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A comparative study, using the light and electron microscope of tissue allograft rejection in W mutant… Collen, Pat 1974

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A COMPARATIVE STUDY, USING THE LIGHT AND ELECTRON MICROSCOPE OF TISSUE ALLOGRAFT REJECTION IN W MUTANT MICE AND THEIR NON-MUTANT LITTERMATES  by B.Sc,  PAT COLLEN University of B r i t i s h Columbia  A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements f o r the Degree of Master of Science In the Department of Zoology  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1974  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  for  of  this  freely  available  for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e  of  the requirements  Columbia,  I agree  reference and copying o f  this  for  that  study. thesis  purposes may be granted by the Head of my Department or  representatives. thesis for  It  financial  i s understood that gain s h a l l  written permission.  Department of The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada  pate  fulfilment  the U n i v e r s i t y of B r i t i s h  s h a l l make i t  scholarly  by h i s  in p a r t i a l  OdbJJMsVnM  Columbia  not  copying or  publication  be allowed without my  ii  ABSTRACT  The p o p u l a t i o n s of c e l l s which  infiltrate  tissue allografts i n  W mutant mice and t h e i r non-mutant l i t t e r m a t e s were i n v e s t i g a t e d the l i g h t and e l e c t r o n m i c r o s c o p e s .  Initially,  attempted b u t t h i s t i s s u e proved u n s a t i s f a c t o r y and s k i n was used i n s t e a d . i s o l a t e d enzymatically  t h y r o i d a l l o g r a f t s were f o r comparative  The c e l l s i n f i l t r a t i n g  and c h a r a c t e r i z e d  In a d d i t i o n , c e l l s i n epon s e c t i o n s  using  using  studies  the s k i n g r a f t s were  the l i g h t  microscope.  o f s k i n g r a f t s were i d e n t i f i e d u s i n g  the e l e c t r o n m i s c r o s c o p e . The frequency o f the v a r i o u s  c e l l types i s o l a t e d from g r a f t s i n  mutant mice d i f f e r e d s i g n i f i c a n t l y from t h a t o f c e l l s i s o l a t e d from i n non-mutant mice.  The e l e c t r o n microscope s t u d i e s  grafts  i n d i c a t e d t h a t the  c e l l types i n f i l t r a t i n g s k i n a l l o g r a f t s are t h e same i n b o t h mutant and non-mutant h o s t s .  iii TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  i i i  LIST OF FIGURES  v  viii  LIST OF TABLES ACKNOWLEDGMENT  ix  INTRODUCTION TRANSPLANTATION THE W MUTANT PROPOSAL  1 IMMUNITY  IMPLANTATION OF THYROID  1 5 8 <  INTRODUCTION MATERIALS AND METHODS Implantation of thyroid RESULTS Normal t h y r o i d T h y r o i d g r a f t s implanted subcutaneously Thyroid eisografts Thyroid a l l o g r a f t s T h y r o i d i s o g r a f t s implanted i n the s p l e e n DISCUSSION TRANSPLANTATION OF SKIN INTRODUCTION MATERIALS AND METHODS Skin g r a f t i n g I s o l a t i o n o f the i n f i l t r a t i n g c e l l s L i g h t microscopy E l e c t r o n microscopy RESULTS I s o l a t i o n o f the i n f i l t r a t i n g c e l l s C e l l types Data and s t a t i s t i c a l a n a l y s i s L i g h t microscopy Epidermis Dermis .• Beneath the dermis E l e c t r o n microscope study o f t h e g r a f t bed o f 6 and 8 day ( s c o r e : 2) and 8 and 10 day ( s c o r e : 3) a l l o g r a f t s  11 11 13 13 15 15 17 17 23 25 28 31 31 34 34 34 36 36 39 39 39 42 50 50 59 60  62  iv T a b l e o f Contents  (cont'd)  O r i e n t a t i o n and i d e n t i f i c a t i o n G e n e r a l c h a r a c t e r i s t i c s o f the g r a f t bed C e l l s o f the g r a f t bed DISCUSSION The s k i n a l l o g r a f t response: General remarks The W mutant BIBLIOGRAPHY  Page  62 66 70 91 91 I l l 118  V  LIST OF FIGURES Page IA.  Normal t h y r o i d  .. ..  IB.  Higher magnification  2A.  Thyroid g r a f t r e c e i v i n g a score of 1  18  2B?  Degenerating f o l l i c l e s  18  3A.  Thyroid g r a f t r e c e i v i n g a score of 2  19  3B.  Composition o f the c e n t r a l a r e a o f glands r e c e i v i n g a  o f normal t h y r o i d t i s s u e  16  score of 2 or 4  '..  4A.  Thyroid graft r e c e i v i n g a score of 5  4B.  Composition o f the c e n t r a l a r e a o f glands r e c e i v i n g a score of 5 T h y r o i d i s o g r a f t implanted i n the s p l e e n and removed 1 day f o l l o w i n g i m p l a n t a t i o n  5A.  5B.  6.  7-20  16  19 24  24 27  T h y r o i d i s o g r a f t implanted i n the s p l e e n and removed 33 days f o l l o w i n g i m p l a n t a t i o n  27  G e n e r a l procedure f o r p r e p a r a t i o n o f t i s s u e and i s o l a t i o n of c e l l s  35  The c e l l  types i s o l a t e d from the s k i n g r a f t beds on both  mutant and non-mutant h o s t s . . . . . . . .  41  21.  6 Day a l l o g r a f t  52  22.  8 Day a l l o g r a f t  53  23A.  E p i d e r m i s o f mouse s k i n  54  23B.  E p i d e r m i s o f a s k i n i s o g r a f t removed 6 days t r a n s p l a n t a t i o n ( s c o r e : 2)  following  E p i d e r m i s o f a s k i n i s o g r a f t removed 8 days t r a n s p l a n t a t i o n ( s c o r e : 2)  following  23C.  24A.  24B.  54  E p i d e r m i s o f a s k i n : a l l o g r a f t removed 4 days t r a n s p l a n t a t i o n ( s c o r e : 1) E p i d e r m i s o f a s k i n a l l o g r a f t removed 6 days t r a n s p l a n t a t i o n ( s c o r e : 2)  54 following 57 following 57  vi L i s t o f F i g u r e s (cont'd) Page 24C.  E p i d e r m i s o f a s k i n a l l o g r a f t removed 8 days  following  t r a n s p l a n t a t i o n ( s c o r e : 3)  57  25.  T r a c i n g of a r e g i o n o f a 6 day s k i n a l l o g r a f t  26.  T r a c i n g o f a r e g i o n o f an 8 day s k i n a l l o g r a f t  27.  Low power e l e c t r o n micrograph o f the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  28.  Low power e l e c t r o n micrograph o f the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  29.  30A.  (score: 2)..  64  (score: 3).  65  68 ...  Low power e l e c t r o n microphage o f the g r a f t bed o f an 8 day s k i n a l l o g r a f t ( s c o r e : 3) Low  69  71  power e l e c t r o n micrograph o f the g r a f t bed o f an 8  day s k i n a l l o g r a f t  ( s c o r e : 3)  30B.  Debris  31A.  Small lymphocyte found i n the g r a f t bed o f a 6 day a l l o g r a f t ( s c o r e : 2)  31B.  Small lymphocyte structure  72 72 skin 76  showing minor changes i n i t s u l t r a 76  32.  Immunoblast  77  33A.  A c t i v a t e d lymphocyte found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  79  A c t i v a t e d lymphocyte found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  79  A c t i v a t e d lymphocyte found i n the g r a f t bed o f a 6 day skin a l l o g r a f t (score: 2).......  81  A c t i v a t e d lymphocyte found i n the g r a f t bed of a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  81  Monocyte, o r " n o n - a c t i v a t e d " form o f macrophage, found i n the g r a f t bed of a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  82  " A c t i v a t e d " macrophage found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  84  33B.  34A.  34B.  35.  36A.  36B.  " A c t i v a t e d " macrophage found i n the g r a f t bed of a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  ...  84  vii L i s t of Figures  (cont'd) Page  36C.  37A.  37B.  37C.  37D.  38A.  38B.  38C.  A c t i v a t e d macrophage i n the p r o c e s s o f p h a g o c y t o s i s w i t h i n the g r a f t bed of a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  84  E o s i n o p h i l found i n the g r a f t bed o f a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2)  85  E o s i n o p h i l f o u n d - i n the g r a f t bed of a 6 day a l l o g r a f t ( s c o r e : 2)  skin 85  N e u t r o p h i l found i n the g r a f t bed o f a 6 day a l l o g r a f t ( s c o r e : 2)  skin .  85  Degenerating n e u t r o p h i l found i n the g r a f t bed o f a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2)  85  F i b r o b l a s t found i n the g r a f t bed o f a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2)  87  Stacks o f rough endoplasmic r e t i c u l u m o f a f i b r o b l a s t i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t  87  F i b r o b l a s t found i n the g r a f t bed o f a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2)  87  viii LIST OF TABLES Page I.  II.  III.  S c o r i n g system r e l a t i n g the degree of d e g e n e r a t i o n of the syngeneic and a l l o g e n e i c t h y r o i d g r a f t s  21  Scores o f d e g e n e r a t i o n of i s o g r a f t s and a l l o g r a f t s of t h y r o i d s removed on d i f f e r e n t days f o l l o w i n g transplantation  22  Number and k i n d o f g r a f t s from which the c e l l s were i s o l a t e d  37  infiltrating  IV.  Number of g r a f t s prepared  V.  Summary o f the c h a r a c t e r i s t i c s o f the c e l l types i s o l a t e d from the s k i n a l l o g r a f t s and viewed w i t h the l i g h t microscope  43  Number and p e r c e n t o f the t o t a l number o f c e l l s counted o f each c e l l type on mutant and non-mutant h o s t s on g i v e n days f o l l o w i n g t r a n s p l a n t a t i o n . . . .  44  Chi-square v a l u e s and p r o b a b i l i t y t h a t the f r a c t i o n o f each c a t e g o r y of l e u k o c y t e i s the same i n s k i n a l l o g r a f t s on mutant and non-mutant h o s t s on g i v e n days f o l l o w i n g transplantation ...  48  S c o r i n g system which i n d i c a t e s the appearance o f the epidermis  56  E s s e n t i a l f e a t u r e s of the u l t r a s t r u c t u r e o f each c e l l type found w i t h i n the s k i n a l l o g r a f t s i t e on both mut a n t and non-mutant h o s t s  89  C e l l types i d e n t i f i e d w i t h i n the s k i n a l l o g r a f t s i t e i n a number of e l e c t r o n microscope s t u d i e s  98  VI.  VII.  VIII.  IX.  X.  f o r e l e c t r o n microscopy  37  ACKNOWLEDGMENT  I would l i k e t o thank Dr. A.B. Acton f o r h i s h e l p and encouragement d u r i n g the course o f t h i s  study.  INTRODUCTION  Despite intensive research our  i n the f i e l d o f t r a n s p l a n t a t i o n  u n d e r s t a n d i n g o f the mechanism of homograft r e j e c t i o n i s s t i l l  plete.  Always a s s o c i a t e d  w i t h the n e c r o s i s of t r a n s p l a n t e d  i n f i l t r a t i o n of mononuclear c e l l s a r i s e s as and  immunity,  i n t o the g r a f t .  tissue i s  However,  the  uncertainty  to the exact i d e n t i t y of these c e l l s , t h e i r mechanism of  a c t u a l r o l e i n the d e s t r u c t i o n of the g r a f t e d t i s s u e .  incom-  The  action  approach  taken i n t h i s i n v e s t i g a t i o n of the phenomenon i n v o l v e s a comparison of D o m i n a n t s - s p o t t i n g mutant and  the  the c o r r e s p o n d i n g non-mutant o f WBB6 mice.  