In some parts of the  world the proportion of seropositive adults may approach 100%, although i n North America the percentage i s probably somewhat l e s s (137) .  The adult population may therefore act as a r e s e r v o i r of  latent v i r u s .  Several modes of transmission are thought to e x i s t  i n c l u d i n g venereal transmission, o r a l transmission, transplacental passage from mother to fetus and transmission during passage through the b i r t h canal (109,137).  HCMV i s a l s o transmitted during blood  transfusions and organ a l l o g r a f t s when the donor i s harbouring l a t e n t v i r u s (36,78).  Although most HCMV i n f e c t i o n are asymptomatic and never develop i n t o c l i n i c a l disease, a small proportion of i n d i v i d u a l s are susceptible to and develop c l i n i c a l symptoms. be divided i n t o three groups:  These i n d i v i d u a l s can  a.) young adults who develop  i n f e c t i o u s mononucleosis, b.) fetuses and newborn c h i l d r e n , and c.)  8  i n d i v i d u a l s p r e d i s p o s e d by i m m u n o l o g i c a l d e f i c i e n c i e s genetic  defects,  immunosuppressive  or lymphomas and p a t i e n t s syndrome  therapy, patients  including w i t h leukemia  s u f f e r i n g from a c q u i r e d immunodeficiency  (25,33,40,44,46,67,69).  Disseminated cytomegalic i n c l u s i o n disease i s characterized l a r g e , s w o l l e n , c y t o m e g a l i c c e l l s which i n d i c a t e virus.  Clinical  symptoms v a r y b u t may i n c l u d e  thrombocytopenia, frequently  involve  j a u n d i c e and pneumonia. the c e n t r a l nervous  by  the p r e s e n c e o f the  hapatosplenomegaly,  Congenital i n f e c t i o n s  system and may r e s u l t i n  mental r e t a r d a t i o n , microencephaly, deafness or b l i n d n e s s (47,88,137).  D i s s e m i n a t e d HCMV i n f e c t i o n has a l s o been c o r r e l a t e d  w i t h t h e o c c u r e n c e o f s e c o n d a r y i n f e c t i o n s , s u g g e s t i n g t h a t the viral  i n f e c t i o n p r e d i s p o s e s the p a t i e n t  to secondary i n f e c t i o n s  (107,110,111).  b.) Murine  Cytomegalovirus  The s u r v i v a l o f mice e x p e r i m e n t a l l y i n f e c t e d w i t h MCMV i s dependent  upon a number o f f a c t o r s i n c l u d i n g i n o c u l a t i o n d o s e , r o u t e  o f i n o c u l a t i o n , s t r a i n o f v i r u s , and the age and genotype  o f the  mouse (17,61,91,101,114).  do so  within  8-9 days  unknown.  Those mice w h i c h d i e g e n e r a l l y  following inoculation.  The a c t u a l cause o f d e a t h i s  9  Experimental infection  i n f e c t i o n s r e s u l t i n death o r a c h r o n i c o r l a t e n t  following  an i n i t i a l a c u t e  phase o f i n f e c t i o n  (101).  Moribund mice s a c r i f i c e d b e f o r e death show a v i r e m i a w i t h n e c r o s i s and  edema o f many t i s s u e s and organs i n c l u d i n g  the l i v e r ,  l u n g s , k i d n e y s , s a l i v a r y g l a n d s , h e a r t , and b r a i n  spleen,  (90,91).  E s p e c i a l l y prominant i n t h i s group a r e t h e l i v e r and s p l e e n , b o t h o f which a r e thought  t o be e a r l y s i t e s o f MCMV r e p l i c a t i o n and  i n f e c t i o n , a f t e r i n o c u l a t i o n by e i t h e r t h e i n t r a p e r i t o n e a l o r intravenous route  (48,91).  Splenomegaly i s a common symptom o f MCMV i n f e c t i o n w i t h t h e organ o f t e n d o u b l i n g i n s i z e .  Infection  o f the s p l e e n i s i n i t i a t e d  i n the p e r i f o l l i c u l a r r e g i o n s and then s p r e a d s and r e d p u l p ; peak t i t e r s infection  (82,90,91).  of virus  i n t o the f o l l i c l e s  o c c u r around days 3-5, a f t e r  L a t e n t v i r u s c a n be r e c o v e r e d by  c o c u l t i v a t i o n with feeder c e l l s , although the a c t u a l c e l l responsible  f o r h a r b o u r i n g the v i r u s i s unknown.  have s u g g e s t e d viral  the macrophage as a c a n d i d a t e which i s p e r m i s s i v e t o  within the l i v e r  recovered.  Kuppfer  (82,91).  t h e r e i s a random d i s t r i b u t i o n o f l e s i o n s  t h e l o b u l e s , from which i n f e c t i o u s v i r u s c a n be The t i t e r  o f recovered v i r u s  i n f e c t i o n and the dose o f v i r u s g i v e n . and  Recent s t u d i e s  r e p l i c a t i o n and which i s h a r b o u r i n g l a t e n t v i r u s  throughout  type  depends upon the r o u t e o f Parenchymal h e p a t i c  c e l l s b o t h become i n f e c t e d w i t h  cells  the v i r u s , however  10  latent virus  o r v i r a l DNA has n o t been d e t e c t e d l n these c e l l s  the a c u t e s t a g e o f i n f e c t i o n l i v e r i s important  (101,114).  Thus, i t appears  as i f the  i n the a c u t e s t a g e o f i n f e c t i o n b u t may n o t be  i n v o l v e d i n the c h r o n i c o r l a t e n t  stage.  A v i r e m i a i s p r e s e n t from t h e time o f i n o c u l a t i o n approximately  after  8 days p o s t i n f e c t i o n .  After this  until  time no f r e e  virus  c a n be r e c o v e r e d from  the serum o f t h e s e a n i m a l s , however v i r u s can  be r e c o v e r e d a t l a t e r  times by c o c u l t i v a t i o n o f b l o o d  with a l l o g e n i c  or syngeneic  feeder c e l l s  c u r r e n t c o n t r o v e r s y over the p a r t i c u l a r  (141) .  lymphocytes  Although  type o f c e l l w h i c h i s  i n f e c t e d by the v i r u s , s e v e r a l s t u d i e s have demonstrated presence  o f l a t e n t v i r u s i n p e r i t o n e a l macrophages  intraperitoneal inoculation.  there i s  the  following  I t has a l s o been r e p o r t e d t h a t  macrophages become a c t i v a t e d d u r i n g the a c u t e s t a g e o f i n f e c t i o n and t h a t macrophages a r e i m p o r t a n t i n p r o t e c t i n g mice a g a i n s t MCMV (11,91,113,114).  A f t e r the a c u t e s t a g e o f i n f e c t i o n a c h r o n i c stage or a l a t e n t stage.  the v i r u s c a n go i n t o  either  The f a c t o r s d e t e r m i n i n g w h i c h o f  these o c c u r s a r e n o t p r e s e n t l y known, b u t i t i s the r o l e o f the immune system  t o p r e v e n t r e a c t i v a t i o n o f the v i r u s .  When l a t e n t l y  i n f e c t e d mice a r e t r e a t e d w i t h immunosuppressive a g e n t s , a n a c u t e and d i s s e m i n a t e d i n f e c t i o n r e o c c u r s  (39,66).  11  VI.  a.)  Immunological  Interactions  Human C y t o m e g a l o v i r u s  HCMV has a complex r e l a t i o n s h i p host. the  The r e s p o n s e o f the h o s t v a r i e s  The v i r u s  been i s o l a t e d  of p a t i e n t s  can p e r s i s t  (37,76).  l a t e n t l y i n human l e u k o c y t e s and  Drew e t a t were a b l e t o grow HCMV i n a l v e o l a r  Thus, HCMV may p e r s i s t  lymphocytes o r b o t h .  o f these c e l l s  What e f f e c t  (41,129).  i n circulatory  i s unknown.  seen i n most  of  In a s m a l l number o f p a t i e n t s w i t h HCMV i n f e c t i o n ,  w h i c h e v e n t u a l l y become l e t h a l .  the i n f e c t i o n  o r whether t h e s e v e r i t y  patient's i n a b i l i t y  the  with severe  is a result  o f the  t o produce a n t i b o d i e s i s n o t c l e a r  (26,97).  t o HCMV i n f e c t i o n  a p p e a r s t o be s u p p r e s s e d i n v i r t u a l l y a l l p a t i e n t groups w i t h  active  HCMV i n f e c t i o n s  (49).  A reversal  ;  Whether t h i s i s a r e s u l t  C e l l m e d i a t e d immunity i s much more s e n s i t i v e and  infected  and i n c l u d e p r o d u c t i o n o f I g M, I g G, and I g A  absence o f a n t i b o d y p r o d u c t i o n c a n be c o r r e l a t e d infections  monocytes  t h i s may have on t h e f u n c t i o n  Normal humoral r e s p o n s e s t o HCMV are individuals  o f the  from washed l e u k o c y t e s as w e l l as from the b l o o d  macrophages ( 3 2 ) . or  w i t h the type o f i n f e c t i o n ,  age and immune competence o f the h o s t and the s e v e r i t y  infection. has  w i t h the immune system o f i t s  o f normal T h e l p e r : T  suppressor c e l l  r a t i o s may be r e s p o n s i b l e  f o r the d e c r e a s e d c e l l  mediated immunity  i n patients  (14,100).  r a t i o s i n o t h e r types o f HCMV i n f e c t i o n have n o t  been  T cell  with infectious  examined.  One  consequence  o f the immunosuppressive  infection i sa predisposition  transplant  patients  e f f e c t o f HCMV  to superinfection  C o n c u r r e n t i n f e c t i o n s from o p p o r t u n i s t i c in  mononucleosis  by o t h e r o r g a n i s m s .  organisms a r e n o t uncommon  o r c h i l d r e n w i t h d e s s i m i n a t e d HCMV i n f e c t i o n s  (20,107).  b.) Murine C y t o m e g a l o v i r u s  The immune r e s p o n s e o f t h e mouse e l i c i t s directed  toward the i n f e c t i n g v i r u s .  a complex  response  Within 3 days, both  humoral  and c e l l m e d i a t e d immune r e s p o n s e s a r e p r e s e n t , and p r o t e c t i v e antibodies  and c y t o t o x i c  T lymphocytes c a n be found i n t h e b l o o d .  A l s o a t t h i s t i m e , a n t i b o d y dependent  cytotoxic  o b s e r v e d and may p l a y a r o l e i n immunity killer  cell  c e l l s have  t o the v i r u s .  been  Natural  a c t i v i t y may b e g i n as e a r l y as 10 hours a f t e r  intraperitoneal  inoculation.  Thus, a complete r e s p o n s e toward the  v i r u s appears t o be p r e s e n t (2,6,52,85,106,124).  Both t h e n a t u r a l  killer  a c t i v i t y , as w e l l  c e l l and a n t i b o d y dependent  as the p r o t e c t i o n  cell  m e d i a t e d by macrophages have been c o r r e l a t e d  the major h i s t o c o m p a t i b i l i t y (6,17,85,114).  cytotoxic  region  (H-2) genotype  o f the mice  with used  13  Despite the response toward the v i r u s , widespread suppression of immune responses toward other immunological targets occurs during the  acute stage of the i n f e c t i o n .  Early experiments  demonstrated  suppressed humoral responses to Newcastle disease virus and sheep erythrocytes i n MCMV i n f e c t e d mice (102).  The suppressive e f f e c t  lasted 9-10 days a f t e r i n o c u l a t i o n , a f t e r which there was a gradual return to normal.  This period coincides with the course o f the  acute stage of i n f e c t i o n (83).  Hamilton et^ al^ i l l u s t r a t e d the  v a l i d i t y o f these observations to the i n vivo s i t u a t i o n by c o i n f e c t i n g mice with MCMV and a second i n f e c t i o u s agent (P. aeruginosa, C. albicans or S. aureus).  The r e s u l t was a s t r i k i n g  increase i n l e t h a l i t y and m u l t i p l i c a t i o n of the bacteria (or yeast) i n the blood and organs of the mice.  In controls inoculated with  bacteria (or yeast) only, the i n f e c t i o u s agent was r a p i d l y cleared from the body and the i n f e c t i o n was non-lethal (47,48).  Howard e t al_ presented evidence that MCMV suppresses both humoral and c e l l mediated immunity.  