TRANSPLANTATION IMMUNITY The  t r a n s f e r of t i s s u e between g e n e t i c a l l y i n c o m p a t i b l e i n d i v i d u a l s  e l i c i t s a number of immunological responses w i t h i n the h o s t and m a l l y r e s u l t i n the d e s t r u c t i o n by  the h o s t of the  immune responses e l i c i t e d are b o t h humoral and which e i t h e r system c o n t r i b u t e s of i n t e n s e  i n v e s t i g a t i o n and  coming c l e a r :  grafted tissue.  cellular.  The  to a l l o g r a f t r e j e c t i o n has  considerable  controversy.  the  c e l l u l a r immune system ( N a j a r i a n  norThe  degree to  been a matter  One  point i s  In many cases the d e s t r u c t i o n of t r a n s p l a n t e d  mediated s o l e l y by  will  and  be-  tissue i s Simmons,  1972;  Winn, 1970). The  first  i n d i c a t i o n s of the  importance of the  sponse i n t r a n s p l a n t a t i o n immunity arose from two The  first  i s that n e c r o s i s  of the  c e l l u l a r immune r e -  separate  observations.  g r a f t i s always a s s o c i a t e d  f i l t r a t i o n of h o s t mononuclear c e l l s i n t o the  transplanted  w i t h the i n -  tissue  ( B i l l i n g h a m and S i l v e r s , 1971).  Secondly, a d o p t i v e  t r a n s f e r o f homograft  immunity can o n l y be e f f e c t e d by v i a b l e lymphocytes ( B i l l i n g h a m e t a l . , 1954;  Mitchison,  1954).  Neither  serum n o r n o n - v i a b l e  c e l l s from s e n s i -  t i z e d animals w i l l s u c c e s s f u l l y t r a n s f e r immunity, thus s u g g e s t i n g t h i s t r a n s f e r i s dependent on t h e continued c e l l s w i t h i n the second h o s t preformed  that  a c t i v i t y o f t h e immunized  and i s n o t due simply  to p a s s i v e  transfer of  antibody.  Other o b s e r v a t i o n s  now demonstrate t h a t the c e l l u l a r immune response  a l o n e can b r i n g about the d e s t r u c t i o n o f some g r a f t e d t i s s u e s . ces where the h o s t  i s unable ( o r has a reduced c a p a c i t y )  In instan-  t o produce humoral  a n t i b o d y , b u t the c e l l u l a r immune response i s " i n t a c t " , as i n the case o f a bursectomized chicken ceeds n o r m a l l y  o r f e t a l lamb, the r e j e c t i o n o f the a l l o g r a f t  ( S i l v e r s t e i n e t a l . , 1964; Niederhuber e t a l . , 1971).  thermore, i t may be noted t h a t d u r i n g  proFur-  the r e j e c t i o n o f g r a f t e d t i s s u e s the  c e l l u l a r immune response, u n l i k e the humoral immune r e s p o n s e , can always be demonstrated as a c t i v e and capable o n l y o f the d e s t r u c t i o n o f the t r a n s planted  tissue.  Often,  depending on the type of t i s s u e g r a f t e d , the produc-  t i o n o f humoral a n t i b o d i e s  i n response t o the a l l o g r a f t cannot be demonstra-  t e d , o r e l s e i t does not c o i n c i d e w i t h g r a f t d e s t r u c t i o n 1973). ated  (Burger e t a l . ,  F u r t h e r , w h i l e t r a n s f e r o f s e n s i t i z e d lymphocytes r e s u l t s i n a c c e l e r  a l l o g r a f t r e j e c t i o n ( B i l l i n g h a m et_ al_. , 1954) o r the breakdown o f i t s  " p r i v i l e g e d " s t a t u s i n a t o l e r a n t host  ( B i l l i n g h a m e t a l . , 1963) , t r a n s f e r  of a s p e c i f i c a n t i b o d y does n o t always have the same e f f e c t on the r e j e c t i o n response ( N a j a r i a n and Simmons, 1972).  The t r a n s f e r r e d a n t i b o d y may a c c e l e r  a t e r e j e c t i o n ( T i n g e t a l . , 1971; S t e t s o n ,  1963; Hasek e t a l . , 1969), o r  3  have no e f f e c t ( S t e i n m u l l e r , 1962; H a r r i s , e t " a l . , 1972), o r r e s u l t i n enhancement (Cohen e t a l . , 1971). t r u c t i o n proceeds u n a l t e r e d ,  Thus, s i n c e t h e tempo o f g r a f t des-  ( H a r r i s , e t a l . , 1971, 1972) w h i l e the  o c c u r r e n c e and a c t i v i t y o f humoral a n t i b o d i e s i s suggested t h a t the humoral a n t i b o d i e s  can v a r y  considerably, i t  are not o b l i g a t o r y p a r t i c i p a n t s  i n the d e s t r u c t i o n o f g r a f t e d t i s s u e s by t h e h o s t .  On the o t h e r hand, i t  has been demonstrated t h a t the presence o f an i n t a c t c e l l u l a r immune s y s tem  i s e s s e n t i a l f o r the a l l o g r a f t response t o proceed n o r m a l l y , b l o c k a g e  of i t s a c t i v i t y r e s u l t i n g i n p r o l o n g e d g r a f t s u r v i v a l ( B i l l i n g h a m and Silvers, The  1971). mechanism by which the c e l l u l a r immune system mediates the des-  t r u c t i o n o f the g r a f t e d t i s s u e i s n o t c l e a r l y understood. questions  uncertainty  Among  e x i s t s as t o the i d e n t i t y o f the c e l l s  other  infiltrating  the g r a f t , t h e i r mechanism o f a c t i o n and a c t u a l r o l e i n t h e d e s t r u c t i o n o f the  grafted tissue.  genous. other  Further,  The p o p u l a t i o n  of i n f i l t r a t i n g c e l l s i s quite  hetero-  the c e l l types and t h e i r numbers v a r y depending on, among  t h i n g s , the time f o l l o w i n g t r a n s p l a n t a t i o n a t which the p o p u l a t i o n i s  examined, the type o f t i s s u e which i s g r a f t e d and the s p e c i e s Furthermore, the exact incomplete.  involved.  i d e n t i f i c a t i o n o f the c e l l s i s d i f f i c u l t and remains  Small lymphocytes, macrophages, plasma c e l l s , b a s o p h i l i c  e o s i n o p h i l s and'neutrophils  have been c i t e d as p a r t o f the c e l l  cells,  population  e n t e r i n g r e n a l (Feldman and Lee, 1967; Kountz e t a l . , 1963; L i n q u i s t e t a l . , 1971), s k i n ( T i t u s and S h o r t e r , 1969; but  1962; Simar and B e t z , 1970; Weiner, e t a l . ,  A l l e g r a e t a l . , 1968) and tumour (Journey and Amos, 1962), a l l o g r a f t s , these may not be p r e s e n t  i n a l l types o f g r a f t s , and o t h e r  o f c e l l s w i l l almost c e r t a i n l y be added to t h i s  list.  categories  4  Thus f a r at l e a s t , the mononuclear e f f e c t o r c e l l p o p u l a t i o n i n f i l t r a t i n g the g r a f t has been s e p a r a t e d i n t o two  subpopulations.  s e n s i t i z e d lymphocyte p o p u l a t i o n , which a r i s e s i n response of  A  specifically  to the  presence  the g r a f t a n t i g e n s ; and, a n o n - s p e c i f i c e f f e c t o r c e l l p o p u l a t i o n , p r o -  b a b l y p r i m a r i l y o f monocyte-macrophage l i n e a g e ( P r e n d e r g a s t ,  1964;  Kongshavin and Lapp, 1973). I n f o r m a t i o n as to the r o l e i n the a l l o g r a f t response a c t i o n of each c e l l  and mechanism o f  type and e l u c i d a t i o n of t h e i r i n t e r a c t i o n w i t h o t h e r  c e l l s a t the g r a f t s i t e a r i s e s from b o t h i n v i v o and i n v i t r o s t u d i e s . However, the e x p e r i m e n t a l t e c h n i q u e s u t i l i z e d have i n h e r e n t problems which prevent a complete understanding response. cell  of the e f f e c t o r phase of the  Many of the i n v i v o s t u d i e s attempt  allograft  to a l t e r the p o o l of  certain  types w i t h i n an animal and then note the e f f e c t o f t h i s change on  allograft  response.  However, some of the techniques which a r e u t i l i z e d  the "background" p h y s i o l o g y of the animal as w e l l as the p o o l of the type under i n v e s t i g a t i o n . to  Thus, f o r example, techniques  alter  cell  commonly employed  i n v e s t i g a t e the r o l e and a c t i v i t y o f the lymphocyte i n the a l l o g r a f t r e -  sponse i n c l u d e t o t a l body x - i r r a d i a t i o n , thymectomy, t h o r a c i c duct and  the  a d m i n i s t r a t i o n of anti-lymphocyte  death due  s e r a , many of which r e s u l t i n animal  to r u n t d i s e a s e ( N a j a r i a n and  S i l v e r s , 1971).  on the a l l o g r a f t  Simmons, 1972;  B i l l i n g h a m and  C a u t i o n must be e x e r c i s e d when a t t e m p t i n g  the e f f e c t o f such treatments  drainage  on a p a r t i c u l a r c e l l  to c o r r e l a t e  type w i t h t h e i r  effect  response.  I n f o r m a t i o n as to the mechanism by which the i n f i l t r a t i n g c e l l s a t the g r a f t s i t e i n t e r a c t w i t h each o t h e r and  the donor t i s s u e , and  mediate  5  the d e s t r u c t i o n  of the g r a f t e d  i n vivo studies,  since  a means of s t u d y i n g  t i s s u e , has  been d i f f i c u l t  to o b t a i n  the e x p e r i m e n t a l systems a v a i l a b l e do not  the c e l l a c t i v i t y i n v i v o .  provide  Because of t h i s , numerous  i n v i t r o systems have been developed i n an attempt to i n v e s t i g a t e e f f e c t o r phase o f c e l l - m e d i a t e d c e l l types. and  S i n c e the  immunity at the  c e l l s are  c e l l s under i n v e s t i g a t i o n are  capable of d o i n g and  they are p e r f o r m i n g w i t h i n I t would be  the  informative  the p r o p e r t y o f one  of the  graft  do not  i n d i c a t e what  then be  able  surrounding t i s s u e s .  the e f f e c t t h i s a l t e r a t i o n has tissue.  o f the  surrounding t i s s u e .  Just  on  the  One  alter-  would  cell-mediated  such an a l t e r a t i o n may  exist  "background"  I f the p h e n o t y p i c c h a r a c t e r i s t i c s  c e l l s a r i s i n g from a mutation r e s u l t e d i n an a l t e r a t i o n i n the  mediated immune response and  i f the manner o f t h i s a l t e r a t i o n c o u l d  mined, t h i s would p r o v i d e i n f o r m a t i o n  as  to the  c e l l n o r m a l l y makes toward the d e s t r u c t i o n b l y , i t s mechanism of  THE  activity  g r a f t s i t e without  i n a m u t a t i o n t h a t a f f e c t s the b l o o d c e l l s w i t h o u t changing the p h y s i o l o g y of the  examples  to manipulate or a l t e r  i n f i l t r a t i n g the  "background" p h y s i o l o g y o f the  immune response to t r a n s p l a n t e d  possible  animal  site.  i n g the  to f o l l o w  removed from the  are o n l y  i f i t were p o s s i b l e  cells  the  l e v e l of s e p a r a t e i n d i v i d u a l  manipulated i n t i s s u e c u l t u r e , these s t u d i e s  of what the  by  contribution  o f the g r a f t e d  the  be  celldeter-  effected  t i s s u e and,  possi-  action.  W MUTANT An  i n t e r e s t i n the homograft response o f the W mutant i s aroused  cause of the known p h e n o t y p i c c h a r a c t e r i s t i c s of the W gene and bility  t h a t the  underlying  defect  may  a l s o a f f e c t the  the  immune system.  be-  possiMuta-  6  t i o n s o f the W l o c u s i n mice a r e common (Green, 1966). of mutant a l l e l e s which e x i s t , two o f which are the W, a l l e l e and the W ,  v i a b l e dominant s p o t t i n g , a l l e l e .  V  There are a number dominant  spotting,  The presence i n mice  o f these a l l e l e s at the d o m i n a n t - s p o t t i n g l o c u s i n the homozygous, W /W V  o r W/W,  o r h e t e r o z y g o u s , W/W, V  V  c o n d i t i o n r e s u l t s i n a " b l a c k - e y e d , white  c o a t " p i g m e n t a t i o n , severe m a c r o c y t i c anemia and s t e r i l i t y .  A l t h o u g h the  exact s t r u c t u r a l or enzymatic d e f e c t caused by the W gene i s unknown, a n a l y s i s of the e x p e r i m e n t a l d a t a c o l l e c t e d thus f a r suggests an a f f e c t on the m i g r a t i o n or p r o l i f e r a t i o n , o r b o t h , o f the p r i m i t i v e c e l l affected  types o f the  tissues.  I t has been shown by t r a n s p l a n t a t i o n experiments  t h a t the cause of the  b l a c k - e y e d , white coat c h a r a c t e r i s t i c o f the W mutant r e s i d e s i n a d e f e c t i n the stem m e l a n o b l a s t .  An i n t r i n s i c p r o p e r t y of t h i s c e l l r e t a r d s or i n -  h i b i t s e i t h e r i t s m i g r a t i o n from the n e u r a l c r e s t , i t s p r o l i f e r a t i o n , i t s f i n a l d i f f e r e n t i a t i o n i n t o a melanocyte, and Green,  1968;  Mayer,  or a combination of these (Mayer  1970).  H i s t o l o g i c a l s t u d i e s i n d i c a t e the s t e r i l i t y of the W mutant may  be  due  to a d e l e t e r i o u s e f f e c t o f the W gene on e i t h e r the m i g r a t i o n or p r o l i f e r a t i o n , o r b o t h , of the p r i m o r d i a l germ c e l l s .  Most o f t h e s e c e l l s do not  m i g r a t e from the y o l k sac to the g e n i t a l r i d g e and, any t h a t do, f a i l p r o l i f e r a t e en r o u t e (Mintz and R u s s e l l ,  to  1957).  The W mutant a l s o shows severe m a c r o c y t i c anemia and i s extremely radiosensitive  ( R u s s e l l and B e r n s t e i n , 1966,  1967).  Both of t h e s e c h a r a c -  t e r i s t i c s have been a t t r i b u t e d to a g e n e t i c d e f e c t i n h e m a t o p o i e s i s , more s p e c i f i c a l l y , i n the p r o l i f e r a t i o n of the h e m a t o p o i e t i c stem c e l l s .  Examina-  t i o n of the b l o o d - f o r m i n g t i s s u e s o f the mutant by t r a n s p l a n t a t i o n  experiments  7 and h i s t o l o g i c a l l y w i t h r a d i o a c t i v e t r a c e r s r e v e a l s a r e t a r d a t i o n i n proliferation  and d i f f e r e n t i a t i o n  t h r o p o i e s i s and l e u k o p o i e s i s  i n hematopoiesis, i n c l u d i n g both e r y -  (Borghese, 1959; R u s s e l l e t a l . , 1953;  Altmann and R u s s e l l , 1964; R u s s e l l and B e r n s t e i n , 1967; M c C u l l o c h e t a l . , 1964<, Bennett e t a l . , 1968; and L e w i s ' e t a l . , 1967). Thus, the e f f e c t  o f the W gene appears e a r l y i n mouse development i n  several differentiating  stem c e l l l i n e s .  The a b i l i t y of these c e l l s t o  m i g r a t e o r p r o l i f e r a t e , o r b o t h , d u r i n g development, the +/+  l i t t e r m a t e s , i s retarded or i n h i b i t e d .  t h a t the W gene a l s o a l t e r s  There i s some  the a c t i v i t y o f some of the more  h e m a t o p o i e t i c c e l l types i n the a d u l t W/W  V  mouse i s : d e f i c i e n t  compared to those o f  mouse.  indication differentiated  F o r example, the  i n i t s response t o e r y t h r o p o e i t i n , i n d i c a t i n g  W/W  V  a defect  i n the e r y t h r o p o e i t i n - r e s p o n s i v e c e l l l i n e ( R u s s e l l and B e r n s t e i n ,  1966).  F u r t h e r , Shearer and Cudkowicz (1967) found the mutant had a reduced number o f p r e c u r s o r c e l l s to the a n t i b o d y - f o r m i n g c e l l s , s u g g e s t i n g reduced p r o l i f e r a t i o n i n the l e u k o c y t i c p r e c u r s o r p o o l . vitro  t h e W/W  V  of W/W  V  lymphocyte  Wong (1969) found t h a t i n  m i g r a t e s s i g n i f i c a n t l y more s l o w l y on a background  c e l l s than the +/+  lymphocytes  on a background  o f +/+ c e l l s .  Thus i t appears that the presence of the W gene, i n an unknown manner, can r e s u l t  i n a r e d u c t i o n o r i n h i b i t i o n i n the p r o l i f e r a t i o n o r m i g r a t i o n  of b l o o d c e l l s a t d i f f e r e n t  stages o f t h e i r  differentiation.  The h o s t e f f e c t o r c e l l s i n f i l t r a t i n g an a l l o g r a f t a r e b l o o d c e l l s which are b o t h m i g r a t o r y and p r o l i f e r a t i v e . not  The q u e s t i o n a r i s e s as t o whether o r  t h e i r p a t t e r n o f response t o an a l l o g r a f t o f t i s s u e a l t e r s  sence o f t h e W gene. response of the W/W  V  McNay (1974) has demonstrated  i n the p r e -  t h a t the s k i n  homograft  mutant d i f f e r s s i g n i f i c a n t l y from t h a t o f i t s W/+,  +/+  8 and W/+ V  littermates.  H i s t o l o g i c a l observations of skin a l l o g r a f t s  indicate that the mutant rejects i t s grafts s i g n i f i c a n t l y faster than i t s non-mutant littermates.  Another experiment, involving the uptake  of t r i t i a t e d thymidine by the peripheral blood c e l l s during the a l l o g r a f t response, indicates that i n the W/W  mouse a larger number of activated  lymphocytes are present i n the blood at an e a r l i e r stage i n graft reject i o n than i n the blood of the W/+, +/+ and W/+ V  It appears, then, that i n the W/W  V  mice at the same stage.  mouse the homograft response i s  s i g n i f i c a n t l y altered r e l a t i v e to the response i n normal mice.  The ques-  t i o n arises as to what the basis of this difference i n responsiveness i s . An answer could provide information as to the c e l l u l a r mechanisms respons i b l e for the r e j e c t i o n of transplanted  tissue i n a l l higher mammals.  PROPOSAL It appears, then, that the W gene affects either the migratory or p r o l i f e r a t i v e properties  of some blood c e l l s , including the lymphocytes.  Further, i n the presence of the W gene the skin a l l o g r a f t response, of which the lymphocyte i s an important component, i s altered s u f f i c i e n t l y to r e s u l t i n reduced graft s u r v i v a l time.  A preliminary  approach to the  investigation of the cause of the altered c e l l u l a r immune response of the mutant w i l l involve a study of c e l l s at the graft s i t e during the a l l o g r a f t response.  In other words, can the difference between the mutant and non-  mutant i n the cell-mediated  immune response be correlated with a difference  i n the c e l l populations i n f i l t r a t i n g the transplanted proposed therefore  tissue.  It i s  to investigate the homograft response at the h i s t o l o g i -  c a l and u l t r a s t r u c t u r a l l e v e l s .  9  Such an i n v e s t i g a t i o n has material  to be  transplanted  which w i l l a l l o w  should  also allow  First,  have a "background" c e l l  f o r the d e t e r m i n a t i o n  the  target  population cell  stimulation.  of t h e i r k i n e t i c s .  Secondly,  s e n s i t i v e enough so that i f a d i f f e r e n c e does  e x i s t between the mutant and  An  problems.  the implant i n response to a n t i g e n i c  the system used must be  the  inherent  f o r the easy i d e n t i f i c a t i o n of the v a r i o u s host  types t h a t i n f i l t r a t e I t should  two  non-mutant i n the types of c e l l s  g r a f t , i n t h e i r movement o r i n t h e i r number, i t would be i d e a l experiment would be  stance i n t o an animal.  to implant an a c e l l u l a r , v e r y  Upon removal o f the m a t e r i a l  c e l l s found w i t h i n i t c o u l d e a s i l y be  infiltrating detected.  a n t i g e n i c sub-  from the host  any  i d e n t i f i e d as h a v i n g i n f i l t r a t e d  the  implant. An  amorphous substance which was  considered  i s collagen.  l i t e r a t u r e review i n d i c a t e d i t would be u n s u i t a b l e  However, a  f o r s e v e r a l reasons.  obtain collagen s u i t a b l e f o r t r a n s p l a n t a t i o n studies i s a tedious r e q u i r i n g i t s i s o l a t i o n , p u r i f i c a t i o n and 1962).  Then, whether o r not  matter of c o n s i d e r a b l e v a r i e t y of host cells,  controversy.  procedure  r e c o n s t i t u t i o n ( G r i l l o and  this p u r i f i e d collagen i s antigeneic Subcutaneous i m p l a n t a t i o n  responses i n c l u d i n g e n c a p s u l a t i o n  To  Gross,  is a  elicits  of the implant by  a  host  f o l l o w e d by replacement or breakdown of the m a t e r i a l w i t h i n 45  days.  However, the h o s t s are not  s e n s i t i z e d to the c o l l a g e n , i n d i c a t i n g the  col-  lagen i s not  t h e r e f o r e not  elici-  ted.  a n t i g e n i c and  In c o n t r a s t , o t h e r  system w h i l e others 1971;  K i r r a n e and  responsible  f o r the response  studies i n d i c a t e i t stimulates  the c e l l u l a r immune  suggest the s t i m u l a t i o n of the humoral ( L i n d s l e y et a l . ,  Glynn, 1968).  10  Thus, two o t h e r t a r g e t t i s s u e s were c o n s i d e r e d whose presence i n a l l o g e n e i c h o s t s are known to e l i c i t and  skin.  vestigate  This  a c e l l u l a r immune r e s p o n s e :  Thyroid  t h e s i s d e a l s w i t h two e x p e r i m e n t a l systems s e t up t o i n -  the cause o f the d i f f e r e n c e  tween the W mutant and the non-mutant.  i n the response t o a l l o g r a f t s beThe f i r s t  involves  t i o n o f t h y r o i d , both subcutaneously and i n t o the s p l e e n .  the i m p l a n t a I t had been  hoped to demonstrate a d i f f e r e n c e between the c e l l u l a r immune responses o f the mutant and non-mutant i n a system d i f f e r e n t from the s k i n a l l o g r a f t . However, as w i l l be e x p l a i n e d  i n the a p p r o p r i a t e  s e c t i o n , t h e r e were many  problems w i t h t h i s system and i t was s u b s e q u e n t l y abandoned. system i n v o l v e s the  transplantation  cells infiltrating  g r a f t beds.  The second  o f s k i n , i n c l u d i n g enzymatic i s o l a t i o n o f  the g r a f t s and an e l e c t r o n microscope study o f the  11 IMPLANTATION OF THYROID  INTRODUCTION The  r e s u l t s of some e a r l y s t u d i e s on the t r a n s p l a n t a t i o n of the  t h y r o i d suggested  t h a t i t might s e r v e as the b a s i s f o r a m i c r o s c o p i c  comparison between the c e l l mediated immune responses mutant and non-mutant h o s t s .  e l i c i t e d by  the  I t had been found i n r a t s and mice t h a t  t h y r o i d i s o g r a f t s c o u l d be t r a n s p l a n t e d to any p a r t of the body and t a i n the p h y s i o l o g y and m o r p h o l o g i c a l transplanted tissues 1955).  (Bennett  c h a r a c t e r i s t i c s found  and Gordman, 1951;  i n the  Dempster and  F u r t h e r , c o n t r a r y to g e n e r a l b e l i e f , a t h y r o i d d e f i c i e n c y i n the  (Dempster and Doniach, 1955;  Gibbs and F i e l d , 1971).  o t h e r hand, a l l o g r a f t s had been found o n l y to succumb l a t e r to an obscure 1955).  