Depression of humoral immunity  was determined by depressed hemagglutinin antibody response to an i n t r a p e r i t o n e a l i n j e c t i o n of sheep erythrocytes.  Both primary and  secondary antibody responses could be suppressed by the v i r u s , but a normal response was seen i f the mice were previously vaccinated to MCMV.  A suppressed c e l l mediated response was i l l u s t r a t e d by  delayed a l l o g r a f t r e j e c t i o n and decreased responses i n mixed lymphocyte  reactions and phytohemagglutinin mitogen stimulation  14  (55,56).  Lymphocytes  i n f e c t e d i n v i v o o r i n v i t r o have a s u p p r e s s e d  response t o s t i m u l a t i o n by e i t h e r T c e l l  or B c e l l  mitogens  (9,10,82,115).  C o n f l i c t i n g r e p o r t s e x i s t as t o the p o p u l a t i o n o f c e l l s responsible cell  f o r m e d i a t i n g the immunosuppression, and f o r the type o f  i n which the v i r u s r e p l i c a t e s .  of v i r u s  Wu and Ho r e p o r t e d the r e c o v e r y  from T lymphocytes b u t n o t B lymphocytes ( 1 4 1 ) , and  S e l g r a d e et_ a l ^ found no s u p p r e s s i v e e f f e c t m e d i a t e d by plastic-adherent c e l l are  populations  (115).  the o b s e r v a t i o n s o f Loh and Hudson.  C o n t r a r y t o these r e p o r t s They found the  plastic-adherent population of spleen c e l l s  to mediate  immunosuppression and a l s o t o r e p l i c a t e v i r u s  (81,82,83).  Maximum  t i t e r s were found i n the macrophage r i c h , a d h e r e n t p o p u l a t i o n and the  number o f i n f e c t i o u s c e n t e r s i n the s p l e e n was  the  degree o f s u p p r e s s i o n .  p r o p o r t i o n a l to  I n f e c t e d a d h e r e n t c e l l s mixed w i t h  u n i n f e c t e d non-adherent c e l l s  r e s u l t e d i n a population of c e l l s  which had a s u p p r e s s e d m i t o g e n r e s p o n s e .  Thus, the a d h e r e n t  p o p u l a t i o n o f s p l e e n c e l l s , and p o s s i b l y the macrophage specifically,  i s r e s p o n s i b l e f o r m e d i a t i n g the o b s e r v e d  immunosuppression.  cell  15  VII.  Purpose o f the Study  The  purpose  o f the p r e s e n t s t u d y was t o i n v e s t i g a t e t h e  mechanism by which MCMV i n d u c e d immunosuppression takes p l a c e . S i n c e t h i s i s a p a r t o f the p a t h o g e n e s i s  o f MCMV, i t s h o u l d be  i n v e s t i g a t e d i n o r d e r t o a t t a i n t h e g o a l o f a complete o f the p a t h o g e n e s i s  of this virus.  Also, virus  understanding  induced  immunosuppression i s a phenomenon which o c c u r s i n s e v e r a l human v i r u s i n f e c t i o n s and t h e r e f o r e i s a r e l e v a n t m e d i c a l p r o b l e m . a l s o hoped t h a t the r e s u l t s o f t h i s s t u d y w i l l i n t o the r e g u l a t i o n o f the immune  response.  It is  p r o v i d e some i n s i g h t  CHAPTER I I  MATERIALS AND METHODS  I.  MATERIALS  1.  Mice  SWR/J mice were obtained from Jackson Laboratories, Bar Harbour, Maine and were maintained by the UBC Animal Care Center.  The mice  were allowed free access to mouse chow and water during t h e i r maintenance and during experiments.  Random bred pregnant Swiss white mice f o r the purposes of mouse embryos were bred by the UBC Animal Care Center.  2.  Cells  Mouse embryo c e l l s were prepared from pregnant random bred Swiss white mice on approximately the fourteenth day of gestation.  Spleen c e l l s were prepared from SWR/J mice i n d i s c r i m i n a t e l y of age or sex.  BHK-21 c e l l s ,  a continuous c e l l l i n e of baby hamster kidney  c e l l s were obtained from D r . D. Vance, Dept. Biochem., UBC.  3.  Viruses  The Smith s t r a i n of murine cytomegalovirus (MCMV) was obtained from the American Type Tissue C o l l e c t i o n , and was propagated i n t e r t i a r y mouse embryo c e l l s .  This s t r a i n was used for the  experiments i n v o l v i n g MCMV.  Sindbis v i r u s was obtained from D r . D. Vance, Dept. Biochem., UBC, and was propagated i n BHK-21 c e l l s .  Bacteriophage TA was obtained from D r . A. Warren, Dept. M i c r o . , UBC.  Bacteriophage PM2 was obtained from D r . U . Kuhnlein, B r i t i s h Columbia Cancer Research Center.  4.  Tissue Culture Medium  Dulbecco's modified Eagle medium (MEM) and RPMI 1640 were obtained i n powder form from Grand Island B i o l o g i c a l Company and dissolved i n deionized water.  MEM A was supplemented with 3.7  NaHC0 , MEM B with 1.5 g/1 NaHCO^, and RPMI 1640 with 2.0 3  g/1  g/1  NaHC0 .  Media were f i l t e r s t e r i l i z e d and supplemented with 50  3  ug/ml gentamicin before use.  5•  F e t a l Bovine Serum  F e t a l bovine serum was obtained from Grand Island B i o l o g i c a l Company and stored at -20°C u n t i l used.  6.  Solutions  a. ) Hanks' balanced s a l t s o l u t i o n (HBSS) consisted o f : NaCl  0.14  KCl  5.40 mM  MgS0 .7 H 0 4  CaCl  2  M  0.81 mM 1.26 mM  2  NaHPO. .7 H„0 4 2 KH-P0. 2 4  0.61 mM 0.44 mM  Glucose  5.56 mM  Phenol red  0.02 g/1  b. ) E l u t i o n buffer f o r Sephadex G-25 column consisted o f : NaCl  0.07  KCl  2.70 mM  MgSO^.7 H 0 2  0.41 mM  M  chromatography  19  CaCl  0.63 mM  2  NaHP0 .7 H 0  0.31 mM  KH P0  0.22 mM  4  2  c.)  2  4  Phosphate buffered s a l i n e consisted of:  NaCl  0.13  KCl  2.70 mM  Na HP0 .7H 0 2  4  2  8.10 mM  CaCl  2  1.00 mM  MgCl  2  0.50 mM  KH P0 2  4  M  1.50 mM  d. ) Electrophoresis running buffer (pH 8.3) consisted o f : Tris-HCl  0.025 M  Glycine  0.192 M  Sodium dodecyl s u l f a t e  1.0%  e. ) Electrophoresis sample buffer (pH 6.8) consisted o f : Tris-HCl Sodium dodecyl s u l f a t e Glycerol 2-mercaptoethanol Bromophenol blue  0.0625 M 2.0% 10.0% 5.0% 0.001%  f . ) Overlay medium f o r plaque assays consisted of MEM A containing 0.5% agarose and 5.0% f e t a l bovine serum.  7.  Chemicals  Product  Distributor  Acrylamide  BioRad Laboratories  Agarose  SeaKem  Concanavalin A  Sigma Chemical Co.  Gentamicin  Sigma Chemical Co.  LB8 Latex Beads  Sigme Chemical Co.  N,N'-bis-methylene acrylamide  BioRad Laboratories  Proteinase K  Beckman  Scintrex  J.T. Baker  Sephadex G-25 3  Pharmacia  Chemicals  New England Nuclear  (methyl- H) Thymidine Difco Laboratories Trypsin  II.  1.  METHODS  Mouse Embryo C e l l Cultures  Embryos were removed from pregnant, random bred, Swiss white mice at approximately the fourteenth day of g e s t a t i o n . The embryos were minced with s c i s s o r s and then suspended i n Hanks' balanced s a l t s o l u t i o n (HBSS) supplemented with 2.5 mg/ml of t r y p s i n and s t i r r e d vigorously f o r 30 minutes.  The  supernatant was drawn o f f and centrifuged a t 900g x 10 minutes.  The undigested tissue fragments were subjected to at  l e a s t two cycles of t r y p s i n digestion and each successive supernatant was centrifuged at 900g x 10 minutes to p e l l e t the c e l l s from the s o l u t i o n .  The c e l l p e l l e t s were combined and  resuspended i n MEM A, containing 10% f e t a l bovine serum (FBS), and then plated i n t o p e t r i dishes •  The cultures were incubated  at 37" C i n an atmosphere of 5.0% C0 , 95.0% a i r and at high 2  humidity.  Mouse embryo c e l l s were passaged by removing the growth medium, r i n s i n g the c e l l s with HBSS, then incubating the c e l l s for 5 minutes i n HBSS, containing 2.5 mg/ml of t r y p s i n , at room temperature.  The HBSS-trypsin was then aspirated and the c e l l s  were removed from the surface of the p e t r i dish by p i p e t t i n g MEM A, containing 10% FBS, vigorously over the surface o f the  dish.  The c e l l s were p e l l e t e d by c e n t r i f u g a t i o n a t 900g x 10  m i n u t e s , resuspended i n MEM A, c o n t a i n i n g 10% FBS, and p l a t e d in  2.  petri  dishes .  BHK-21 C e l l  The  Culture  continuous  cell  line  o f baby hamster c e l l s was c u l t u r e d  in petri  d i s h e s u s i n g MEM A, c o n t a i n i n g 10% FBS, as a growth  medium.  I n c u b a t i o n was a t 37 C i n an atmosphere o f 5% CO2,  95% a i r and a t h i g h h u m i d i t y .  The c e l l s were passaged by  t r y p s i n i z a t i o n and r e p l a t i n g as was d e s c r i b e d f o r mouse embryo cells.  3.  Spleen  Cell  Spleen  c e l l c u l t u r e s were p r e p a r e d  Spleens  Cultures  were removed from the mice and p l a c e d i n p e t r i  c o n t a i n i n g 10 mis o f HBSS . cells  from a d u l t SWR/J m i c e .  The s p l e e n s were b i s e c t e d and t h e  g e n t l y t e a s e d o u t from the s p l e e n u s i n g f o r c e p s .  s u s p e n s i o n was passaged through cells .  dishes  The  a s y r i n g e t o break c l u s t e r s o f  The c e l l s were p e l l e t e d by c e n t r i f u g a t i o n a t 900g x 10  m i n u t e s and then r e s u s p e n d e d i n 0.17 M NH^Cl f o r 5-7 minutes .  The c e l l s were a g a i n p e l l e t e d by c e n t r i f u g a t i o n and  the resuspended i n RPMI 1640.  V i a b l e c e l l s were a s s e s s e d by  counting f o r trypan blue excluding c e l l s  i n a hemocytometer and  the c e l l s u s p e n s i o n was then d i l u t e d w i t h RPMI 1640 t o y i e l d the d e s i r e d c o n c e n t r a t i o n o f c e l l s .  A l l incubation of spleen  c e l l s was a t 37 C, i n a n atmosphere o f 5% CX"^, 95% a i r and a t high  4.  humidity.  P r o p a g a t i o n o f Murine  The  Smith  embryo c e l l s  Cytomegalovirus  s t r a i n o f MCMV was propagated  i n t e r t i a r y mouse  from the embryos o f random b r e d Swiss  mice.  T e r t i a r y mouse embryo c e l l s were grown t o c o n f l u e n c y i n r o l l i n g b o t t l e s i n MEM B, supplemented-with i n f e c t e d with PFU/cell).  5% FBS b e f o r e b e i n g  MCMV a t a low m u l t i p l i c i t y o f i n f e c t i o n (<0.1  V i r u s was h a r v e s t e d a f t e r  3-4 days i n c u b a t i o n a t 37  C d u r i n g which time t h e r o l l i n g b o t t l e s were r o t a t e d a t a speed o f 0.25 r e v o l u t i o n s per m i n u t e .  The medium was c o l l e c t e d and  c e n t r i f u g e d a t 4000g x 10 minutes t o remove c e l l u l a r The  debris.  s u p e r n a t a n t was then c e n t r i f u g e d a t I9,000g x. 4 hours t o  pellet  the v i r u s .  The v i r a l  p e l l e t was resuspended  i n a small  volume o f MEM B, c o n t a i n i n g 5% FBS, and d i s p e n s e d i n t o one ml a l i q u o t s and s t o r e d u n t i l u s e a t -70 C.  5.  Propagation o f Sindbis Virus  S i n d b i s v i r u s was propagated cells  i n p e t r i d i s h e s o f BHK-21  i n MEM A, c o n t a i n i n g 10% FBS.  P e t r i dishes o f confluent  BHK-21 c e l l s were i n f e c t e d by removing the medium and i n o c u l a t i n g the c e l l s w i t h a v i r a l suspension f o r one hour, a f t e r which the inoculum was removed and the c e l l s were o v e r l a i n w i t h f r e s h MEM A, c o n t a i n i n g 10% FBS.  After  completion o f the l y t i c c y c l e , the medium was removed and c e n t r i f u g e d a t 900g x 10 minutes t o remove the c e l l u l a r debris.  