non-  Doniach,  h o s t i s not e s s e n t i a l f o r the take and s u r v i v a l of the t h y r o i d implant  re-  Concerning  to "take, r e g e n e r a t e  On  the  and f u n c t i o n ,  r e a c t i o n " (Dempster and  Doniach,  t h i s r e a c t i o n of the host to the a l l o g e n e i c t h y r o i d  t i s s u e , i t i s s t a t e d t h a t "lymphocytic  i n f i l t r a t i o n i s the  More r e c e n t work done by Gibbs and F i e l d o f the t h y r o i d subcutaneously h o s t r a t s and  tissue  rule."  (1971) i n v o l v e d i m p l a n t a t i o n  i n the t h i g h of both syngeneic  and a l l o g e n e i c  d e t e r m i n a t i o n of the v i a b i l i t y o f the g r a f t by i t s a b i l i t y  to c o n c e n t r a t e r a d i o i o d i n e .  N i n e t y p e r c e n t o f the i s o g r a f t s t r a n s p l a n t e d  by the i n c i s i o n method (used here) were f u n c t i o n i n g a f t e r 29 days i n the host. and  T h i s i s i n c o n t r a s t to the method i n v o l v i n g the use o f a t r o c h a r  cannula which showed o n l y f i f t y - t h r e e p e r c e n t s u c c e s s .  a l l o g r a f t s were f u n c t i o n i n g a f t e r 19 days i n the h o s t . t r a n s p l a n t i n g the t h y r o i d w i t h r e a s o n a b l e  None of the  Thus, a method o f  s u r v i v a l of the i s o g r a f t s i s  12  available.  F u r t h e r , the homograft response  19 days which i s s u f f i c i e n t  occurs w i t h i n a p e r i o d of  time to view the v a r i o u s s t a g e s o f i n f i l t r a -  tion. The  c e l l u l a r c o m p o s i t i o n o f the t h y r o i d p r o v i d e s good background  material f o r studying i n f i l t r a t i o n . are f o l l i c l e s which a r e packed f a i r l y an o u t e r and i n n e r c a p s u l e .  The s t r u c t u r a l u n i t s o f the t h y r o i d c l o s e l y t o g e t h e r and surrounded by  The i n n e r c a p s u l e c o n s i s t s o f f i b r o e l a s t i c  c o n n e c t i v e t i s s u e and forms the septae o f the g l a n d , c a r r y i n g b l o o d and lymphatic v e s s e l s and nerves  throughout  the organ.  The e p i t h e l i a l w a l l s  of the f o l l i c l e s a r e composed p r i m a r i l y o f low c u b o i d a l e p i t h e l i a l i n v o l v e d i n c o l l o i d s e c r e t i o n and a few p a r a f o l l i c u l a r c e l l s The  cells  (Ham, 1969).  types o f c e l l s b e l i e v e d t o be i n v o l v e d i n immune responses  a r e not  n o r m a l l y p r e s e n t i n the t h y r o i d , and any i n f i l t r a t i n g c e l l s moving i n t o the f o l l i c l e s would be e a s i l y i d e n t i f i e d as f o r e i g n to the gland  tissue.  However, as w e l l be shown i n the r e s u l t s s e c t i o n , there were so many p r o blems w i t h t h i s system t h a t i t was abandoned.  13  MATERIALS AND METHODS  Implantation of thyroid Male and female mice o f the s t r a i n s DBA/2 and WBB6, which in  differ  the a l l e l e s a t the H-2 l o c u s , were used i n t h e s e experiments.  The  r e c i p i e n t s , always o f the same sex as the donors, were 14 to 16 weeks of  age a t the time of the experiment. While the donor animal was under Nembutal a n a e s t h e s i a , the l a r y n x  and p a r t o f the t r a c h e a , w i t h the t h y r o i d a t t a c h e d , were removed through an i n c i s i o n a l o n g the v e n t r a l m i d l i n e i n the neck r e g i o n and p l a c e d i n Ringers s o l u t i o n .  Then, under a d i s s e c t i n g m i c r o s c o p e , t h e t h y r o i d t o be  used f o r t r a n s p l a n t a t i o n was d i s s e c t e d away from t h e t r a c h e a . of  Two s e r i e s  experiments were c a r r i e d o u t . I.  The f i r s t  s e r i e s o f experiments i n v o l v e d i m p l a n t a t i o n o f DBA/2  t h y r o i d l o b e s subcutaneously i n the t h i g h o f (1) DBA/2 ( s y n g e n e i c g r a f t s ) or  (2) WBB6 ( a l l o g e n e i c g r a f t s ) mice.  A t h y r o i d l o b e was p l a c e d i n a sub-  cutaneous pocket made by p l a c i n g s h a r p l y p o i n t e d s c i s s o r s i n t o a s m a l l i n c i s i o n i n the s k i n and opening the b l a d e s t o s e p a r a t e the s k i n from the u n d e r l y i n g body w a l l .  The wound was c l o s e d w i t h M i c h e l c l i p s .  The g r a f t s  were h a r v e s t e d , f o r (1) t h e s y n g e n e i c g r a f t s , a t 4, 6, 8 and 12 days  fol-  lowing t r a n s p l a n t a t i o n and, f o r (2) the a l l o g e n e i c g r a f t s , a t 3, 4, 6, 8, 10, 12, 13, 14 and 17 days f o l l o w i n g t r a n s p l a n t a t i o n . g r a f t s were f i x e d i n Bouin's f l u i d ,  When removed, the  embedded i n p a r a f f i n wax, s e c t i o n e d ,  and s t a i n e d w i t h haematoxylin and e o s i n . II.  The second s e r i e s o f experiments i n v o l v e d i m p l a n t a t i o n o f t h y r o i d  l o b e s i n t o the s p l e e n s (see page 25) o f r e c i p i e n t mice o f t h e same s t r a i n  14  as the donor (syngeneic g r a f t s ) . and  i n the u n d e r l y i n g  The  surface  The  f i n wax, For  10,  the  and  35,  The  spleen.  inserted  into  g r a f t s were removed  f i x e d i n Bouin's f l u i d , embedded i n  s t a i n e d w i t h haematoxylin and  same manner as the  skin  s t i t c h e d c l o s e d , w h i l e the more s u p e r f i -  comparison, a t h y r o i d gland was  pared i n the  t h y r o i d l o b e was  closed with Michel c l i p s .  18-21, 33 and  sectioned  cut and  body w a l l was  c i a l s k i n wound was days 1-6,  body w a l l to a l l o w f o r the exposure of the  of the s p l e e n was  the opening.  S m a l l i n c i s i o n s were made i n the  thyroid  paraf-  eosin.  removed from a DBA grafts.  on  donor and  pre-  15  RESULTS  Normal  thyroid  A b r i e f d e s c r i p t i o n i s g i v e n here o f the m i c r o s c o p i c s t r u c t u r e o f the normal t h y r o i d microscopy.  gland removed from a DBA donor and p r e p a r e d f o r l i g h t  The t h y r o i d  gland c o n s i s t s  of i r r e g u l a r lobules,  called  f o l l i c l e s , which a r e packed c l o s e t o g e t h e r and a r e surrounded by a f i b r o u s c a p s u l e ( f i g u r e 1A).  G e n e r a l l y , the c e n t r a l f o l l i c l e s a r e s m a l l e r i n  s i z e than the f o l l i c l e s i n the p e r i p h e r a l cle consists  a r e a o f the g l a n d .  Each  o f a s i n g l e l a y e r o f e p i t h e l i a l c e l l s which r e s t on a base-  ment membrane and surround a c e n t r a l lumen f i l l e d w i t h c o l l o i d The  e p i t h e l i a l w a l l i s composed o f 2 c e l l t y p e s ; f o l l i c u l a r  and  a few p a r a f o l l i c u l a r ( l i g h t ) c e l l s .  cubodial  o r columnar.  region  the b a s a l  There are one to two c l e a r l y d e l i n e a t e d geneous and i s s l i g h t l y  region  nucleoli.  Their  chromatin.  The cytoplasm i s homo-  basophilic.  membrane between the f o l l i c l e c e l l s .  a l o n g the basement  They show no p o l a r i t y :  open to the lumen o f the f o l l i c l e s .  c o l l o i d , within  region  and o c c u p y i n g two  f i n e l y dispersed  There a r e o n l y a few p a r a f o l l i c u l a r c e l l s l o c a t e d  l a r g e r than the f o l l i c l e  cells  The f o l l i c l e c e l l s a r e e i t h e r  t h i r d s o f the c e l l , a r e s p h e r i c a l and c o n t a i n  The  (dark)  next to the basement membrane.  nuclei, situated centrally or within  the c e l l s u r f a c e  (figure IB).  They have a d e f i n i t e p o l a r i t y : an a p i c a l  toward the lumen and b a s a l  is  folli-  c e l l s and have a c l e a r e r  A t no p o i n t  These c e l l s a r e  cytoplasm.  the lumen o f the f o l l i c l e , i s an amorphous, homo-  geneous f l u i d which i s a c i d o p h i l i c .  Cellular inclusions within  the c o l l o i d  16  F i g u r e LA: This  Normal  gland was  microscopy. thyroid  thyroid removed from a DBA  donor and  I t i s c o n s i d e r e d to show the  t i s s u e , which i s d e s i g n a t e d by  F i g u r e IB: Higher m a g n i f i c a t i o n N o t i c e the  organization  prepared f o r  "normal" appearance of  a score of 0 (table I ) . X  of normal t h y r o i d  of the  light  follicles  (F).  51.  tissue Each c o n s i s t s  of  a s i n g l e l a y e r o f e p i t h e l i a l c e l l s which surround a c e n t r a l lumen f i l l e d with c o l l o i d c e l l s and and  (C).  Note a l s o the  d e f i n i t e p o l a r i t y of  t h e i r spherical n u c l e i containing  1 or 2 c l e a r l y delineated  nucleoli. X  f i n e l y dispersed 138.  the chromatin  17  are sometimes p r e s e n t but The  separation  a serrated O f t e n the  of the  are p r o b a b l y due  c o l l o i d from the  c o l l o i d of the  follicles  to f o l l i c u l a r  follicle cells,  edge, i s an a r t i f a c t produced by central f o l l i c l e s  c o l l o i d of the p e r i p h e r a l The  only  damage.  g i v i n g the  the p r e p a r a t i o n  procedure.  s t a i n s more d a r k l y  than  and  t h y r o i d the  Thyroid  are surrounded by  The DBA  nerves throughout the components o f the  g r a f t s implanted  an i n n e r c a p s u l e , or septum, which  (1.)  Thyroid  septum are d i f f i c u l t  mice, and  syngeneic g r a f t s show v a r y i n g  few  of  follicles  both s y n g e n e i c g r a f t s , t h y r o i d to WBB6 mice.  t h a t of the  'normal u n t r a n s p l a n t e d t h y r o i d  degrees o f n e c r o s i s .  Generally,  the gland.  only  t h e r e s i n g l y or i n groups of 6 to 8 ( f i g u r e 2A and  follicles  a  at the p e r i p h e r y of the gland appear s t r u c t u r a l l y  found i n a normal t h y r o i d g l a n d are u s u a l l y  g r a f t s , replaced  an amorphous m a t e r i a l  and  by  e i t h e r degenerating f o l l i c l e s  a few  That some of these i n n e r gested by  made i n c l u d e d  between the m i c r o s c o p i c appearance of  The  from the  see.  isografts  t h y r o i d i s o g r a f t and  inner  of normal  subcutaneously.  DBA  normal, o c c u r r i n g  to  a l l o g e n e i c g r a f t s , DBA  There i s a marked c o n t r a s t  the  In t i s s u e s e c t i o n s  lymphatic  subcutaneously  These g r a f t s were implanted  The  organ.  f i r s t s e r i e s of t r a n s p l a n t s  t h y r o i d to DBA  the  follicles.  c o n s i s t s of f i b r o e l a s t i c c o n n e c t i v e t i s s u e , c a r r y i n g b l o o d and vessels  colloid  scattered  follicles  t h e i r s t r u c t u r a l appearance.  cells  (figure  appear to be The  3A).  absent  ( f i g u r e 2B)  or  3B).  d e g e n e r a t i n g i s sug-  e n d o t h e l i a l w a l l no  longer  18  F i g u r e 2A:  Thyroid  graft r e c e i v i n g a score of 1  Note t h a t o n l y the f o l l i c l e s a t the p e r i p h e r y of the gland  appear  s t r u c t u r a l l y normal, o c c u r r i n g t h e r e s i n g l y or i n groups of 6 to The  inner f o l l i c l e s  found i n normal t h y r o i d ( f i g u r e 1A)  r e p l a c e d by d e g e n e r a t i n g  F i g u r e 2B: Notice  follicles,  Degenerating  X  51.  t h a t the e n d o t h e l i a l w a l l no l o n g e r e n c l o s e s  tened, w i t h very l i t t l e 138.  are absent,  follicles  i s disrupted at various p o i n t s .  