The supernatant c o n t a i n i n g the v i r u s was then  c e n t r i f u g e d a t 36,400g x 3 hours t o p e l l e t the v i r u s . The v i r u s p e l l e t was resuspended i n a s m a l l volume o f MEM A, c o n t a i n i n g 10% FBS, and s t o r e d a t -70 C.  6.  Plaque Assay of Virus  Virus preparations were t i t e r e d by plaque assay.  MCMV and  Sindbis v i r u s were t i t e r e d on t e r t i a r y mouse embryo and BHK-21 cells respectively.  Confluent  p e t r i dishes of c e l l s were  i n o c u l a t e d w i t h appropriate d i l u t i o n s o f the v i r u s preparation i n MEM A, c o n t a i n i n g 10% FBS, by removing the medium from the c e l l s and r e p l a c i n g i t w i t h the v i r a l inoculum. incubated  f o r one hour a t 37 C, 5% C 0  2 >  The c e l l s were  95% a i r and a t high  humidity, before the inoculum was removed and replaced by s e m i - s o l i d o v e r l a y medium.  The c e l l s were then incubated  plaques became v i s i b l e and were counted.  until  7•  C o l l e c t i o n of Mouse Serum  Serum was c o l l e c t e d from SWR/J mice by heart puncture immediately  f o l l o w i n g s a c r i f i c e o f the mice.  Infected mouse  serum (IMS) was c o l l e c t e d on the f o u r t h day f o l l o w i n g an i n t r a p e r i t o n e a l i n j e c t i o n o f 5 x 10^ PFU o f MCMV. Normal mouse serum (NMS)  was c o l l e c t e d from uninfected mice.  After  the blood was c o l l e c t e d from the punctured h e a r t , i t was pooled and allowed to c l o t a t 4 C. The u n c l o t t e d f r a c t i o n was removed and c e n t r i f u g e d at 500g f o r 10 minutes t o remove any r e s i d u a l cells.  The supernatant was then c e n t r i f u g e d a t 60,000g x 90  minutes t o remove any i n f e c t i o u s v i r u s from the serum. The supernatant  8.  was taken and stored at 4 C u n t i l used.  Preparation o f Conditioned Media  Spleen c e l l s a t a concentration o f 5 x 10^ c e l l s / m l were i n f e c t e d w i t h MCMV i n v i t r o at m u l t i p l i c i t y o f i n f e c t i o n (MOI) of 10 P F U / c e l l and incubated a t 37 C i n RPMI 1640, c o n t a i n i n g 2% FBS, f o r 24-48 hours.  The medium was removed, c e n t r i f u g e d  at 900g x 10 minutes to remove the c e l l s and then c e n t r i f u g e d at 60,000g x 90 minutes to sediment any i n f e c t i o u s v i r u s . The supernatant was taken and stored at 4 C u n t i l used. m a t e r i a l was termed c o n d i t i o n e d medium.  This  Control conditioned  medium was produced i n an i d e n t i c a l manner except the spleen  c e l l s were u n i n f e c t e d .  Conditioned  media from S i n d b i s  T4, and PM2 i n f e c t e d s p l e e n c e l l s were p r e p a r e d  virus,  i n an I d e n t i c a l  manner, each u s i n g an MOI o f 10 P F U / c e l l o f the a p p r o p r i a t e v i r u s ; LB8 c o n d i t i o n e d medium employed l a t e x beads w i t h a d i a m e t e r o f 0.8 um t o mimic i n f e c t i o n a t a dose o f 10 l a t e x particles/cell.  9.  Mitogen S t i m u l a t i o n  F r e s h l y prepared  Test  s p l e e n c e l l s were a s s a y e d f o r t h e i r  r e s p o n s e to the T c e l l m i t o g e n , c o n c a n a v a l i n presence o r absence o f v a r i o u s s u b s t a n c e s .  A (Con A) i n the Spleen  c o n c e n t r a t i o n o f 5 x 10^ c e l l s / m l were d i s p e n s e d Lindbro  p l a t e s a t a volume o f 0.2 m l / w e l l .  cells at a  i n t o 96 w e l l  Con A was added t o  a c o n c e n t r a t i o n o f 2.0 ug/ml t o w e l l s which were used t o measure m i t o g e n s t i m u l a t i o n . cells  r e c e i v e d no Con A.  Wells  containing  unstimulated  A f t e r i n c u b a t i o n f o r 24 hours a t 37  3 C, 2.5 u C i o f m e t h y l - H-thymidine were added t o each w e l l . The  p l a t e s were i n c u b a t e d  the c e l l s were p r o c e s s e d  f o r an a d d i t i o n a l 18-24 h o u r s onto g l a s s f i b e r f i l t e r s  and washed  f o u r times w i t h e q u a l volumes o f phosphate b u f f e r e d (PBS) The  u s i n g a MASH I I H a r v e s t e r  saline  (Microbiological Associates).  f i l t e r d i s c s were a i r d r i e d and r a d i o a c t i v i t y was  by l i q u i d  scintillation  before  counting.  counted  The e f f e c t s of various conditioned media, extracts , column f r a c t i o n s and sera on the stimulation of spleen c e l l s was determined by i n i t i a l l y suspending the spleen c e l l s i n media containing each substance.  This was achieved by adjusting the  concentration of spleen c e l l s to 5 x IO" c e l l s / m l and then 7  d i l u t i n g the c e l l s to 5 x 10^ c e l l s / m l with the substance to be tested.  10.  U l t r a f i l t r a t i o n of Conditioned  Media  U l t r a f i l t r a t i o n of conditioned media was performed using an Amicon S t i r r e d U l t r a f i l t r a t i o n C e l l , Model 12, with D i a f l o membrane f i l t e r s YM2 and YMLO (molecular weight exclusion l i m i t s of 1000 and 10,000 daltons r e s p e c t i v e l y ) .  The assembled  c e l l was s t e r i l i z e d by passing 70% ethanol through the f i l t e r , followed by a volume o f s t e r i l e d i s t i l l e d water.  U l t r a f i l t r a t i o n was performed by p i p e t t i g 10 mis of conditioned medium i n t o the c e l l and f i l t e r i n g under ^ gas pressure u n t i l a small volume remained.  The cycle was repeated  u n t i l the desired amount of conditioned medium had been passed through the f i l t e r and only a small volume of l i q u i d remained inside the c e l l .  The remaining  r e s i d u a l l i q u i d contained a  concentration of molecules excluded by the f i l t e r and was used as concentrated conditioned medium.  The concentrated  c o n d i t i o n e d medium was removed from t h e c e l l , measured v o l u m e t r i c a l l y and a d j u s t e d d i l u t i n g w i t h RPMI 1640.  t o the d e s i r e d c o n c e n t r a t i o n by  The f i l t r a t i o n procedure was c a r r i e d  out under c o n d i t i o n s o f up t o 50 p s i p r e s s u r e with constant the c e l l .  stirring  of ^  gas and  p r o v i d e d by a magnetic s t i r r e r  Ultrafiltration  inside  was performed a t 4 C, and the  f i l t r a t e s and r e s i d u e s produced were s t o r e d a t 4 C u n t i l  11.  Extraction of Lipids  A modified lipids  from C o n d i t i o n e d  used.  Media  F o l c h e x t r a c t i o n p r o c e d u r e was used t o e x t r a c t  from c o n d i t i o n e d media ( 3 8 ) .  Ten mis o f c o n d i t i o n e d  medium were shaken v i g o r o u s l y w i t h 40 mis o f a 2:1 chloroform:methanol mixture to  settle.  i n a separatory  f u n n e l and a l l o w e d  The upper phase was drawn o f f and d i s c a r d e d .  q u a r t e r volume (8.2 m i s )  One  o f 1:1 d i s t i l l e d water:methanol was  added t o t h e lower phase and a g a i n  the m i x t u r e  v i g o r o u s l y and a l l o w e d  The lower phase was drawn  o f f and reduced  to s e t t l e .  t o dryness  under r o t a r y e v a p o r a t i o n u s i n g a  Savant Speed Vac C o n c e n t r a t o r . i n 10 mis o f RPMI 1640.  was shaken  The p r e c i p i t a t e was resuspended  This suspension  was used as a s o u r c e  of e x t r a c t i n mitogen s t i m u l a t i o n a s s a y s .  12.  Sephadex G-25  F r a c t i o n a t i o n of Conditioned  C o n d i t i o n e d media were c o n c e n t r a t e d ultrafiltration  through  a YM2  f r a c t i o n a t i o n ; serum was  1 0 - f o l d by  membrane f i l t e r  used n e a t .  Media  One  ml  prior  to  samples  of  c o n d i t i o n e d media ( c o n c e n t r a t e d ) o r mouse serum were a p p l i e d to a  20 x 1.6  cm  Sephadex G-25  column w i t h a bed volume o f  36.2  3 cm  •  A f t e r a p p l i c a t i o n , each sample was  bed and e l u t i o n was a constant  was  13.  mis/minute.  from the column.  performed a t 4  The  fractionation  growth and  A s t i m u l a t i o n o f s p l e e n c e l l s by  S i x u l o f 8X RPMI 1640  10  7  stimulation  restoring  n u t r i t i o n a l status  A stimulation test.  were added to 84 u l o f the sample  10 u l o f s p l e e n c e l l s a t a c o n c e n t r a t i o n o f 5 x  c e l l s / m l i n RPMI 1640.  suspension  for  then u s i n g the r e s u l t a n t s o l u t i o n as a  growth medium f o r s p l e e n c e l l s i n a Con  f r a c t i o n and  procedure  column were a s s a y e d  each f r a c t i o n to p r o p e r i o n i c s t r e n g t h and for c e l l  fraction  Fractions  F r a c t i o n s from the Sephadex G-25 on Con  Three ml  C.  Assay o f Sephadex G-25  their effect  matrix  c a r r i e d out w i t h a s a l i n e e l u t i o n b u f f e r a t  f l o w r a t e o f 0.30  were c o l l e c t e d  r u n i n t o the  which was tests.  T h i s was  dispensed  used as  the  cellular  i n t o L i n d b r o p l a t e s f o r Con  A  30  14.  Proteinase  One  ml  K  Digestion  samples o f c o n d i t i o n e d  media, c o n c e n t r a t e d  YM2  u l t r a f i l t r a t i o n were d i g e s t e d w i t h 1.0  for  30 minutes a t 37  C.  The  chromatography was  In s e c t i o n  15.  was  gel  electrophoresis  used f o r the a n a l y s i s o f mouse serum c o n d i t i o n e d (75).  vertical  g e l e l e c t r o p h o r e s i s a p p a r a t u s (BioRad) and  The  g e l s were c a s t i n a 25 cm  0.375 M T r i s - H C l (pH  8.6)  i n a l i n e a r gradient  o f 10/0.05 to 20/0.1.%  and  0.1%  A s t a c k i n g g e l , 3 cm  3.0/0.08% a c r y l a m i d e / B i s ,  0.125  g e l s was  achieved  x 1.5  (SDS)  0-3.9%  c o n s i s t i n g of  M T r i s - H C l (pH gel.  mm  contained  sodium d o d e c y l s u l f a t e  i n height  p r e c e e d e d the main s e p a r a t i o n  media  x 15 cm  a c r y l a m i d e / N , N ' - b i s - m e t h y l e n e a c r y l a m i d e ( B i s ) and  and  described  Electrophoresis  proteins  SDS,  until  Sephadex  the samples as  Laemmli method o f p o l y a c r y l a m i d e  glycerol.  K  12.  Polyacrylamide Gel  The  F r a c t i o n a t i o n by  performed on  by  of proteinase  samples were s t o r e d a t 4 C  f r a c t i o n a t i o n c o u l d be p e r f o r m e d . G-25  mg  10X  6.8)  and  Polymerization  0.1% of  the  by a d d i n g 0.001% t e t r a m e t h y l e t h y l e n e d i a m i m e  0.0001% ammonium p e r s u l f a t e to the g e l s o l u t i o n i m m e d i a t e l y  p r i o r to the c a s t i n g o f the  gels.  Samples o f mouse serum and 10X c o n c e n t r a t e d  conditioned  media were d i l u t e d 50% i n e l e c t r o p h o r e s i s sample b u f f e r and placed  i n a b o i l i n g water b a t h f o r two m i n u t e s .  samples were l o a d e d  i n t o the w e l l s  o f the g e l and  e l e c t r o p h o r e s i s was c a r r i e d out a t a c u r r e n t the bromophenol b l u e  Twenty-five u l  o f 15 mAmps  until  i n d i c a t o r dye from the sample b u f f e r  r e a c h e d t h e bottom o f the g e l . v  E l e c t r o p h o r e s i s was  performed  u s i n g e l e c t r o p h o r e s i s r u n n i n g b u f f e r ( s e e s e c t i o n 6 d.) i n the electrode  16.  chambers, and the system was water  cooled.  