nuclei.  X  8.  the c o l l o i d  Most o f the f o l l i c l e c e l l s a r e  cytoplasm, and  contain i r r e g u l a r l y  and  flat-  shaped  19  F i g u r e 3A:  T h y r o i d g r a f t r e c e i v i n g a score o f 2  Only the f o l l i c l e s a t the p e r i p h e r y of the gland  appear s t r u c t u r a l l y  normal, o c c u r r i n g t h e r e s i n g l y o r i n groups of 6 to 8. licles  The i n n e r  fol-  found i n normal t h y r o i d a r e absent, r e p l a c e d by an amorphous ma-  t e r i a l and a few s c a t t e r e d c e l l s .  F i g u r e 3B:  X 51.  Composition of the c e n t r a l a r e a o f glands  receiving a  score of 2 or 4 Here the c e n t r a l r e g i o n of the gland terial  and a few s c a t t e r e d c e l l s .  X 138.  c o n s i s t s of an amorphous ma-  20  e n c l o s e s the c o l l o i d , and i s d i s r u p t e d at v a r i o u s p o i n t s . cells  are f l a t t e n e d , w i t h v e r y l i t t l e v i s i b l e  r e g u l a r l y shaped, f l a t t e n e d n u c l e i  cytoplasm  and  follicle  contain i r -  ( f i g u r e 2B), a marked c o n t r a s t to the  d i s p e r s e d , round n u c l e i o f the normal f o l l i c l e c e l l s the c o l l o i d n o r m a l l y  The  ( f i g u r e 1A).  Further,  c o n t a i n e d w i t h i n the lumen i s e i t h e r g r a i n y and  weakly s t a i n e d ( f i g u r e 2B) o r absent  ( f i g u r e 3A and  very  3B).  In o t h e r i s o g r a f t s the c e n t r a l r e g i o n c o n s i s t s o f an amorphous, v e r y weakly s t a i n e d m a t e r i a l .  L o c a t e d w i t h i n i t are a few  sometimes, c e l l u l a r d e b r i s ( f i g u r e 3B). easily visible  Areas o f c o n n e c t i v e t i s s u e ,  i n the normal t h y r o i d , are now  r e s t o f the t i s s u e  scattered c e l l s  and, not  e a s i l y d i s t i n g u i s h e d from the  present.  Based on these v a r i o u s s t r u c t u r a l changes i n the i s o g r a f t s , a method of  s c o r i n g the degree o f d e g e n e r a t i o n  r o i d g r a f t s was  of the s y n g e n e i c and a l l o g e n e i c t h y -  established (table I ) .  The  two main a s p e c t s  were (1) the number o f s t r u c t u r a l l y normal f o l l i c l e s p h e r a l r e g i o n o f the gland as a percentage the c o m p o s i t i o n from 0 to  considered  p r e s e n t i n the p e r i -  of the t o t a l r e g i o n and  o f the c e n t r a l r e g i o n of the g l a n d .  The  (2)  s c o r e s range  5.  Based on t h i s system the t h y r o i d i s o g r a f t s were s c o r e d and recorded i n t a b l e I I . degeneration.  the  results  Note t h a t a l l of the i s o g r a f t s show some degree o f  In almost  every gland the c e n t r a l r e g i o n , which i n a normal,  u n t r a n s p l a n t e d gland c o n s i s t s o f f o l l i c l e s an amorphous m a t e r i a l and  a few  ( s c o r e : 0 ) , now  scattered c e l l s  contains only  ( s c o r e s : 2 or 4 ) .  Another c h a r a c t e r i s t i c of these t h y r o i d i s o g r a f t s i s the presence a cellular  c a p s u l e p r o b a b l y a r i s i n g from the host which surrounds  the  of  21  Table I  Score  S c o r i n g System R e l a t i n g the Degree o f D e g e n e r a t i o n of Syngeneic and A l l o g e n e i c T h y r o i d G r a f t s .  Illustrated i n :  % normal f o l l i c l e s present i n periphera l area  the  Composition of the c e n t r a l area of the gland  0  F i g u r e 1A and 5B  100%  normal  1  Figure 2 and 5A  ->50%  degenerating follicles  2  Figure  >50%  amorphous m a t e r i a l with scattered c e l l s  3  <50%  degenerating follicles  4  <50%  amorphous m a t e r i a l with scattered c e l l s  5  3  Figure 4  <50% (usually  410%)  follicles  Table  I I Scores o f d e g e n e r a t i o n o f i s o g r a f t s and a l l o g r a f t s o f t h y r o i d s removed on d i f f e r e n t days f o l l o w i n g t r a n s p l a n t a t i o n  Days G r a f t s were Removed F o l l o w i n g T r a n s p l a n t a t i o n Day Thyroid  graft  1  2  3  Implanted subcutaneous l y (1.) DBA to DBA  5  2  (2.) DBA t o WBB6  Implanted i n t h e spleen (1.) DBA t o DBA or WBB6 t o WBB6  4  3  1  1  1,2  2,3  1  2  6  8  10  12  1,2,4 2,4  2  2,2,2 2,2 2,2 4  2,5  4,4  4,2  13  1  14  17  18  19  20  21  3  0  0  33  2,5 2.2  2,3  3  0.1  ro  23  s u b c u t a n e o u s l y implanted gland.  The  c e l l s are e l o n g a t e d and  orientated  such t h a t t h e i r long axes  are p a r a l l e l to the s u r f a c e  The  around the g r a f t s removed soon a f t e r t r a n s -  capsule  i s not  p l a n t a t i o n but  present  of the  gland.  appear^ r e g u l a r l y around the g r a f t s removed on days 8  and  12. (2.) The the  Thyroid a l l o g r a f t s t h y r o i d a l l o g r a f t s show the  thyroid i s o g r a f t s discussed  these g r a f t s were scored are r e c o r d e d  same s t r u c t u r a l c h a r a c t e r i s t i c s as  i n the p r e v i o u s  section.  as o u t l i n e d i n t a b l e I and  Therefore,  the v a l u e s  instable II.  E x a m i n a t i o n of t a b l e IT r e v e a l s two  important p o i n t s about the  o f d e g e n e r a t i o n o f the a l l o g r a f t s .  The  to the l e n g t h of time the g r a f t has  remained i n the h o s t .  first  trum of i n c r e a s i n g c e l l u l a r i n f i l t r a t i o n and the day  obtained  4 g r a f t through to the day  17  i s t h a t i t i s not  follicular  g r a f t , was  not  related  Thus, a s p e c -  c e l l death, from  found.  Some o f  g r a f t s c o n s i s t e d p r i m a r i l y of an amorphous m a t e r i a l w i t h s c a t t e r e d ( s c o r e : 4 or 5) w h i l e o t h e r s ,  f o r example the 17 day  many s t r u c t u r a l l y normal f o l l i c l e s  (score: 2).  The  degree  graft,  the  cells  contained  second important  point  about the degree of d e g e n e r a t i o n of the a l l o g r a f t s i s t h a t the range i s almost comparable to t h a t o f the i s o g r a f t s .  With the a l l o g r a f t s however  the range i s extended to i n c l u d e a stage of complete t h y r o i d d e g e n e r a t i o n ( s c o r e : 5)  ( r e f e r to f i g u r e s 4A and  region' of two and  debris  scores  4B).  The  p e r i p h e r a l and  of the a l l o g r a f t s c o n s i s t o f a d i s o r g a n i z e d  ( f i g u r e 4B).  r e c e i v e d by  T h i s never o c c u r r e d  central  mass o f  w i t h the i s o g r a f t s .  the r e s t of the a l l o g e n e i c g r a f t s were on a par  cells The with  24  F i g u r e 4A:  Thyroid g r a f t r e c e i v i n g a score of 5  T h i s degree o f t h y r o i d t i s s u e d e g e n e r a t i o n was o n l y found i n t h e allografts. licles.  There i s almost a complete absence o f normal t h y r o i d  Both the p e r i p h e r a l  i z e d mass o f c e l l s  and d e b r i s .  s u r r o u n d i n g the g l a n d .  F i g u r e 4B:  and c e n t r a l r e g i o n  fol-  consist of a disorgan-  N o t i c e the c e l l u l a r  c a p s u l e (CAP)  X 51.  Composition o f the c e n t r a l a r e a o f glands  receiving  a s c o r e o f 5. The debris.  c e n t r a l region  consists  of a disorganized  mass o f c e l l s and  25  those r e c e i v e d by the syngeneic  grafts.  Thus, f o r example, t h e two 17  day a l l o g r a f t s show the same degree o f n e c r o s i s as t h e 4 day i s o g r a f t and even the 4 day a l l o g r a f t . As w i t h the syngeneic g r a f t s , e n c a p s u l a t i o n o f the a l l o g r a f t s occurs.  However, i t i s most f r e q u e n t l y p r e s e n t i n , and t h i c k e s t around,  the g r a f t s removed a f t e r l o n g e r p e r i o d s o f t r a n s p l a n t a t i o n ( f i g u r e 4A).  T h y r o i d i s o g r a f t s implanted i n the s p l e e n Because the s c o r e s o b t a i n e d and r e c o r d e d i n t a b l e I I i n d i c a t e t h a t subcutaneous i m p l a n t a t i o n o f the t h y r o i d l o b e s i n v o l v e s marked degenerat i o n of the glands another procedure was f o l l o w e d .  I t was hoped t h a t  i m p l a n t a t i o n o f the glands i n t o the s p l e e n , because o f i t s r i c h b l o o d s u p p l y , would e l i m i n a t e the marked degrees  o f d e g e n e r a t i o n and the en-  c a p s u l a t i o n o f the g r a f t s . The  t h y r o i d i s o g r a f t s , implanted  i n the h o s t ' s s p l e e n and removed  1-6, 10, 18-21 and 33 days f o l l o w i n g t r a n s p l a n t a t i o n , show the same s t r u c t u r a l c h a r a c t e r i s t i c s as seen i n the syngeneic and a l l o g e n e i c g r a f t s imp l a n t e d subcutaneously, occur.  except  t h a t e n c a p s u l a t i o n of the lobes does not  Thus, the system o u t l i n e d i n t a b l e I i s used  to s c o r e these  syn-  g e n e i c g r a f t s and the r e s u l t s a r e r e c o r d e d i n t a b l e I I . A l l of the i s o g r a f t s implanted  i n t h e s p l e e n and removed from 1 t o  14 days f o l l o w i n g t r a n s p l a n t a t i o n show t h e same degree o f d e g e n e r a t i o n as o c c u r r e d when the t h y r o i d l o b e s were implanted s u b c u t a n e o u s l y .  Again,  t h e r e i s no c o r r e l a t i o n between the degree o f d e g e n e r a t i o n and the l e n g t h o f time  the g r a f t remained i n the h o s t .  Indeed, 3 o f the g r a f t s removed  26  on days 20, 21 and 33 r e c e i v e d normal ( f i g u r e  5B).  scores  o f 0, s i n c e they were  structurally  F i g u r e 5A: T h y r o i d i s o g r a f t implanted i n the s p l e e n and removed 1 day f o l l o w i n g i m p l a n t a t i o n . X 51  F i g u r e 5B; T h y r o i d i s o g r a f t implanted i n the s p l e e n and removed 33 days f o l l o w i n g i m p l a n t a t i o n . X 49  28 DISCUSSION  G e n e r a l l y , the t h y r o i d i s o g r a f t s and a l l o g r a f t s , which were imp l a n t e d subcutaneously, and  showed, w i t h a few e x c e p t i o n s , the same degree  range o f f o l l i c l e t i s s u e d e g e n e r a t i o n .  l i c l e d e g e n e r a t i o n was expected  not  to be so e x t e n s i v e nor to be p r e s e n t a t a l l the stages examined N e i t h e r d i d the a l l o g r a f t s show the  of i n c r e a s e i n d e g e n e r a t i o n w i t h  Much of the f o l l i c u l a r  d e g e n e r a t i o n o c c u r r i n g i n the syngeneic  T h i s i s suggested  most s e r i o u s l y a f f e c t e d .  expected  time.  e a r l y a l l o g e n e i c g r a f t s implanted subcutaneously was necrosis.  some degree of f o l -  expected w i t h the e a r l i e r i s o g r a f t s , i t was  following transplantation. spectrum  Although  probably  and  ischemic  s i n c e the c e n t r a l f o l l i c l e s were always the  The  c o n n e c t i v e t i s s u e o f the subcutaneous  i s p o o r l y v a s c u l a r i z e d and, a c c o r d i n g to B i l l i n g h a m and  space  Silvers  (1971)  does not f a c i l i t a t e v e r y prompt v a s c u l a r i z a t i o n of an e n d o c r i n e  organ.  Some of the t i s s u e damage may The may  be due  a l s o to the h a n d l i n g of the  a p p a r e n t l y good c o n d i t i o n of the l o n g - s t a n d i n g t h y r o i d  be accounted  f o r i n two ways.  c e l l s , p r o b a b l y of h o s t o r i g i n the implanted  tissue.  First,  i t appears  ( B i l l i n g h a m and  allografts  t h a t a c a p s u l e of  S i l v e r s , 1971),  surrounds  T h i s h o s t response would cover t h e a n t i g e n s  thereby h i n d e r the h o s t ' s r e j e c t i o n response. proposed  by Woodruffs ( B i l l i n g h a m and S i l v e r s , 1971)  that regeneration  f o l l i c l e s by the s u r v i v i n g g r a f t c e l l s  of  the s u r r o u n d i n g h o s t t i s s u e s to form the stroma o f the g l a n d .  is  the case t h i s would f u r t h e r reduce thus p r o l o n g g r a f t  survival.  and  Secondly, i t has been  of  t i o n and  tissue.  i s accompanied by an i n d u c t i o n If this  the magnitude o f a n t i g e n i c s t i m u l a -  29  In o r d e r to minimize i s c h e m i c n e c r o s i s the problem o f e n c a p s u l a t i o n the spleen.  C e l l u l a r e n c a p s u l a t i o n o f the  range of  g r a f t s d i d not  f o l l i c u l a r d e g e n e r a t i o n as  planted subcutaneously. (Gibbs and  F i e l d , 1970)  hopefully,  eliminate  t h y r o i d i s o g r a f t s were implanted i n  except f o r the much l o n g e r s u r v i v a l , the and  and,  occur.  a l l o g r a f t s and  Since r e j e c t i o n occurs w i t h i n a f t e r g r a f t i n g , i t would not  be  the  the h a n d l i n g of the  f i r s t 19  possible  i n g the  transplantation  o r , most l i k e l y , This  the  21 and  three thyroid  33 days r e c e i v e d  e i t h e r that  due  to  i s o g r a f t s which  a score (table  surprising "return  the  cellular  studied  by  t i s s u e was  not  to normal" suggests t h a t ,  t h y r o i d i s o g r a f t s can  occurred  quickly  themselves.  g i v e n enough time,  (Billingham,  would i n v o l v e  following  the  r e - e s t a b l i s h themselves suggests  et a l . , 1963).  steps:  (1.)  The  and  procedure  that  be  adoptively  followed  T o l e r a n c e to donor a n t i g e n s i s  induced i n host mice at b i r t h ( B i l l i n g h a m  and  (2.)  a t h y r o i d a l l o g r a f t and  the p r o p e r age  the  themselves.  a v a i l a b l e methods to induce t o l e r a n c e  t r a n s f e r immunity  At  The  immune response o f a h o s t to t h y r o i d a l l o g r a f t s might  u s i n g the  II)  damaged d u r -  enough time to r e - e s t a b l i s h  thyroid isografts w i l l re-establish That the  the  procedure, or r e v a s c u l a r i z a t i o n  g r a f t s had  days  to  which i n d i c a t e s h i s t o l o g i c a l c h a r a c t e r i s t i c s o f a normal t h y r o i d . e x p l a n a t i o n of t h i s c o u l d be  im-  tissue.  I t i s v e r y i n t e r e s t i n g to note that remained i n the h o s t f o r 20,  same degree  isografts  s e p a r a t e immunological c e l l death from "background" n e c r o s i s i s c h e m i a and  However,  i s o g r a f t s showed the the  the  these mice r e c e i v e  B r e n t , 1958;  S e l l e r , 1968); the  30  g r a f t i s allowed bearing  to " r e - e s t a b l i s h " i t s e l f ;  the "normal a p p e a r i n g "  syngeneic antigens  (3.) The t o l e r a n t mice  t h y r o i d a l l o g r a f t s are i n j e c t e d with  lymphocytes, normal o r s p e c i f i c a l l y s e n s i t i z e d t o the donor ( B i l l i n g h a m e t a l . , 1963);  followed.  (4.)  A t the time t h i s was c o n s i d e r e d  c o u l d be induced  The r e j e c t i o n procedure i s i t was not known i f t o l e r a n c e  i n WBB6 mice t o DBA a n t i g e n s .  Sometimes, i f the mice  d i f f e r markedly a t t h e i r major h i s t o c o m p a t i b i l i t y l o c i , ble to  to induce  tolerance.  determine proper  with  the p o s s i b i l i t y  impossible,  i t i s not p o s s i -  S i n c e a g r e a t d e a l o f time would be r e q u i r e d  c e l l dosages and procedure to induce  t o l e r a n c e , and  t h a t i t s i n d u c t i o n between these 2 s t r a i n s may be  i t was decided not to attempt the i m p l a n t a t i o n o f t h y r o i d  as a means o f i n v e s t i g a t i n g the c e l l u l a r immune response o f t h e W mutant.  31  TRANSPLANTATION OF  SKIN  INTRODUCTION  Several serve  f a c t o r s suggested t h a t the s k i n a l l o g r a f t  response  as the b a s i s o f a comparison o f the c e l l - m e d i a t e d  e l i c i t e d by  the mutant and  demonstrated w i t h a d o p t i v e periments t h a t the h o s t somehow r e s p o n s i b l e 1963;  Najarian  and  p a r t i c i p a n t s and mediated one  non-mutant h o s t s .  immune responses  F i r s t , i t has  t r a n s f e r s t u d i e s and  could  been c l e a r l y  lymphocyte d e p l e t i o n  c e l l s which m i g r a t e i n t o the s k i n a l l o g r a f t  exare  f o r i t s subsequent d e s t r u c t i o n ( B i l l i n g h a m et_ a l . , Simmons, 1972).  Humoral a n t i b o d i e s  are not  obligatory  thus the s k i n a l l o g r a f t response i s , b a s i c a l l y , a  cell-  (Winn, 1970).  Secondly, p r e l i m i n a r y work by McNay (1974) has  established  that  t h e r e i s a d i f f e r e n c e i n the c e l l u l a r response of the W mutant to s k i n a l l o g r a f t s when compared to i t s non-mutant l i t t e r m a t e s . j e c t s i t s g r a f t s s i g n i f i c a n t l y f a s t e r and t i v a t e d lymphocytes p r e s e n t graft  i n i t s blood  there  mutant r e -  are l a r g e r numbers of  at an e a r l i e r stage i n the  ac-  allo-  response. Another f a c t o r f a v o r i n g the s k i n a l l o g r a f t  background i n f o r m a t i o n vestigations. ing  The  out  on i t b i s a l r e a d y  Such i n f o r m a t i o n  the p r e l i m i n a r y  studies.  provided  The  first  by o t h e r m i c r o s c o p i c  a i d s i n s e t t i n g up  experiments and  L i g h t microscope o b s e r v a t i o n s  the s k i n a l l o g r a f t response develops i n t h r e e 1944).  response i s t h a t some  successive  stage i n v o l v e s would r e p a i r and  stages  in-  carry-  reveal  that  (Medawar,  h e a l i n g phenomena,  32  similar to the response seen i n i s o g r a f t s .  I t i s characterized by  p r o l i f e r a t i o n of f i b r o b l a s t s and graft epithelium, formation of new c a p i l l a r i e s i n the graft bed and a large i n f i l t r a t i o n of polymorphonuclear leukocytes.  The second stage i s marked by the i n f i l t r a t i o n  of mononuclear c e l l s into the graft s i t e .  These c e l l s migrate from  the blood venules of the graft bed and small veins overlying the panniculus carnosus into the surrounding graft tissues (Weiner et a l . , 1969) .  The c e l l s spread from the graft bed through the dermis  the epidermis (Weiner, et a l . , 1964). are no signs of epidermal degeneration.  toward  During this second stage there The t h i r d and f i n a l stage i s  marked by degeneration of the epidermis and a large i n f i l t r a t i o n of polymorphonuclear  leukocytes.  The entire process requires 10 to 12  days i n mice (Simar and Betz, 1970). Another favorable factor i n skin transplantation i s that the c e l l u l a r "background" composition of the dermis into which the i n f i l t r a ting c e l l s migrate consists only of f i b r o b l a s t s , macrophages and blood c e l l s , none of them numerous (Montagne, 1956; Ham,  1969).  Although d i f -  f i c u l t y i n i d e n t i f i c a t i o n can arise due to the problem of c l e a r l y i d e n t i f y i n g any c e l l i n a section of skin graft prepared for l i g h t microscopy  (often the cytoplasm of c e l l s i n the dermis cannot be d i s -  tinguished from the surrounding tissue components and i t i s impossible to i d e n t i f y or describe a c e l l by i t s nuclear structure alone) proper i d e n t i f i c a t i o n of these c e l l s can be made with the electron microscope. Furthermore,  the h i s t o l o g i c a l appearance of the epidermis provides  a means by which the degree of graft degeneration may be scored.  This  \  33  a l l o w s a c o r r e l a t i o n to be made between the stage o f r e j e c t i o n , the degree of c e l l u l a r i n f i l t r a t i o n ,  and the c e l l types c o m p r i s i n g t h i s  population. F i n a l l y , a method i s a v a i l a b l e t h a t p r o v i d e s a means o f t i n g the i n f i l t r a t i n g comprising i t .  c e l l population  The c e l l s move f i r s t  quantita-  and o f d e t e r m i n i n g the c e l l  types  i n t o the r e g i o n between the t i s s u e s  o f the h o s t and those o f the s k i n g r a f t .  For l a c k of a b e t t e r  term,  t h i s r e g i o n w i l l be r e f e r r e d to as the g r a f t bed.  A method,  enzyme d i g e s t i o n , has been developed by J a k o b i s i a k  et^ a l . (1971) i n  which the c e l l s from the g r a f t bed and p o s s i b l y s e p a r a t e d from the s u r r o u n d i n g t i s s u e . study the d i f f e r e n t c e l l types t h a t both the mutant and non-mutant h o s t .  This  using  the lower dermis can be  then makes i t p o s s i b l e to  infiltrate  the s k i n a l l o g r a f t s on  34  MATERIALS AND METHODS  Skin grafting Male and female mice o f the s t r a i n s WBB6 and DBA/2 were used i n these experiments. the  The WBB6 r e c i p i e n t s were 14 t o 24 weeks o f age a t  time o f the experiment and were always o f the same sex as the donor. The s k i n g r a f t i n g technique employed  and Medawar (1951). pared by s c r a p i n g  was as o u t l i n e d i n B i l l i n g h a m  P i e c e s o f donor s k i n , 10 mm i n diameter, were p r e -  away both the f a t t y l a y e r and the periniculus carnosus  beneath the dermis.  The g r a f t s were then p l a c e d i n an i n t a c t bed o f  p e n n i c u l u s carnosus i n the l a t e r a l t h o r a c i c w a l l o f the h o s t . were covered w i t h a square o f gauze covered i n v a s e l i n e was  encased i n a p l a s t e r c a s t .  and the thorax  These experiments i n c l u d e d  two types o f  s k i n g r a f t s : (1) a l l o g r a f t s , i n v o l v i n g t h e t r a n s p l a n t a t i o n DBA/2 mice  t o WBB6 mice; and,  o f s k i n from  (2) i s o g r a f t s , i n v o l v i n g the t r a n s p l a n t a t i o n  of s k i n from a WBB6 mouse t o i t s WBB6 l i t t e r m a t e s . the  Grafts  I n o r d e r t o compare  c e l l u l a r response o f the mutant w i t h t h a t o f the non-mutant, the  g r a f t i n g was done i n p a i r s such t h a t the s k i n from DBA/2 donor was t r a n s p l a n t e d t o a p a i r c o n s i s t i n g o f a W/W  V  +/W  V  littermates.  mouse and one o f i t s +/+, +/W o r  When the g r a f t was l a t e r removed from the h o s t , h a l f  of i t was used f o r the e x t r a c t i o n o f c e l l s i n f i l t r a t i n g the  remaining h a l f was p r e p a r e d f o r e l e c t r o n microscopy.  