S t a i n i n g o f Gels  The  u l t r a s e n s i t i v e s i l v e r s t a i n method o f Goldman e t a l was  used f o r the s t a i n i n g o f the p o l y a c y l a m i d e g e l s  ( 4 5 ) . The g e l s  were soaked f o r 30 m i n u t e s i n a s o l u t i o n o f 50% methanol and 12%  a c e t i c a c i d t o f i x the p r o t e i n s , and then soaked  hours i n 10% e t h a n o l  and 5% a c e t i c a c i d , u t i l i z i n g  changes o f the s o l u t i o n d u r i n g  this  time p e r i o d .  18-24  several The g e l s were  then soaked f o r 5 minutes i n 0.0034 M p o t a s s i u m dichromate and 0032 N n i t r i c a c i d , washed f o u r times f o r 30 seconds each i n d e i o n i z e d w a t e r and p l a c e d minutes.  T h i s was  followed  i n 0.012  M s i l v e r n i t r a t e f o r 30  by two r a p i d r i n s e s w i t h 0.28  sodium c a r b o n a t e and 0.067% commercial f o r m a l i n .  M  The g e l s were  then a l l o w e d t o d e v e l o p i n a t h i r d p o r t i o n o f the sodium  32  carbonate-formalin s o l u t i o n , with gentle a g i t a t i o n u n t i l the development had reached the desired i n t e n s i t y .  Development was  stopped by placing the gels i n a 1% a c e t i c a c i d s o l u t i o n .  17.  L i q u i d S c i n t i l l a t i o n Counting of R a d i o a c t i v i t y  The glass f i b e r f i l t e r discs containing  the a i r - d r i e d  samples from mitogen stimulation tests were placed i n s c i n t i l l a t i o n v i a l s and 2 mis of s c i n t i l l a t i o n f l u i d were added. 3  The v i a l s were capped and counted f o r r a d i o a c t i v i t y  ( H) on channel one i n an Isocap/300 L i q u i d S c i n t i l l a t i o n System (Searle A n a l y t i c , Inc.).  Liquid s c i n t i l l a t i o n  fluid  consisted of toluene containing e i t h e r 4.2% ( v o l / v o l ) Spectroflour  PPO-POPOP or 5.0% ( v o l / v o l ) Scintrex  PP0-P0P0P.  CHAPTER I I I  RESULTS I  Evidence f o r the Presence of a Soluble Immunosuppressive Factor  The i n i t i a l step i n determining the mechanism whereby MCMV induces a state o f immunosuppression on the i n f e c t e d host was to d i f f e r e n t i a t e between a mechanism i n v o l v i n g e x c l u s i v e l y c e l l to c e l l contact and one i n v o l v i n g an e x t r a c e l l u l a r e f f e c t o r molecule.  Some  previous studies have suggested that i t may be the l a t t e r mechanism (9,10,83);  thus, the p o s s i b i l i t y of a soluble immunosuppressive  f a c t o r was chosen as the i n i t i a l area f o r i n v e s t i g a t i o n i n t h i s study.  I.  Immunosuppressive Effect o f Serum from Murine  Cytomegalovirus  Infected Mice  Serum c o l l e c t e d from MCMV i n f e c t e d mice, four days a f t e r an i n t r a p e r i t o n e a l i n j e c t i o n , and from uninfected mice were added i n varying concentrations to Con A stimulated spleen c e l l s .  Previous  studies have found serum from Infected mice to suppress the response to Con A, although the authors a t t r i b u t e t h i s to the presence o f free virus i n the serum (9,10).  In order to eliminate the  p o s s i b i l i t y o f suppression due to free  34  Figure 1:  E f f e c t o f Normal and Infected Mouse Serum On Con A  Stimulation o f Spleen  Cells.  A mitogen s t i m u l a t i o n test was performed to evaluate the e f f e c t of various concentrations of normal mouse serum ( i n f e c t e d mouse serum (—"O  •  )and MCMV  ) on the response of spleen c e l l s to  stimulation by Con A. Serum from uninfected mice and from mice which had been given an i n t r a p e r i t o n e a l i n j e c t i o n of 5 x 10^ PFU of MCMV 4 days e a r l i e r was added to spleen c e l l s a t the time o f a d d i t i o n o f Con A. Values are expressed as percentages  o f the  c o n t r o l which contained no mouse serum, and represent the mean o f four samples ± standard d e v i a t i o n s .  F i g u r e 1;  E f f e c t of Normal and I n f e c t e d Mouse Serum On Con A S t i m u l a t i o n of Spleen  Cells  7o Mouse Serum  LO Ln  36  v i r u s , the serum was c e n t r i f u g e d a t 60,000g x 90 m i n u t e s . i n f e c t i o u s v i r u s c o u l d be d e t e c t e d by plaque c e l l s a f t e r the u l t r a c e n t r i f u g a t i o n . are presented suppressed  i n f i g u r e 1.  t h e response  No  a s s a y on mouse embryo  The r e s u l t s o f t h i s  experiment  Both i n f e c t e d and u n i n f e c t e d mouse serum  o f the s p l e e n c e l l s  t o Con A, however t h e  MCMV i n f e c t e d mouse serum showed a s i g n i f i c a n t l y g r e a t e r degree o f s u p p r e s s i o n when p r e s e n t a t c o n c e n t r a t i o n s o f 2% t o 15% ( S t u d e n t ' s T t e s t , p < 0.05).  The most marked s u p p r e s s i o n o c c u r e d a t 5% and 10%  mouse serum.  II.  In V i t r o P r o d u c t i o n o f A V i r u s Induced S u p p r e s s o r  The  presence  mice i n d i c a t e d probably  o f a suppressive substance  such a molecule  i n the serum o f i n f e c t e d  t h a t t h e immunosuppression observed  due t o a s o l u b l e s u p p r e s s o r  factor.  Factor  i n v i v o was  In v i t r o production o f  would g r e a t l y f a c i l i t a t e s t u d i e s c o n c e r n i n g i t s  p r o d u c t i o n , s o u r c e , p h y s i c a l n a t u r e and mode o f a c t i o n , t h e r e f o r e a n attempt was made t o i n d u c e induced suppressor  factor  t h e p r o d u c t i o n o f the p u t a t i v e v i r u s (VISF) in_ v i t r o .  S p l e e n c e l l s were chosen  chosen a s t h e c e l l s most l i k e l y t o produce VISF in_ v i t r o s i n c e c o n t a i n a complete p o p u l a t i o n o f l e u k o c y t e s and the s p l e e n i s a major t a r g e t o f the MCMV i n f e c t i o n .  they  S p l e e n c e l l s were i n f e c t e d w i t h  MCMV a t a n MOT o f 10 P F U / c e l l .  The medium was removed a f t e r 24-48 h o u r s and the c e l l s and f r e e v i r u s were removed by u l t r a c e n t r i f u g a t i o n .  The s u p e r n a t a n t was  termed c o n d i t i o n e d medium and was u s e d a s a s o u r c e o f VISF by t e s t i n g f o r suppressive a c t i v i t y  i n a Con A s t i m u l a t i o n t e s t .  C o n t r o l and MCMV c o n d i t i o n e d mediun were compared w i t h n o r m a l , s t i m u l a t e d s p l e n o c y t e s and MCMV I n f e c t e d s p l e n o c y t e s i n f i g u r e 2.  Although  t h e c o n t r o l c o n d i t i o n e d medium d i d n o t s u p p o r t  s t i m u l a t i o n t o t h e degree seen i n t h e RPMI 1640 o n l y sample, i t i s still  c o n s i d e r a b l y g r e a t e r than the response  medium sample.  MCMV c o n d i t i o n e d medium appears t o a c c o u n t  the s u p p r e s s i o n o b s e r v e d  Table  o f the MCMV c o n d i t i o n e d  I illustrates  i n t h e MCMV i n f e c t e d  for a l l  cells.  the r e l a t i o n s h i p between u n s t i m u l a t e d and  s t i m u l a t e d s p l e e n c e l l s suspended i n c o n d i t i o n e d media o r i n f e c t e d with  MCMV.  A l l cells  used i n the e x p e r i m e n t were o b t a i n e d from a  common p o o l and t h e r e f o r e a n y s u p p r e s s i o n seen i n the u n s t i m u l a t e d c u l t u r e s must be due t o t h e e f f e c t s o f t h e medium ( o r v i r u s , i f present).  Although  the s t i m u l a t i o n i n d i c e s r e f l e c t  the general  p a t t e r n o f s t i m u l a t i o n , t h e s u p p r e s s i o n seen i n t h e u n s t i m u l a t e d samples masks t h e degree o f s u p p r e s s i o n which i s p r e s e n t i n the  38  Figure 2:  Suppression  of Con A Stimulation of Spleen Cells by  Murine Cytomegalovirus and Murine Cytomegalovirus Conditioned  Medium  Spleen c e l l s were suspended i n RPMI 1640 or conditioned medium and subjected to a Con A stimulation t e s t .  MCMV i n f e c t e d spleen  c e l l s were i n f e c t e d at an MOI of 10 PFU/cell.  Values represent  means from quadruplicate samples ± standard d e v i a t i o n s .  39  Figure 2;  Suppression of Con A Stimulation of Spleen C e l l s by  Murine Cytomegalovirus and Murine Cytomegalovirus Conditioned Medium  140 • -  120  m-  100  80  60  40  20  RPMI 1640  R P M I 1640 f MCMV  Control Conditioned Medium  MCMV Conditioned Medium  TABLE I ; and  E f f e c t o f C o n d i t i o n e d Media on the Uptake o f H-dThd by U n s t i m u l a t e d  Con A S t i m u l a t e d Spleen  Medium  CPM  Cells  H-dThd I n c o r p o r a t e d  Unstimulated  RPMI 1640  20392 ±,2951  Stimulation 2 Index  Con A  109240 ± 32936  Uninfected/ 3 Infected  5.36  _  6356 ± 1619  1.23  17.19  56014 ± 11099  1.86  _  0.50  11.44  RPMI 1640 +  -  MCMV MOI 10.0  5164 ± 586  Control Conditioned  30069 ± 11318  Medium MCMV Conditioned  9847 ± 5088  4895 ± 6 6 2  Medium  1. CPM  H-dThd i n c o r p o r a t e d by u n s t i m u l a t e d o r Con A s t i m u l a t e d s p l e e n c e l l s  i n t h e d e s c r i b e d medium.  suspended  Values a r e means ± s t a n d a r d d e v i a t i o n s from q u a d r u p l i c a t e samples.  2. S t i m u l a t i o n Index i s the r a t i o o f s t i m u l a t e d t o u n s t i m u l a t e d 3. R a t i o o f Con A s t i m u l a t e d u n i n f e c t e d counts t o i n f e c t e d  counts.  counts.  41  stimulated samples.  Direct comparison of the stimulated samples, as  seen i n the uninfected to i n f e c t e d r a t i o s or by d i r e c t comparison o f stimulated CPM (figure 2 ) , provides a more accurate representation of the degree of suppression which i s a c t u a l l y taking place. Therefore, rather than stimulation i n d i c e s , d i r e c t comparisons o f the stimulated samples are used throughout this  study.  To further determine whither the suppressive a c t i v i t y was due to a suppressor  f a c t o r (VISF) or simply r e s i d u a l undetected  virus  p a r t i c l e s , the conditioned media was tested f o r suppressive at each stage of preparation.  activity  In table I I , c o n t r o l conditioned  medium and RPMI 1640 are compared with the MCMV conditioned medium before and a f t e r the u l t r a c e n t r i f u g a t i o n step.  The u l t r a c e n t r i f u g e d  MCMV conditioned medium gave a s i g n i f i c a n t l y greater response than the untreated MCMV conditioned medium (Student's  T t e s t , p < 0.025)  yet both were s i g n i f i c a n t l y suppressed when compared with the c o n t r o l medium.  Resuspension o f the u l t r a c e n t r i f u g e p e l l e t produced  a medium which was suppressive but not nearly as much so as the u l t r a c e n t r i f u g e d supernatant.  Thus, i t appeared that the  suppressive a c t i v i t y i s a soluble q u a l i t y and cannot be removed by ultracentrifugation.  TABLE I I : E f f e c t o f t h e P u r i f i c a t i o n o f C o n d i t i o n e d Media on t h e S u p p r e s s i o n of Con A S t i m u l a t i o n o f Spleen  Supernatant RPMI 1640  None  C o n t r o l CM MCMV CM  Treatment"*"  Ultracentrifuged None  MCMV CM  Ultracentrifuged  MCMV CM  Resuspended UC Pellet  2  Cells  CPM H-dThd 3  Incorporated  3  %  Suppression  522,513 ± 24,287  0.00  214,996 ± 83,598  58.80  19,198 ± 6,749  96.33  44,535 ± 17,216  91.48  125,121 ± 27,987  76.05  1.  Ultracentrifugation  was a t 60,000g X 90 minutes.  2.  The p e l l e t from t h e u l t r a c e n t r i f u g a t i o n was resuspended i n an i d e n t i c a l volume of RPMI 1640. 3  3.  CPM  H-dThd i n c o r p o r a t e d i n t o Con A s t i m u l a t e d s p l e e n c e l l s  the a p p r o p r i a t e c o n d i t i o n e d media. from q u a d r u p l i c a t e samples.  Values  suspended i n  a r e means ± s t a n d a r d d e v i a t i o n s  43  III.  S p e c i f i c i t y o f the V i r u s Induced S u p p r e s s o r  The  p r e v i o u s l y d e s c r i b e d e x p e r i m e n t s e s t a b l i s h e d the presence o f  a v i r u s induced  suppressor  VISF was n o t d e t e r m i n e d .  f a c t o r ( V I S F ) , however the s p e c i f i c i t y o f I n f i g u r e 3 the degree o f s u p p r e s s i o n  a v a r i e t y o f c o n d i t i o n e d media was examined. incubated with  Factor  with  Spleen  from  c e l l s were  one o f s e v e r a l v i r u s e s a t an MOI o f 10 P F U / c e l l o r  l a t e x beads a t a c o n c e n t r a t i o n o f 10 l a t e x b e a d s / c e l l , and  c o n d i t i o n e d media were p r e p a r e d .  PM2 and T4 r e p r e s e n t e n v e l o p e d and  non-enveloped p a r t i c l e s , both o f which a r e n o n - i n f e c t i o u s i n t h e eukaryote c e l l . although  S i n d b i s v i r u s i s an e n v e l o p e d RNA v i r u s a n d ,  i n f e c t i o u s f o r some mouse c e l l s  not appear t o i n v o k e  ( i e . fibroblasts), i t did  c y t o l y s i s o f the l e u k o c y t e s .  were s p h e r i c a l l a t e x p a r t i c l e s a p p r o x i m a t e l y  In f i g u r e 3 each type various  dilutions.  LB8 l a t e x beads  800 nm i n d i a m e t e r .  o f c o n d i t i o n e d medium was t e s t e d a t  A l l c o n d i t i o n e d media s u p p o r t e d  a  decreased  r e s p o n s e a t 90%, when compared t o t h e c o n t r o l medium, however, MCMV c o n d i t i o n e d medium s u p p o r t e d than t h e o t h e r  a much g r e a t e r degree o f s u p p r e s s i o n  four conditioned media.  A l s o , the a c t i v i t y o f t h e  MCMV c o n d i t i o n e d media was p r e s e n t when d i l u t e d t o 20%; the non-MCMV c o n d i t i o n e d media d i s p l a y e d no s i g n i f i c a n t  suppression  when d i l u t e d  44  to o n l y 50%.  I f a s i m i l a r suppressor  f a c t o r I s present  f o u r c o n d i t i o n e d m e d i a , i t I s a t v e r y low c o n c e n t r a t i o n s  I n the o t h e r r e l a t i v e to  MCMV c o n d i t i o n e d medium.  IV.  F a c t o r s A f f e c t i n g the P r o d u c t i o n  Suppressor  o f the V i r u s  Induced  Factor  In o r d e r  to f a c i l i t a t e  the c h a r a c t e r i z a t i o n o f VISF I t was  n e c e s s a r y t o determine the o p t i m a l c o n d i t i o n s f o r p r o d u c t i o n i n vitro.  Previous  e x p e r i m e n t s had d e t e r m i n e d t h a t a low MOI (< 3  P F U / c e l l ) d i d n o t c o n s i s t e n t l y produce a s i g n i f i c a n t  degree o f  Immunosuppression, and t h a t t h e degree o f immunosuppression was p r o p o r t i o n a l t o t h e MOI ( 8 2 ) .  The MOI o f 10 P F U / c e l l was chosen  because i t c o n s i s t e n t l y demonstrated c o n s i d e r a b l e s u p p r e s s i o n was p o s s i b l e t o o b t a i n v i r u s i n h i g h enough t i t e r s an  and i t  to c a r r y out such  infection.  To d e t e r m i n e whether VISF was produced o v e r a l o n g p e r i o d o f time o r produced r a p i d l y over a s h o r t p e r i o d o f t i m e , and i f s o , a t what time a f t e r i n o c u l a t i o n , c o n t r o l and MCMV c o n d i t i o n e d media were collected a t various the p r o d u c t i o n  times a f t e r i n o c u l a t i o n .  F i g u r e 4 demonstrates  o f VISF over a p e r i o d o f s e v e r a l d a y s .  A f t e r 24  hours t h e MCMV c o n d i t i o n e d medium d i d n o t demonstrate a f u r t h e r d e c r e a s e i n s t i m u l a t i o n , thus t h e p r o d u c t i o n within  24 hours a f t e r i n f e c t i o n .  o f VISF was completed  The d e c r e a s e i n s t i m u l a t i o n  45  F i g u r e 3: of  Spleen  E f f e c t o f Various  Conditioned  Media On Con A S t i m u l a t i o n  Cells  C o n d i t i o n e d media from u n i n f e c t e d , v i r u s i n f e c t e d and l a t e x bead i n o c u l a t e d s p l e e n c e l l s were compared w i t h RPMI 1640 f o r t h e i r e f f e c t on s p l e e n c e l l substituted was  stimulation.  C o n d i t i o n e d medium was  f o r RPMI 1640 t o the percentage  added t o the samples .  Values  described before  are expressed  RPMI 1640 sample w h i c h was a r b i t r a r i l y  as a p e r c e n t  termed t h e maximum  Con A o f the  response.  Each v a l u e r e p r e s e n t s the mean from f o u r samples; e r r o r b a r s were too s m a l l t o be i n c l u d e d .  Figure 3:  .  Effect of Various Conditioned Media On Con A Stimulation of Spleen Cells  R P M I Control M C M V 1640  Sindbis  PM2  T4  LB8  ON  47  observed  i n the c o n t r o l c u r v e was p r o b a b l y  n u t r i e n t s and e x c r e t i o n o f m e t a b o l i c I n t e r e s t i n g l y , the MCMV c u r v e  due t o d e p l e t i o n o f  byproducts  d i d not d e c r e a s e  i n t o the medium. after  a l t h o u g h media d e p l e t i o n and r e l e a s e o f m e t a b o l i c have taken  place.  24 hours  byproducts  must  T h i s may have been a r e s u l t o f d e g r a d a t i o n o f  VISF, o r VISF may have masked a n y e f f e c t s o f such f a c t o r s p r e s e n t i n the medium.  Many c e l l s r e q u i r e serum f o r in_ v i t r o c u l t i v a t i o n a n d f o r the production o f c e l l spleen c e l l s  products.  To examine the serum r e q u i r e m e n t s o f  f o r the p r o d u c t i o n o f VISF, MCMV i n f e c t e d and  u n i n f e c t e d c o n t r o l c u l t u r e s were p r e p a r e d c o n c e n t r a t i o n o f f e t a l bovine  serum.  using varying  The r e s u l t s o f s t i m u l a t i o n  t e s t s u s i n g the c o n d i t i o n e d mdia a r e p r e s e n t e d  i n f i g u r e 5.  Quite  c l e a r l y t h e r e i s no dependence o f immunosuppressive a c t i v i t y upon FBS  concentration i neither  the c o n t r o l o r t h e MCMV samples.  F o r the p r o d u c t i o n o f VISF, a serum c o n c e n t r a t i o n o f 2% FBS was chosen f o r two r e a s o n s . made i t more d i f f i c u l t  F i r s t , a h i g h e r c o n c e n t r a t i o n would have t o i s o l a t e and p u r i f y the s u p p r e s s o r  Secondly,  the v i r u s p r e p a r a t i o n s used t o i n f e c t the s p l e e n  contained  serum, t h e r e f o r e a c h o i c e o f 0% FBS,  serum would r e s u l t  factor. cells  o r v e r y low l e v e l s o f  i n an u n d e r s i r a b l e d i f f e r e n c e between the two  media .  An i n c u b a t i o n time o f 24-48 hours was chosen because  a l l o w e d s u f f i c i e n t time f o r the p r o d u c t i o n o f VISF b u t n o t s i g n i f i c a n t amounts o f s u p p r e s s i v e s u b s t a n c e s i n the c o n t r o l conditioned  medium.  49  Figure 4: K i n e t i c s o f the Production o f the Virus Induced Suppressor Factor  Conditioned medium was assayed f o r suppressive (VISF) a c t i v i t y a f t e r various lengths of i n c u b a t i o n .  Control (•—•—) and MCMV  (•—O^—) c o n d i t i o n e d media were prepared a f t e r v a r y i n g lengths o f i n c u b a t i o n time w i t h the spleen c e l l s and assayed f o r a c t i v i t y by d i l u t i n g f r e s h spleen c e l l s a t 5 x 1 0 c e l l / m l i n conditioned 7  medium t o y i e l d 5 x 10^ c e l l s / m l .  Con A s t i m u l a t i o n t e s t s were  then performed on these spleen suspension.  Values represent means -  standard d e v i a t i o n s from quadruplicate samples.  F i g u r e 4:  P  K i n e t i c s of P r o d u c t i o n of the V i r u s - I n d u c e d  24  48  72  Time  96  (hours)  120  Suppressor  144  Factor  Ln O  51  F i g u r e 5:  E f f e c t o f F e t a l B o v i n e Serum On the P r o d u c t i o n  Induced S u p p r e s s o r  Conditioned  Factor  media were p r e p a r e d  MCMV i n f e c t e d s p l e e n c e l l s p e r c e n t a g e s o f FBS.  by i n c u b a t i n g u n i n f e c t e d and  i n RPMI 1640 supplemented w i t h  Production  represent  varying  o f VISF was measured by s u p p r e s s i o n  o f Con A s t i m u l a t i o n o f f r e s h s p l e e n c e l l s ( — )  o f Virus  suspended i n u n i n f e c t e d  and MCMV i n f e c t e d (—•-O—«•) c o n d i t i o n e d m e d i a . the means from q u a d r u p l i c a t e  samples  Values  ± standard  deviations.  Figure 5:  Effect of Fetal Bovine Serum on the Production of Virus-Induced Suppressor Factor  2  4  %  6  FBS  8  10  CHAPTER IV  RESULTS I I  C h a r a c t e r i z a t i o n o f the V i r u s Induced S u p p r e s s o r  The  previous  a c t i v i t y present mice.  chapter  presented  evidence  i n v i r u s - f r e e supernatants  Factor  o f immunosuppressive from MCMV i n f e c t e d  T h i s e f f e c t was due t o the p r e s e n c e o f a p u t a t i v e  immunosuppressive m o l e c u l e termed V I S F .  The e v i d e n c e  presented  demonstrates the a c t i v i t y b u t does n o t i n d i c a t e t h a t i t i s due t o a s i n g u l a r molecular presenting evidence  species.  This chapter  i s devoted  f o r the e x i s t e n c e o f a s u p p r e s s o r  toward m o l e c u l e and  the c h a r a c t e r i z a t i o n o f some o f i t s p h y s i c a l p r o p e r t i e s .  I  Effect  o f Indomethacin on the P r o d u c t i o n  o f the V i r u s  Induced  Suppressor Factor  Prostaglandins  o f the E s e r i e s a r e known t o be  immunosuppressive, I n v o l v e d macrophages ( 4 2 ) .  i n immune r e g u l a t i o n and produced by  I n a d d i t i o n , a p r o s t a g l a n d i n has been i m p l i c a t e d  as a m e d i a t o r o f dengue v i r u s i n d u c e d  immunosuppression ( 2 3 ) ,  t h e r e f o r e the p o s s i b i l i t y o f VISF b e i n g a p r o s t a g l a n d i n was considered.  To i n v e s t i g a t e t h i s  p o s s i b i l i t y , c o n d i t i o n e d media were  54  prepared  from c o n t r o l and MCMV i n f e c t e d spleen c e l l cultures  supplemented with indomethacin, an i n h i b i t o r o f prostaglandin synthesis .  As i l l u s t r a t e d i n figure 6, indomethacin f a i l e d t o  a f f e c t the production of suppressive a c t i v i t y , suggesting that prostaglandins play no role i n the observed immunosuppression unless the mechanism involved the release o f preformed prostaglandins.  To i n v e s t i g a t e the p o s s i b i l i t y o f preformed prostaglandins  being  released, a t o t a l l i p i d e x t r a c t i o n from MCMV and c o n t r o l conditioned media was performed using a modified Folch e x t r a c t i o n technique (38).  The extracted l i p i d s were resuspended i n RPMI 1640 at a  volume equivalent to the o r i g i n a l volume.  Table I I I demonstrates  the e f f e c t of the resuspended l i p i d s on the stimulation o f spleen cells .  