the g r a f t w h i l e The procedure  i s summarized i n f i g u r e 6. I s o l a t i o n o f the i n f i l t r a t i n g  cells  The procedure f o r enzymatic i s o l a t i o n o f t h e i n f i l t r a t i n g the g r a f t bed and dermis i s o u t l i n e d by J a k o b i s i a k  cells  et_ a l . (1971).  from  The  35  F i g u r e 6:  G e n e r a l procedure of  cells.  f o r p r e p a r a t i o n o f t i s s u e and  isolation  DONOR  ,  (DBA or W B B 6 )  Q  HOST  (WBB6)  COMPARED  w/w  TO  o y  v  lisp™ *  +l  w/ +  or +  5  EPIDERMIS  GRAFT  "^oo O o C2J  FATTY LAYER  P. C A R N O S U S  FIXATIVE  EPON  EM  ENZYMATIC  MIXTURE  ISOLATED CELLS  IDENTIFICATION a n d CELL  COUNT  36 grafts were removed and the loose tissue, lying beneath the dermis and above the pannicuius carnosus, was scraped away, minced and incubated with an enzymatic mixture (0.25% collagenase and 0.25% trypsin ( c r y s t a l l i n e ) , i n Hanks balanced s a l t solution)) at 37°C f o r 30 minutes.  The  p e l l e t of c e l l s obtained was smeared on a microscope s l i d e and stained with Wright's s t a i n .  In some instances the p e l l e t was resuspended i n  Ringer's and c e l l v i a b i l i t y was determined by trypan blue exclusion tests.  The number and kind of grafts from which the i n f i l t r a t i n g c e l l s  were i s o l a t e d are shown i n table I I I . The c e l l s were separated into categories at the time they were counted.  Each c e l l was placed i n a category depending on (1) size of  nucleus, (2) proportion of nucleus to cytoplasm, (3) color of cytoplasm and (4) chromatin d i s t r i b u t i o n i n the nucleus.  These were l a t e r r e -  grouped on the basis of blood c e l l c l a s s i f i c a t i o n and l i g h t microscope studies of c e l l s i n tissue culture.  Light microscopy lu sections of the skin grafts prepared f o r electron microscopy were cut and stained with toluidine blue f o r examination with the l i g h t microscope.  This made i t possible to orient within the skin grafts and  characterize the tissue layers found there.  The results allow for es-  tablishment of a scoring system f o r categorizing the grafts according to their degree of degeneration. Electron microscopy The skin grafts were removed on the appropriate day (table IV) and prepared for electron microscopy.  Care was taken to cut away the edge  37 Table III Number and kind of grafts from which the i n f i l t r a t i n g were i s o l a t e d .  GENOTYPE  Host  DAYS AFTER TRANSPLANTATION  Donor  0  4  6  8  10  5  5  5  3  3  WBB6  (W/+, w /+, +/+) v  DBA/2  WBB6 (W /W) v  DBA/2  -  5  5  5  no host  DBA/2  5  -  -  -  Table IV Number of grafts prepared for electron microscopy  HOST GENOTYPE  DAYS AFTER TRANSPLANTATION  Allografts  4  64  7  8  9  10  13  WBB6 (WV/w)  5  6  2  6  2  3  1  5  6  2  6  2  4  1  WBB6 (WV/+, +/+, W/+)  Isografts WBB6 (W /+, +/+, W/+) v  3  3  cells  38  of the g r a f t and i n c l u d e o n l y the p i e c e s f i x a t i o n procedures were t r i e d : f i x e d i n 6.25% g l u t a r a l d e h y d e  from the c e n t r a l r e g i o n .  Two  (1) F o r some, the g r a f t m a t e r i a l was  i n 0.1M phosphate b u f f e r a t pH 7.2 f o r 2  hours, r i n s e d i n b u f f e r f o r 15 minutes and then p o s t - f i x e d i n 1% osmium t e t r o x i d e f o r 1 hour; o r , (2) the g r a f t was f i x e d i n Karnovsky's tive  fixa-  (Karnovsky, 1965) f o r 2 to 5 h o u r s , r i n s e d i n 0.1M phosphate b u f f e r  f o r 3 to 7 hours and then p o s t - f i x e d i n 1% osmium t e t r o x i d e f o r 1 hour. D i r e c t l y f o l l o w i n g e i t h e r f i x a t i o n procedure, t h e m a t e r i a l was w i t h 2% aqueous u r a n y l a c e t a t e The  a t 60°C o v e r n i g h t  stained  (Lock, e_t al_. , 1971) .  t i s s u e s were then dehydrated i n a l c o h o l s and embedded i n epon from  propylene oxide.  S e c t i o n s were cut on an LKB u l t r a t o n e and examined  w i t h a H i t a c h i HS7S and AEI 801.  39  RESULTS  I s o l a t i o n o f the i n f i l t r a t i n g (1.) C e l l Based page 34)  cells  types  on the c r i t e r i a o u t l i n e d i n M a t e r i a l s and Methods (see  the c e l l s i s o l a t e d from the g r a f t bed were s e p a r a t e d i n t o  the f o l l o w i n g  categories:  (a.) Small  lymphocyte  The nucleus of t h i s c e l l has a diameter o f 5 to 6 u and, w i t h Wright's s t a i n , appears dark p u r p l e .  The  chromatin i s clumped and i s  surrounded by a narrow r i m o f cytoplasm which e i t h e r l a c k s c o l o r o r s t a i n s v e r y p a l e b l u e or p i n k .  The diameter o f the e n t i r e c e l l i s us-  u a l l y 6 to 7 u ( f i g u r e 7 ) . (b.) Large  lymphocyte  The n u c l e u s o f t h i s c e l l has a diameter o f 8 to 10 u and dark p u r p l e .  The  chromatin i s clumped, but not as markedly  the s m a l l lymphocyte,  The  diameter  the e n t i r e c e l l i s 9 to 11 u ( f i g u r e 8 ) . (c.)  "Transformed"  These c e l l s may In  as t h a t o f  and i s surrounded by a narrow r i m o f cytoplasm  which e i t h e r l a c k s c o l o r o r s t a i n s v e r y p a l e b l u e or p i n k . of  stains  one  15 u.  lymphocyte  be s e p a r a t e d i n t o two  groups based on t h e i r  size.  the n u c l e a r diameter i s 8 to 10 u w h i l e i n the o t h e r i t i s 10 to The  chromatin i s e v e n l y d i s p e r s e d and i s surrounded by v a r y i n g  amounts o f cytoplasm, a p p e a r i n g as a wide r i m as shown i n f i g u r e 10 o r i n an amount a p p r o x i m a t e l y twice the w i d t h o f the n u c l e u s .  Similarly,  40  although the c o l o r of the cytoplasm i s always b l u e , i t v a r i e s markedly i n depth of c o l o r .  The  c o l o r ranges  from a p a l e sky b l u e to a v e r y  almost b l a c k , b l u e as i n f i g u r e s 9 and  10  dark,  respectively,  (d.) Macrophage T h i s category i n c l u d e s a r a t h e r m o r p h o l o g i c a l l y d i v e r s e p o p u l a t i o n of  cells.  C h a r a c t e r i s t i c s common among these c e l l s  of  cytoplasm r e l a t i v e to the s i z e of the n u c l e u s , and  of p a l e p i n k , gray o r deep p i n k . somewhat between c e l l s .  The  i n c l u d e a l a r g e amount cytoplasmic color  appearance o f the n u c l e u s  The n u c l e a r chromatin  sometimes appears  clumped w h i l e i n o t h e r i n s t a n c e s i t i s e v e n l y d i s p e r s e d . transformed  lymphocyte, two  on t h e i r n u c l e a r s i z e . y while others f a l l  groups of c e l l s  Some f a l l  varies  As w i t h  loosely the  can be d i s t i n g u i s h e d based  i n t o a n u c l e a r s i z e range from 8 t o  i n a range from 10 t o 12 y  ( f i g u r e s 15, 16 and  10  17).  (e.) B a s o p h i l i c c c e l i The b a s o p h i l i c lymphocytes a r e s e p a r a t e d i n t o two  c a t e g o r i e s based  on the amount of cytoplasm s u r r o u n d i n g the n u c l e u s : B l which has a r i m of  cytoplasm, and B2 which has a l a r g e amount of cytoplasm.  t e g o r i e s the n u c l e a r diameter  ranges  from 8 to 10 y.  The  clumped but i s somewhat open as i n the medium lymphocyte.  I n both  ca-  chromatin i s The  c o l o r of  the cytoplasm v a r i e s from b l u e to v e r y dark b l u e or dark p u r p l e , as demons t r a t e d i n f i g u r e s 12, 13 and ( f . ) Polymorphonuclear  14.  leukocyte  Both e o s i n o p h i l s and n e u t r o p h i l s were p r e s e n t and n u c l e i are lobed and s t a i n e d dark p u r p l e . by r e d c y t o p l a s m i c g r a n u l e s  ( f i g u r e s 19 and  The  counted.  The  eosinophil i s characterized  20).  41 The c e l l types I s o l a t e d from the s k i n g r a f t beds on b o t h mutant and non-mutant h o s t s . X 270.  F i g u r e 7:  Small  F i g u r e 8:  L a r g e lymphocyte  F i g u r e s 9, 10 and  lymphocyte  11:  Transformed  lymphocytes.  Note the v a r y i n g degrees of c y t o p l a s m i c b a s o p h i l i a .  F i g u r e s 12,  13 and  14: B a s o p h i l i c c e l l s  F i g u r e s 15,  16 and  17: Macrophages  F i g u r e 18:  Fibroblast  F i g u r e s 19 and  4  20:  Neutrophils  42 (g.) The  Fibroblast f i b r o b l a s t nucleus has  a diameter  ed by a l a r g e amount of cytoplasm. cytoplasm  The  of 8 to 12 y and i s surround-  c e l l appears  elongated,  the  extending as s t r a n d s from the a r e a of the n u c l e u s as demon-  s t r a t e d i n f i g u r e 18.  The  c o l o r o f the cytoplasm  i s usually pale blue,  though sometimes i t i s deeper b l u e . The number of m i t o t i c f i g u r e s , b a s o p h i l s and plasma c e l l s i n the cell  counts were extremely  small.  The e s s e n t i a l f e a t u r e s o f each  type i s o l a t e d from the g r a f t - s i t e a a r - e s s u m m a r i z e d i i n l t a b l e  (2.) Data and s t a t i s t i c a l  cell  V.  analysis  (a.) Donor s k i n The  c e l l s o f normal s k i n were i s o l a t e d i n o r d e r to get an i d e a as  to the c o n t r i b u t i o n the donor s k i n might make to the o v e r a l l c e l l i n the a l l o g r a f t s and  to see the types of c e l l s n o r m a l l y p r e s e n t  to the i n f i l t r a t i o n by h o s t c e l l s .  Only a few  c e l l s c o u l d be  from u n g r a f t e d s k i n , r e l a t i v e to the number o f c e l l s i s o l a t e d skin allografts.  counts prior  isolated from  the  Of those i s o l a t e d , the m a j o r i t y were f i b r o b l a s t s  and  macrophages, w i t h a few b l o o d  cells.  (b.) S k i n a l l o g r a f t s removed f o l l o w i n g t r a n s p l a n t a t i o n . F i v e a l l o g r a f t s were removed from mutant h o s t s and  f i v e from  non-  mutant h o s t s a t 4, 6, 8 and 10 days f o l l o w i n g t r a n s p l a n t a t i o n . The below the g r a f t dermis were e x t r a c t e d and 500 were i d e n t i f i e d and counted. o r d e r to determine  of t h e s e c e l l s per  graft  The v a l u e s are r e c o r d e d i n t a b l e VI.  i f the f r e q u e n c i e s of the 9 c e l l types c o m p r i s i n g  i n f i l t r a t i n g p o p u l a t i o n were the same f o r both  cells  In the  the mutant and non-mutant  Table V  Summary o f the c h a r a c t e r i s t i c s o f the c e l l types i s o l a t e d from t h e s k i n a l l o g r a f t s and viewed w i t h the l i g h t microscope  Color (Wright's stain)  Fig:ures  Cytoplasm  Nucleus Chromatin  Color (Wright's stain)  Amount  Illustrated i n :  Cell-. Types  Size  Small Lymphocyte  5y-6y  dark p u r p l e  clumped ( o f t e n notched)  no c o l o r o r p a l e p i n k or b l u e  r i m around the nucleus  7  Large 1 Lymphocyte  8y-10y  dark p u r p l e  clumped, but somewhat open  no c o l o r o r p a l e pink or blue  rim around the nucleus  8  Transformed Lymphocyte  8y-10y or 10y-15y  dark b l u e  dispersed  b l u e to very dark b l u e  wide r i m t o t w i c e area of the nucleus  9,10,11  Macrophage  8y-10y or 10y-15y  dark p u r p l e  dispersed or some clumping  gray, p a l e or deep p i n k  At l e a s t twice the a r e a o f t h e nucleus  15,16,17  Basophilic Cell I  8y-10y  dark p u r p l e  clumped, but open  b l u e , deep b l u e o r deep p u r p l e  r i m around the nucleus  12,14  Basophilic Cell II  8y-lly  dark p u r p l e  clumped, but open  b l u e , deep b l u e o r deep p u r p l e  a t l e a s t twice the area o f t h e nucleus  13  dark p u r p l e  lobed  gray ( e o s i n o p h i l have r e d granules  r i m o f cytoplasm  19,20  Polymorphonuclear Leukocytes  T a b l e VI  Day Following Transplantation  Number and p e r c e n t of the t o t a l number o f c e l l s counted of each c e l l type on mutant and nonmutant h o s t s on g i v e n days f o l l o w i n g t r a n s p l a n t a t i o n . Chi-square and p r o b a b i l i t y v a l u e s t h a t the f r e q u e n c i e s o f the v a r i o u s c e l l types w i t h i n the g r a f t bed on mutant and non-mutant h o s t s are t h e same a r e i n c l u d e d  Small Large Transformed Lymphocyte Lymphocyte Lymphocyte # % # % # %  Baso. I #  %  Baso I I Macrophage # % # %  NeutroEosinophil phil # % # %  Fibroblast # %  Total #  2497  Day 4 a.  