No s i g n i f i c a n t d i f f e r e n c e was observed a t e i t h e r of the two  concentrations tested (Student's  T test, p  0.05).  When tested a t  a concentration of 80% of the o r i g i n a l concentration, the c o n t r o l 3 suspension  demonstrated a considerable decrease i n  H-dThd  incorporation r e l a t i v e to the c o n t r o l conditioned medium.  This may  have been due to chemical modification of some of the extracted substances to produce toxic or suppressive species. The corresponding  MCMV sample gives a very s i m i l a r value, a value which  i s s i g n i f i c a n t l y greater than that o f the MCMV conditioned medium i n d i c a t i n g that e i t h e r the suppressor  f a c t o r was not extracted or  i t s b i o l o g i c a l a c t i v i t y was destroyed i n the e x t r a c t i o n procedure.  55  Figure 6:  E f f e c t of Indomethacin On the Production of Virus Induced  Suppressor Factor Conditioned media were prepared by incubating uninfected and MCMV Infected spleen c e l l s i n RPMI 1640 containing 2% FBS, and with or without  indomethacin.  The production of VISF i n the conditioned  medium was assayed by measuring suppression of Con A stimulation of fresh spleen c e l l s suspended i n the conditioned media.  Values  represent the means from quadruplicate samples ± standard d e v i a t i o n s .  F i g u r e 6;  E f f e c t o f Indomethacin On the P r o d u c t i o n o f the V i r u s - I n d u c e d  Suppressor  Factor  Indomethacin (molarity) ' Ln ON  57  II.  Concentration of the Virus Induced Suppressor Factor by  Ultra f i l t r a t i o n  A method of concentrating VISF was needed i n order to obtain sufficient  q u a n t i t i e s of the factor f o r f r a c t i o n a t i o n studies .  Attempts to do so were made by u l t r a f i l t r a t i o n through Amicon membrane f i l t e r s .  I f VISF was l a r g e r than the molecular exclusion  l i m i t of the f i l t e r then i t would be retained i n the r e s i d u a l volume and could be concentrated i n this manner.  Since the suppressive  e f f e c t of the MCMV conditioned medium was not due to a prostaglandin, the most l i k e l y a l t e r n a t i v e was a protein or peptide.  I f t h i s was the case the molecule would be large enough to  be concentrated by u l t r a f i l t r a t i o n .  Conditioned medium was passed through membrane f i l t e r s with defined exclusion l i m i t s and the r e s i d u a l l i q u i d and f i l t r a t e were analysed f o r the presence of suppressive a c t i v i t y i n a Con A stimulation t e s t .  Using a membrane f i l t e r with a molecular  exclusion l i m i t of 10,000 daltons (Amicon YMLO f i l t e r ) , no a c t i v i t y was retained by the f i l t e r  ( r e s u l t s not shown) , however when an  Amicon YM2 f i l t e r (molecular exclusion l i m i t of 1000 daltons) was used the suppressor a c t i v i t y was retained by the f i l t e r and was not present i n the f i l t r a t e (see table IV) .  The r e s i d u a l f r a c t i o n was  i n i t i a l l y a ten-fold concentrate of the retained molecules and was  58  TABLE I I I ;  Effect  o f L i p i d s E x t r a c t e d from C o n d i t i o n e d  On t h e S t i m u l a t i o n o f S p l e e n  Medium  CPM  RPMI 1640  C e l l s by Con A  H-dThd I n c o r p o r a t e d  Control  Media  Uninfected:Infected  MCMV  150,716  24,518  ±24,516  ±3,998  80% E x t r a c t  36,536  36,138  Suspension  ±5,584  ±5,672  20% E x t r a c t  114,743  96,641  Suspension  ±12,697  ±21,583  6.15  RPMI 1640 +  1  1.01  RPMI 1640 +  1.  1  E x t r a c t suspension  1.19  i s the resuspension  of materials  extracted  from c o n t r o l and MCMV c o n d i t i o n e d media by F o l c h e x t r a c t i o n . 3 2.  CPM  H-dThd i n c o r p o r a t e d i n t o Con A s t i m u l a t e d  suspended i n t h e d e s c r i b e d medium. standard 3.  Values represent  d e v i a t i o n s from q u a d r u p l i c a t e  R a t i o o f c o n t r o l CPM t o MCMV CPM.  spleen  samples.  cells means ±  59  diluted  t o the c o n c e n t r a t i o n shown i n t a b l e I V .  No s i g n i f i c a n t  d i f f e r e n c e was o b s e r v e d between the MCMV n o n - f i l t e r e d and t h e MCMV r e s i d u e I X medium ( S t u d e n t ' s the VISF was r e c o v e r e d  Figure  7 presents  T t e s t , p > 0.05), i n d i c a t i n g t h a t a l l  from the c o n c e n t r a t e d  a more a c c u r a t e  VISF from t h e 10X c o n c e n t r a t e d  a c t i v i t y , and t h e r e b y  e s t i m a t i o n o f the r e c o v e r y o f  residue.  the u l t r a f i l t r a t i o n was d i l u t e d  residue.  Concentrated  to find  the end p o i n t o f s u p p r e s s i v e  a c t i v i t y was  U n f i l t e r e d MCMV c o n d i t i o n e d medium c o u l d be d i l u t e d t o detectable suppressor  r e s u l t s o f these  activity  ( s e e f i g u r e 3 ) . The  e x p e r i m e n t s I n d i c a t e d t h a t VISF had a m o l e c u l a r  weight between 1000 and 10,000 d a l t o n s and c o u l d be r e c o v e r e d h i g h e f f i c i e n c y and c o n c e n t r a t e d  III.  The  a c t i v i t y a t d i l u t i o n s a s low as 0.4X  the o r i g i n a l c o n c e n t r a t i o n , b u t a t 0.2X no s u p p r e s s o r  0.2X w i t h  from  q u a n t i t a t e the amount o f VISF p r e s e n t .  r e s u l t s demonstrate s u p p r e s s i v e  detected.  residue  with a  by u l t r a f i l t r a t i o n .  F r a c t i o n a t i o n o f Conditioned  Medium by G e l F i l t r a t i o n  Chromatography  The  results  o f the u l t r a f i l t r a t i o n  the i s o l a t i o n o f V I S F .  p r o v i d e d an i m p o r t a n t  Not o n l y d i d they  define a molecular  step i n weight  range f o r the f a c t o r b u t u l t r a f i l t r a t i o n a l s o p r o v i d e d a means o f c o n c e n t r a t i n g VISF, a l l o w i n g f r a c t i o n a t i o n s t u d i e s by methods which  60  normally Involve a degree of d i l u t i o n . f i l t r a t i o n chromatography. experiments suggested  One such method i s gel  The r e s u l t s of the  ultrafiltration  that the molecular weight o f VISF was between  1000 and 10,000 daltons, therefore Sephadex G-25 was selected as a gel  f i l t r a t i o n matrix.  Samples of conditioned medium were concentrated 10X by u l t r a f i l t r a t i o n and were separated on a Sephadex G-25 column.  Three  ml f r a c t i o n s were c o l l e c t e d and assayed f o r suppressor a c t i v i t y .  A  comparison of the f r a c t i o n s from c o n t r o l and MCMV conditioned media i s presented i n figure 8. Fractions 4 and 13 are s i g n i f i c a n t l y lower i n the MCMV curve; repeat experiments showed that f r a c t i o n 4 was not c o n s i s t e n t l y suppressive r e l a t i v e to the c o n t r o l f r a c t i o n . Fraction 13 was c o n s i s t e n t l y suppressive i n repeat experiments and therefore was considered to contain VISF.  The l o c a t i o n of i t s  f r a c t i o n a t i o n indicated a molecular weight l e s s than 1400 daltons, thus agreeing with the molecular weight provided by the ultrafiltration  data.  TABLE IV: E f f e c t of U l t r a f i l t r a t i o n Through a YM2 Membrane  6]  F i l t e r On the A b i l i t y of Conditioned Media to Suppress Con A S t i m u l a t i o n of Spleen C e l l s CPM  H-dThd  Conditioned  Filtration  Medium  Fraction  Factor  Control  Filtrate  1.0  417,413 + 42,052  Residue  1.0  632,035 + 86y687  Residue  2.0  556,568 + 36,595  Residue  3.0  255,267 + 16,712  Residue  4.0  178,140 + 16,499  Non-filtered  1.0  10,537 + 7168  Filtrate  1.0  154,734 + 22,852  Residue  1.0  12,099 + 4596  Residue  2.0  9,169 + 2798  Residue  3.0  5,831 + 3214  Residue  4.0  4,119 + 1665  MCMV  1.  Concentration 1  Incorporated^  Concentration f a c t o r r e l a t e s the concentration of the contents of the f r a c t i o n r e l a t i v e to the u n f i l t e r e d conditioned medium. 3  2.  CPM  H-dThd incorporated i n t o spleen c e l l s suspended i n each  f i l t r a t i o n fraction.  Values represent means ± standard d e v i a t i o n s  from quadruplicate samples.  62  Figure 7;  E f f e c t of the Concentration of the Virus Induced  Suppressor Factor on the Stimulation of Spleen  Conditioned medium from uninfected ( ("••••O  ) spleen c e l l s was concentrated  through on Amicon YM2 D i a f l o f i l t e r . r e s i d u a l volume.  Cells  ) and MCMV i n f e c t e d  t e n f o l d by u l t r a f i l t r a t i o n VISF was retained i n the  This concentrated medium was added to spleen c e l l s  i n RPMI 1640 to y i e l d the appropriate concentration f a c t o r and Con A stimulation tests were then performed to assay the e f f e c t o f VISF, at d i f f e r e n t concentrations, on stimulation o f the spleen c e l l s . The concentration f a c t o r was the r a t i o of the YM2 retained molecules to t h e i r concentration i n u n f i l t e r e d conditioned medium.  Values  represent means from quadruplicate samples ± standard d e v i a t i o n s .  Figure 7;  Effect of the Concentration of Virus-Induced Suppressor Factor On Con A Stimulation of Spleen C e l l s  0.2  04  0.6  0.8  1.0  1.2  1.6  Concentration Factor  2.0  64  IV.  Fractionation  o f Mouse Serum by G e l F i l t r a t i o n  Chromatography  To compare the in_ v i t r o produced VISF t o the s u p p r e s s o r produced i n v i v o  and found i n the serum o f i n f e c t e d m i c e , samples o f  serum from MCMV i n f e c t e d and u n i n f e c t e d the  factor  Sephadex G-25 column.  Fractions  mice were f r a c t i o n a t e d on  were c o l l e c t e d and a s s a y e d f o r  immunosuppressive a c t i v i t y .  The r e s u l t s p r e s e n t e d i n f i g u r e 9  illustrate  a complex p a t t e r n  w i t h many f r a c t i o n s  containing  suppressive a c t i v i t y .  f r a c t i o n s 8-12 o f t h e u n i n f e c t e d the  MCMV i n f e c t e d mouse serum.  from b o t h  sera  Of p a r t i c u l a r i n t e r e s t a r e mouse serum and f r a c t i o n s Fractions  6-13 o f  6,7 and 13 demonstrate  s i g n i f i c a n t l y g r e a t e r s u p p r e s s i o n i n t h e i n f e c t e d mouse serum c u r v e ( S t u d e n t ' s T t e s t , p < 0.0005).  These f r a c t i o n s may r e p r e s e n t  a d d i t i o n a l s u p p r e s s i v e s u b s t a n c e s i n t h e i n f e c t e d mouse serum, o r they may r e p r e s e n t  the t a i l s o f a d i s t r i b u t i o n c u r v e o f a s u b s t a n c e  present i n higher q u a n t i t y  i n the i n f e c t e d mouse serum.  was n o t p o s s i b l e  the f r a c t i o n responsible  difference sera the  to define  i n immunosuppressive c a p a c i t i e s  ( s e e f i g u r e 1 ) , t h e data i n f i g u r e l o c a t i o n o f VISF i n f i g u r e 8.  Although i t  f o r the  o f the two u n f r a c t i o n a t e d  9 are not inconsistent  with  65  Figure 8;  Sephadex G-25 F r a c t i o n a t i o n o f Control and MCMV  Conditioned Media  Conditioned media from uninfected ( — ) (  o  and MCMV i n f e c t e d  •) spleen c e l l s was concentrated 10X by YM2 u l t r a f i l t r a t i o n .  One ml samples were loaded onto a 20 x 1.6 cm Sephadex G-25 column and eluted with a s a l i n e b u f f e r a t a rate of 0.3 ml/minute.  Three  ml f r a c t i o n s were c o l l e c t e d and assayed i n quadruplicate f o r suppression of Con A stimulation o f spleen c e l l s •  Values represent  the means from quadruplicate samples ± standard d e v i a t i o n s . The small arrows mark the positions o f two molecular weight standards, a p r o t i n i n (MW 6300), and angiotensin (MW 1400). marks the p o s i t i o n o f VISF.  The large arrow  Figure 8:  Sephadex G-25 Fractionation of Control and Murine Cytomegalovirus Medium  100  Fraction Number  Conditioned  67  Figure 9:  Sephadex G-25 F r a c t i o n a t i o n of Normal and Infected Mouse  Serum  Serum was c o l l e c t e d from normal, uninfected mice (—•—•) and from mice infected with 5 x 10 infection (  O  •) •  6  PFU of MCMV, at 4 days post  One ml samples were loaded onto a 20 x 1.6 cm  Sephadex G-25 column and eluted with a s a l i n e buffer at a rate of 0.3 ml/minute.  Three ml samples were c o l l e c t e d and assayed for  suppression of Con A s t i m u l a t i o n of spleen c e l l s .  Values represent  the means from quadruplicate samples ± standard d e v i a t i o n s .  F i g u r e 9:  Sephadex G-25 F r a c t i o n a t i o n o f Normal and I n f e c t e d Mouse Serum  • NMS  2  4  6  8  10  12  F r a c t i o n Number  14  16  18  69  V.  F r a c t i o n a t i o n o f S i n d b i s V i r u s and LB8 C o n d i t i o n e d Media by G e l  Filtration  Chromatography  Two a d d i t i o n a l samples were f r a c t i o n a t e d on the G-25 column t o a g a i n a s s e s s t h e s p e c i f i c i t y o f VISF t o MCMV i n f e c t i o n s . f r a c t i o n a t i o n and a s s a y p r o c e d u r e s were i d e n t i c a l the p r e c e e d i n g two s e c t i o n s . spleen c e l l s  The  t o those used i n  C o n d i t i o n e d media were p r e p a r e d  i n f e c t e d w i t h S i n d b i s v i r u s o r LB8 l a t e x b e a d s .  from The  c o n d i t i o n e d media were c o n c e n t r a t e d 10X by u l t r a f i l t r a t i o n and f r a c t i o n a t e d on t h e Sephadex G-25 column. s u p p r e s s i v e f r a c t i o n s were observed f r a c t i o n 13 were a p p r o x i m a t e l y These data s u g g e s t e d  No low m o l e c u l a r  weight,  i n e i t h e r c u r v e and v a l u e s f o r  the same a s the c o n t r o l ( f i g u r e 1 0 ) .  t h a t t h e immunosuppressive a c t i v i t i e s  o f these  c o n d i t i o n e d media ( f i g u r e 3) were n o t due t o the same m o l e c u l a r s p e c i e s which was found i n f r a c t i o n 13 o f the MCMV c o n d i t i o n e d medium.  VI.  P r o t e i n a s e K D i g e s t i o n o f the V i r u s Induced  The m a j o r i t y o f s o l u b l e immunoregulatory  Suppressor  molecules a r e p r o t e i n s  o r p e p t i d e s , a l t h o u g h o t h e r c l a s s e s o f immunoregulatory have been d e s c r i b e d (15,28,42).  Factor  The m o l e c u l a r weight  i n d i c a t e d was c o n s i s t e n t w i t h i t b e i n g a p e p t i d e .  molecules range  To determine  whether o r n o t t h i s was the c a s e , s u s c e p t i b i l i t y t o d e g r a d a t i o n by  70  the p r o t e o l y t i c enzyme, proteinase K was examined.  When c o n t r o l and  MCMV conditioned media were digested with proteinase K, the r e s u l t i n g s o l u t i o n supported  no stimulation o f lymphocytes by Con A,  therefore the e f f e c t of the enzyme on VISF could be determined. To overcome this problem the digested conditioned medium was fractionated on the Sephadex G-25 column and the f r a c t i o n s were assayed f o r suppressive a c t i v i t y ; the r e s u l t s are presented i n figure 11.  The proteinase K digestion r e s u l t e d i n a number o f strongly suppressive f r a c t i o n ( f r a c t i o n s 2-7) possibly due to the production of smaller suppressive fragments from the d i g e s t i o n of some o f the l a r g e r molecules present i n the conditioned medium.  Fraction 13,  the f r a c t i o n shown to contain VISF (figure 8 ) , displayed an increased support  f o r Con A stimulation o f spleen c e l l s , presumably  due to p r o t e o l y t i c degradation o f VISF.  Table V presents the numerical values f o r the undigested and proteinase K digested f r a c t i o n s 13.  No s i g n i f i c a n t difference was  observed between the proteinase K treated and untreated controls , however the digested MCMV f r a c t i o n was s i g n i f i c a n t l y greater (Student's  T t e s t , p < 0.005) than the undigested  f r a c t i o n suggested  that VISF i s l a b i l e to proteinase K d i g e s t i o n and i s therefore a peptide.  71  Figure 10:  Sephadex G-25 F r a c t i o n a t i o n of Sindbis Virus and LB8  Conditioned Msdia  Conditioned media from Sindbis v i r u s ( — A — ) and LB8 l a t e x bead (  'A  ) treated spleen c e l l s were concentrated tenfold by  ultrafiltration.  One ml samples were loaded onto a 20 x 1.6 cm  Sephadex G-25 column and eluted with a s a l i n e buffer at 0.3 ml/minute.  Three ml samples were c o l l e c t e d and assayed for  suppression of Con A stimulation of spleen c e l l s .  The large arrow  indicates the f r a c t i o n i n which VISF e l u t e s ; the small arrow indicates values of this f r a c t i o n from uninfected ( infected (  O  ) conditioned media.  •  ) and MCMV  Values represent the means  from quadruplicate samples ± standard d e v i a t i o n s .  Figure 10:  Sephadex G-25  Fractionation of Sindbis and LB8 Conditioned Media  73  Figure 11:  Sephadex G-25 F r a c t i o n a t i o n of Proteinase K Digested  Control and MCMV Conditioned Medium  Conditioned media from uninfected (•—-•—) and MCMV i n f e c t e d (  -O  ) spleen c e l l s were concentrated tenfold by u l t r a f i l t r a t i o n .  One ml samples were digested with 1 mg o f proteinase K f o r 30 minutes a t 37 C. The samples were then loaded onto a 20 x 1.6 cm Sephadex G-25 column and eluted with a s a l i n e b u f f e r at 0.3 ml/minute.  Three ml f r a c t i o n s were'collected and assayed f o r  suppression o f Con A s t i m u l a t i o n o f spleen c e l l s .  The large arrow  indicated the value from the f r a c t i o n a t i o n o f undigested MCMV conditioned medium.  Values represent the means from quadruplicate  samples ± standard d e v i a t i o n s .  Figure 11; Sephadex G-25 Fractionation of Proteinase K Digested Control and Murine Cytomegalovirus Conditioned Medium  Fraction Number  TABLE V: E f f e c t of Proteinase K On the F r a c t i o n a t i o n of VISF by Sephadex G-25 Chromatography Conditioned Medium  Treatment  Control  None  282,493 ± 17,937  Control  Pro K  259,625 ± 37,467  MCMV  None  MCMV  Pro K  1.  CPM H-dThd Incorporated  56,493 ± 23,160 134,311 ± 38,707  Conditioned medium was concentrated 10X and f r a c t i o n a t e d on a Sephadex G-25 column.  2.  Samples t r e a t e d w i t h P r o t e i n a s e K were digested p r i o r t o fractionation. 3  3.  Values represent CPM H-dThd incorporated i n t o spleen c e l l s i n a Con A s t i m u l a t i o n t e s t . 13 from each e l u t i o n .  C e l l s were suspended i n F r a c t i o n  Values are means ± standard d e v i a t i o n s  from quadruplicate samples.  VII.  Electrophoresis of Mouse Serum and Conditioned  Medium  Proteins and peptides are resolvable by electrophoresis when present  i n s u f f i c i e n t quantity to allow detection.  Therefore an  attempt was made to resolve VISF i n t o a detectable band by electrophoresis•  Samples o f normal and i n f e c t e d mouse serum, and MCMV and c o n t r o l conditioned media were dissolved i n an SDS sample buffer and aliquots of each sample were applied to an SDS-polyacrylamide g e l . Following electrophoresis of the samples, the gels were stained by an u l t r a s e n s i t i v e s i l v e r s t a i n capable of detecting proteins a t  3 concentrations  as low as 0.01 ng/mm .  In neither the serum nor the conditioned media samples was a protein band, s p e c i f i c for the MCMV i n f e c t e d sample, observed i n the appropriate molecular weight range to be considered as candidate for VISF.  A possible explanation  f o r this i s very low concentrations of  VISF i n both the serum and conditioned medium.  CHAPTER V  DISCUSSION  Many v i r u s e s have been implicated as immune modulators, most commonly as suppressors of the immune system (116).  In most cases  the extent o f suppression has been characterized by t e s t i n g the response of i n f e c t e d animals or leukocytes to a series o f immune challenges.  In c o n t r a s t , very few data have accumulated  a mechanism f o r the observed immunosuppression.  to present  As a r e s u l t , this  aspect o f the pathogenesis of the v i r a l i n f e c t i o n i s poorly understood.  The object o f the present study was to i n v e s t i g a t e the  mechanism whereby MCMV can induce a state of immunosuppression i n i n f e c t e d mice.  The r e s u l t s presented i n the preceeding two chapters i n d i c a t e d the presence o f a soluble immunosuppressive f a c t o r , termed VISF, which i s released during MCMV i n f e c t i o n of spleen c e l l s .  Previous  studies have presented some evidence to suggest the presence of a soluble immunosuppressive f a c t o r .  Booss and Wheelock presented  evidence that serum from i n f e c t e d mice was immunosuppressive but were unable to conclude whether or not this was due to free v i r u s i n the serum (9,10).  Loh and Hudson presented evidence f o r  immunosuppression i n spleens with as few as 100 i n f e c t i o u s centers per spleen (82) .  They also showed a c o r r e l a t i o n between i n f e c t i o u s  centers and the degree of immunosuppression.  The low number of  i n f e c t i o u s centers indicated that a c e l l to c e l l contact event was probably not the mechanism of immunosuppression; an a l t e r n a t i v e explanation was the release of a lymphokine or monokine-like f a c t o r .  The r e s u l t s i n chapter I I I demonstrate that a immunosuppressive factor i s present i n both the serum from i n f e c t e d mice as w e l l as supernatants (conditioned media) from i n v i t r o cultures of i n f e c t e d spleen c e l l s , both of which were free of i n f e c t i o u s v i r u s .  The  e f f e c t of i n f e c t e d mouse serum on Con A stimulation of spleen c e l l s , shown i n figure 1, demonstrates a suppressed response when 2-10% mouse serum was added to the c u l t u r e s •  Higher or lower percentages  of mouse serum r e s u l t e d i n values which were not s i g n i f i c a n t l y d i f f e r e n t from those using normal mouse serum.  Suppression by high  concentrations of normal mouse serum was presumably due to immunoregulatory factors such as normal immunosuppressive p r o t e i n , immunoregulatory alpha g l o b u l i n , C reactive protein and alpha fetoprotein (15). The i n a b l i b t y of Booss and Wheelock to show consistent suppression due to IMS may have been due to low l e v e l s of suppressive a c t i v i t y i n the serum or use of too low or too high a concentration of mouse serum f o r optimal r e s u l t s .  Assessment of suppressive a c t i v i t y from i n v i t r o cultures of i n f e c t e d spleen c e l l s proved to be much less complicated than the studies on i n f e c t e d mouse serum.  MCMV conditioned medium showed  79  s i g n i f i c a n t suppression r e l a t i v e to the c o n t r o l conditioned medium at concentrations of 20% or greater and evidence was presented  that  the observed immunosuppression was not due to free v i r u s i n e i t h e r the serum or the conditioned medium ( f i g u r e s 1,2; t a b l e I I ) .  Two parameters of the production o f VISF which were examined were the e f f e c t of the concentration o f f e t a l bovine serum on production o f VISF, and the time a f t e r i n f e c t i o n a t which VISF was produced ( f i g u r e s 4,5). Although f e t a l bovine serum content had no e f f e c t on the production o f VISF i n v i t r o , 2% FBS was chosen f o r the standard production o f VISF i n order to keep the serum content low, but a l s o to prevent the serum present i n the v i r u s inoculum from making a q u a l i t a t i v e or l a r g e q u a n t i t a t i v e d i f f e r e n c e between the MCMV and c o n t r o l c o n d i t i o n e d media.  Figure 4 i n d i c a t e s that VISF i s  produced w i t h i n 24 hours o f i n f e c t i o n .  This r e l a t i v e l y short p e r i o d  of production suggests that the mechanism does not i n v o l v e c e l l t o c e l l I n t e r a c t i o n s to t r i g g e r the production o f VISF.  Although  changes i n the r a t i o of T helper c e l l s : T suppressor c e l l s have been a s s o c i a t e d w i t h v i r u s induced immunosuppression, i n c l u d i n g HCMV mononucleosis (3,14,108,128), the short time period r e q u i r e d f o r the production of VISF would not a l l o w time f o r the necessary i n t e r a c t i o n s and d i f f e r e n t i a t i o n to take place f o r changes i n c e l l ratios.  I t i s therefore concluded  that the production o f VISF i s  not r e l a t e d to changes i n T c e l l r a t i o s , although such changes may occur i n v i v o concurrent w i t h or as r e s u l t o f the production o f VISF.  80  Previous studies have implicated the macrophage c e l l as playing a major r o l e i n harbouring l a t e n t v i r u s and being permissive f o r MCMV r e p l i c a t i o n (11,81).  These studies have shown that the  macrophage i s p r i n c i p a l l y responsible f o r the r e p l i c a t i o n o f MCMV i n spleen c e l l populations and most o f the input v i r u s i s taken up by macrophages during i n f e c t i o n .  This information, coupled with the  time of production of VISF suggests that VISF i s a product o f the macrophage which i s released a short time a f t e r the uptake of the v i r u s by the macrophage•  Many immunoregulatory factors have been  reported, and macrophage i s responsible f o r the production of many of them (28,132).  In a d d i t i o n , Loh and Hudson have demonstrated  that the adherent population of spleen c e l l s mediate MCMV induced immunosuppressi on o f Con A and LPS stimulation o f spleen c e l l s  (82);  B i x l e r and Booss have reported a s i m i l a r f i n d i n g using hemolytic plaque assays to test immune function ( 8 ) .  Although-many viruses may induce immunosuppression (116), this observation i s not u n i v e r s a l for a l l viruses nor i s i t a r e s u l t of simple phagocytosis  of p a r t i c l e s , v i r a l or otherwise, as i s  demonstrated by the r e s u l t s o f figure 3. The production o f VISF appears to be s p e c i f i c f o r MCMV, although i t i s possible that c e r t a i n other viruses may a l s o induce i t s production.  Figure 8 demonstrated that VISF was i n fact a molecular  entity  by i s o l a t i n g i t i n a single f r a c t i o n from the Sephadex G-25 column.  81  S i m i l a r f r a c t i o n s from Sindbis v i r u s and LB8 l a t e x bead  conditioned  media showed no degree o f immunosuppression, thus reemphasizing the s p e c i f i c i t y o f VISF production f o r MCMV ( f i g u r e 10).  I t i s possible  however, that the small degree o f immunosuppression observed i n the non-MCMV samples i n f i g u r e 3 was due t o low q u a n t i t i e s o f VISF which were not detected i n f i g u r e 10. Wainberg and I s r a e l have reported the production o f a s o l u b l e immunosuppressive f a c t o r from lymph node c e l l s i n f e c t e d with any o f a v a r i e t y o f v i r u s e s (64,133).  Their  f a c t o r was apparently a product o f the macrophages but d i f f e r e d from VISF by the n o n s p e c i f i c i t y o f the v i r u s needed to stimulate i t s production.  Although many v i r u s e s induce immunosuppression, the  mechanisms i n d i c a t e d f o r d i f f e r e n t v i r u s - h o s t systems are v a r i e d . Included among the mechanisms which have been reported are a l t e r a t i o n s i n T c e l l r a t i o s (3,108,111), production o f prostaglandins ( 2 1 ) , production o f a n o n s p e c i f i c suppressor f a c t o r (64,133), i n h i b i t i o n o f helper f a c t o r production (13) and c e l l associated  suppressor mechanisms ( 1 8 ) . I t may w e l l be that  immunosuppression i s simply a common outcome o f a v a r i e t y of different virus-host  interactions.  The p o s i t i o n o f VISF on the Sephadex G-25 f r a c t i o n a t i o n ( f i g u r e 8 ) , and the r e t e n t i o n o f VISF during u l t r a f i l t r a t i o n define a s i z e range of 1000-1400 daltons f o r VISF.  A d d i t i o n a l information  to the  p h y s i c a l nature o f VISF was i t s l a b i l i t y to the p r o t e o l y t i c enzyme, proteinase K, suggesting that the molecule i s , a t l e a s t i n p a r t , a  82  peptide.  Whether or not i t contains other moieties i s yet unknown.  This d e s c r i p t i o n does not correspond t o any f a c t o r produced i n other systems o f v i r u s induced immunosuppression w i t h the exception o f the f a c t o r d e s c r i b e d by Wainberg and I s r a e l (64,133), on which no i n f o r m a t i o n i s a v a i l a b l e on the p h y s i c a l or chemical nature o f the molecule, and t h e r e f o r e the two cannot be compared.  Numerous  immunosuppressive f a c t o r s have been described from systems not i n v o l v i n g v i r u s e s , some o f which are comparable t o VISF.  Cultures  of a c t i v a t e d macrophages have been reported to secrete a d i a l i z a b l e suppressor f a c t o r which was not a p r o s t a g l a n d i n , c o r t i c o s t e r o i d , cAMP or thymidine (72,79,134,139).  These c u l t u r e s could be  a c t i v a t e d w i t h a v a r i e t y o f substances i n c l u d i n g t h i o g l y c o l l a t e and Corynebacterium parvum.  Studies o f MCMV i n f e c t i o n o f macrophages  have i n d i c a t e d that the v i r u s a c t i v a t e s macrophages and t h i s i s an important step i n c o n t r o l l i n g the spread and r e p l i c a t i o n o f the v i r u s (91,113).  S e v e r a l a l t e r n a t i v e s to a macrophage  produced  f a c t o r e x i s t i n c l u d i n g the low molecular weight peptide which was found i n T c e l l l y s a t e s and i n h i b i t e d mitogenesis ( 9 8 ) , the spleen derived immunosuppressive peptide found i n bovine spleen homogenates (34,80), thymic hormones (4,5) and immunoregulatory (93,128).  alpha g l o b u l i n s  Because of the incomplete c h a r a c t e r i z a t i o n o f these  molecules and VISF, i t i s not p o s s i b l e compare or c o n t r a s t them further.  83  The i n v i t r o studies have demonstrated the presence o f a low molecular weight peptide produced i n low quantities by MCMV i n f e c t e d spleen c e l l s .  This peptide, VISF, i n h i b i t s the incorporation of  3 H-dThd i n t o unstimulated and Con A stimulated (figure 2, table I ) .  spleen  cells  Con A stimulation o f spleen c e l l s was chosen  as a test f o r immune competence throughout these s t u d i e s , although i t i s not n e c e s s a r i l y i n d i c a t i v e of many other immunological functions.  The Con A stimulation test was chosen for i t s  consistency  and r a p i d i t y .  Many previous studies on MCMV induced  immunosuppression have a l s o used the Con A stimulation t e s t (9,10,82,83,115) although other studies have u t i l i z e d a v a r i e t y o f other immune challenges  (8,47,55-57,68,102).  Since v i r t u a l l y a l l  immune functions are impaired, I t was assumed that one mechanism was responsible, rather than d i f f e r e n t mechanisms for d i f f e r e n t functions.  Suppression o f unstimulated spleen c e l l s by MCMV  conditioned medium (table I) supports the one mechanism theory by i l l u s t r a t i n g that the suppressive  a c t i v i t y o f VISF i s not r e s t r i c t e d  to Con A s t i m u l a t i o n , however complete b i o l o g i c a l c h a r a c t e r i z a t i o n of the a c t i v i t y of VISF should be c a r r i e d out a f t e r the complete p u r i f i c a t i o n of VISF has been achieved. The studies presented i n figures 1 and 9 attempt to demonstrate the relevancy  of the i n v i t r o studies with the i n vivo s i t u a t i o n .  Figure 1 demonstrates the presence of a soluble suppressor factor i n the serum of i n f e c t e d mice.  Attempts to i s o l a t e this f a c t o r in.a  84  p a r t i c u l a r Sephadex G-25 f r a c t i o n were complicated by the presence of normal suppressive substances present i n the serum o f both i n f e c t e d and u n i n f e c t e d mice ( f i g u r e 9 ) .  F r a c t i o n 13 o f the  i n f e c t e d mouse serum was suppressive r e l a t i v e to the normal mouse serum f r a c t i o n and therefore i s c o n s i s t e n t w i t h the p o s i t i o n o f VISF i n f r a c t i o n 13 i n f i g u r e 8. The presence o f other suppressive f r a c t i o n s i n f i g u r e 9 complicate the p i c t u r e , making i t impossible to i m p l i c a t e the contents o f f r a c t i o n 13 as being r e s p o n s i b l e f o r the suppressive c a p a c i t y o f the i n f e c t e d mouse serum seen i n f i g u r e 1.  Further s t u d i e s are necessary to determine whether or not VISF  i s present i n the i n f e c t e d mouse serum.  One o f the goals o f t h i s study was to use the MCMV system as a model t o provide i n s i g h t i n t o the pathogenesis o f HCMV i n f e c t i o n . I t i s d i f f i c u l t t o r e l a t e the f i n d i n g s o f t h i s study t o the observed suppression o f immune responses i n HCMV i n f e c t e d humans because l i t t l e work has been done on the mechanism o f suppression i n HCMV i n f e c t i o n s , except HCMV mononucleosis (14,111).  The r e v e r s a l o f T  c e l l r a t i o s (T helper c e l l s : T suppressor c e l l s ) during HCMV mononucleosis (14) does not appear to be c o n s i s t e n t w i t h the production o f VISF unless the generation o f T suppressor c e l l s i s a r e s u l t o f the presence o f VISF.  I t i s a l s o p o s s i b l e t h a t the change  i n T c e l l r a t i o s i s a response to p r o l i f e r a t i o n o f lymphocytes r a t h e r than a d i r e c t e f f e c t o f the v i r u s .  A soluble  immunosuppressive factor may be present but, as of yet undetected, i n patients with ongoing HCMV i n f e c t i o n s .  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