Mutant  316  12.7  178  7.1  456  18.3  338  13.5  177 7.1 586 23.5 427  17.1  7  12  b.  Non-mutant  411  16.5  280 11.7? 377  15.1  379  15.2  149 5.9 592 23.7 283  11.3  17  7  „ 2495 x = 82.12 P< .005  Day 6 a.  Mutant  519  20.8  281111.2  333  13.3  306  12.2  150 6.0 503 20.1 315  12.6  18  14  b.  Non-mutant  597  23.9  225  9.0  269  10.8  325  13.0  184 7.4 520 20.8 320  12.8  44  14  2499 2498 x = 28.56 P< .005  Day 8 a.  Mutant  414  16.6  190  7.6  219  8.8  265  10.6  104 4.2 731 29.2 539  21.4  30  13  b.  Non-mu-  605  24.3  196  7.9  155  6.2  207  8.3  135 5.4 605 24.3 516  20.7  61  14  tant  2500 2494 x = 79.04 P< .005 2  Day 10 a.  Mutant  249  16.6  78  5.2  130  10.6  131  8.7  49 3.3 359 23.9 474  31.6  27  3  1500  b.  Non-mutant  239  16.0  90  6.0  81  8.3  107  7.2  52 3.5 345 23.1 539  36.2  28  9  1490 x = 22.4 P< .005  45  h o s t s a c h i - s q u a r e t e s t i s used.  Recorded i n t a b l e VI a r e the c h i - s q u a r e  v a l u e s o b t a i n e d and the p r o b a b i l i t i e s t h a t t h e f r e q u e n c i e s o f the l e u k o c y t e s w i t h i n the s k i n g r a f t s on mutant and non-mutant h o s t s on g i v e n days f o l l o w i n g t r a n s p l a n t a t i o n a r e the same. of  I t i s c l e a r t h a t the d i s t r i b u t i o n  the v a r i o u s l e u k o c y t e s w i t h i n the mutant g r a f t s i t e i s s i g n i f i c a n t l y  d i f f e r e n t from t h a t o f the non-mutant on a l l days f o l l o w i n g t r a n s p l a n t a t i o n ( i n a l l cases, P <  .005).  In o r d e r to o b t a i n some i d e a as t o the k i n e t i c s o f c e l l movement a t the g r a f t s i t e a percentage o f each c e l l  type w i t h i n the t o t a l number o f  c e l l s were c a l c u l a t e d and a r e i n c l u d e d i n t a b l e V I .  These v a l u e s have  been p l o t t e d a g a i n s t days f o l l o w i n g t r a n s p l a n t a t i o n i n c h a r t I and c h a r t II. and  Some trends which a r e i l l u s t r a t e d i n c l u d e the f o l l o w i n g :  The s m a l l  l a r g e lymphocytes and b a s o p h i l i c c e l l s peak i n c o n c e n t r a t i o n on day  6 w h i l e the h i g h e s t p e r c e n t o f macrophages o c c u r s on day 8 w h i l e the p e r centage o f polymorphonuclear l e u k o c y t e s i n c r e a s e s from day 6 to day 8 and day 8 to day 10. In o r d e r t o get some i d e a as to where, among the 9 c e l l  types, the  s i g n i f i c a n t d i f f e r e n c e between the mutant and non-mutant host c e l l l a t i o n s a r i s e s , i n d i v i d u a l c h i - s q u a r e t e s t s were done f o r each c e l l gory.  popucate-  I n t a b l e V I I a r e recorded the c h i - s q u a r e v a l u e s and p r o b a b i l i t y  t h a t the f r a c t i o n of each c a t e g o r y o f l e u k o c y t e i s the same i n s k i n  grafts  on mutant and non-mutant h o s t s on g i v e n days f o l l o w i n g t r a n s p l a n t a t i o n . These c a l c u l a t i o n s suggest where the d i f f e r e n c e i n t h e f r e q u e n c i e s o f t h e l e u k o c y t e s o f mutant and non-mutant h o s t s may l i e and may be summarized as f o l l o w s :  46  Chart I :  Percentage o f lymphocytes ("normal" and b a s o p h i l i c ) out o f the t o t a l number of c e l l s counted (per 5 g r a f t s ) on days 4, 6, 8 and 10 f o l l o w i n g t r a n s p l a n t a t i o n . F o r the e x p l a n a t i o n of I and I I ( b a s o p h i l i c c e l l s ) see page 4 0 ) .  4 Days  6 Following  8 10 Transplantation  47  Chart I I : P e r c e n t of macrophages and n e u t r o p h i l s out o f the t o t a l number of c e l l s counted (per 5 g r a f t s ) on days 4, 6, 8 and 10 f o l l o w i n g t r a n s p l a n t a t i o n .  40  48 Table VII  Chi-square v a l u e s and p r o b a b i l i t y t h a t the f r a c t i o n o f each c a t e g o r y o f l e u k o c y t e i s the same i n s k i n a l l o g r a f t s on mutant and non-mutant h o s t s on g i v e n days f o l l o w i n g t r a n s p l a n tation. Days F o l l o w i n g T r a n s p l a n t a t i o n 4  Cell Types Transformed Lymphocyte  2 X  6 P  values  7.49  2 X  6.80  < .01  < .05  < .005  6.198  Small Lymphocyte  12.41  < .005  0.23  Basophilic Cell I  2.34  0.572  Basophilic Cell II  2.40  Macrophage  0.03  <.005  10  2  P  X  values  < .01  22.72  29.2  P  values  Large Lymphocyte  Neutrophil  8  10.95  <  .005  0.093  x values 2  11.38  .005  0.240  7.128  <  .01  2.42  3.46  4.02  <  .05  0.09  0.28  11.88  <  .005  0.28  0.039  0.309  <.005  0.857  <  35.8  P  4.170  < .05  49  1.  Transformed  lymphocyte  T h e i r number i s s i g n i f i c a n t l y h i g h e r (P <.01 o r P < .005) i n the skin graft  on the mutant h o s t on a l l o f t h e days f o l l o w i n g t r a n s p l a n t a t i o n .  2. Large  lymphocyte  The number o f these c e l l s i s s i g n i f i c a n t l y and  s i g n i f i c a n t l y h i g h e r (P <  lower on day 4 (P <.005)  .05) on day 6 i n t h e s k i n a l l o g r a f t  on the  mutant h o s t . 3.  Small  lymphocyte  The number o f s m a l l lymphocytes i s s i g n i f i c a n t l y i n the s k i n g r a f t  lower  (P <  .005)  on the mutant h o s t on days 4 and 8 f o l l o w i n g t r a n s p l a n t a -  tion. 4.  Basophilic c e l l  The number o f these c e l l s i s s i g n i f i c a n t l y tween the mutant h o s t and non-mutant host:. h i g h e r (P < 5.  different  The B l c e l l s a r e s i g n i f i c a n t l y  .01) w h i l e the B2 c e l l s a r e s i g n i f i c a n t l y  lower  (P < .05).  Macrophage  There are a h i g h e r number i n the mutant (P < 6.  on day 8 be-  Polymorphonuclear  A significant  .005) on day 8.  leukocytes  d i f f e r e n c e i n the number o f n e u t r o p h i l s between mutant  and non-mutant h o s t s occurs on days 4 (P <  .005) and 10 (P <  .05). On day  4 the numbers a r e h i g h e r i n the mutant w h i l e on day 10 they a r e lower the number f o r the non-mutant h o s t .  than  50  L i g h t Microscopy Whenever p o s s i b l e included  the  underlying  two  the  1 u s e c t i o n s o f the epon-embedded  l a y e r s o f the  t i s s u e s of the host  s k i n g r a f t (epidermis and ( g r a f t bed,  l u s carnosus) ( r e f e r to f i g u r e s 21 and characterize thin  each l a y e r and  the  pannicu-  T h i s made i t p o s s i b l e  to  tissue block i n preparation  for  sectioning. The  g e n e r a l appearance o f the  degree of g e n e t i c the  dermis) and  p a n n i c u l u s adiposus and  22).  to o r i e n t the  material  g r a f t had  underlying  t i s s u e v a r i e d depending on  d i s p a r i t y between donor and  remained on  t i s s u e s w i l l be  (1.)  grafted  the h o s t .  The  considered  h o s t and  various  the  length  of  components o f the  the  time  graft  and  separately.  Epidermis  A b r i e f h i s t o l o g i c a l d e s c r i p t i o n of the s i n c e a comparison of the graft'febed h o s t s must be  g r a f t e p i d e r m i s i s important  c e l l s between the mutant and  l i m i t e d to t i s s u e between mutant and  non-mutant b l o c k s which  show the same degree of g r a f t d e g e n e r a t i o n , a l l o w i n g p o p u l a t i o n s p r e s e n t at s i m i l a r s t a g e s of the c o n d i t i o n of the e p i d e r m i s , r a t h e r d i c a t i v e of the  f o r comparison of  a l l o g r a f t response, and  than t h a t of the  degree of t h i s d e g e n e r a t i o n .  c r i t e r i a established  d i f f e r e n t categories  of d e g e n e r a t i o n .  cell  i t is  the  dermis, t h a t i s most i n -  Thus, the  c h a r a c t e r i s t i c s of  the e p i d e r m i s o f u n g r a f t e d mouse s k i n , s k i n i s o g r a f t s and c a r e f u l l y noted, and  non-mutant  for separating  a l l o g r a f t s were the  allografts into  (a.) E p i d e r m i s of mouse s k i n The wall  e p i d e r m i s of normal mouse s k i n removed from the  lateral  thoracic  c o n s i s t s of 3 to 6 i l l - d e f i n e d l a y e r s o f e p i t h e l i a l c e l l s which morpho-  l o g i c a l l y may  be  divided into various  s t r a t a ( f i g u r e 23A).  The most  con-  51  spicuous o f these i s the stratum germiriativum from the dermis;  t h i s has a s i n g l e l a y e r o f c u b o d i a l o r columnar  which r e s t on the basement membrane. of  s e p a r a t i n g the epidermis  There a r e from one t o t h r e e l a y e r s  rounded c e l l s above t h i s which do not have any o t h e r  c h a r a c t e r i s t i c s than t h e i r l o c a t i o n .  cells  distinguishing  The c e l l s o f the outermost  layer,  the stratum granulosum, c o n t a i n b a s o p h i l granules and a r e e l o n g a t e d t h e i r l o n g a x i e s p a r a l l e l to the s u r f a c e o f the s k i n . stratum corneum above t h i s l a y e r a r e dead. p r e s e n t and, l e s s f r e q u e n t l y , sebaceous (b.)  The  with  The c e l l s of the  Numerous h a i r  f o l l i c l e s are  glands.  Epidermis o f i s o g r a f t s removed 6 days and 8 days f o l l o w i n g transplantation epidermis o f the 6 day i s o g r a f t i s two t o t h r e e times  than the e p i d e r m i s ( f i g u r e 23B).  thicker  o f u n g r a f t e d mouse s k i n removed from t h e same r e g i o n  Furthermore, t h e r e i s a marked i n c r e a s e i n the k i n d and  number o f c e l l l a y e r s .  Above the stratum germinativum t h e r e are s e v e r a l  l a y e r s o f c e l l s which a r e r e f e r r e d t o as t h e s t r a t u m spinosum.  This layer  i s normally  found o n l y i n mammalian t h i c k s k i n .  b u t , except  f o r narrow c y t o p l a s m i c c o n n e c t i o n s , a r e s e p a r a t e d from one  another by a wide space.  With the connections  The c e l l s a r e p o l y h e d r a l  t h i s g i v e s the c e l l s a  " p r i c k l y appearance", c l e a r l y v i s i b l e i n f i g u r e 23B. &.more d i s t i n c t is  The stratum granulosum i  l a y e r than the stratum granulosum p r e v i o u s l y d e s c r i b e d .  c l e a r l y 2 to 3 c e l l s t h i c k and many o f the c e l l s o f the lower  tum  corneum i s the same as t h a t o f the e p i d e r m i s F i g u r e 23C i s a micrograph  It. •  layers, a l l  of which c o n t a i n g r a n u l e s , are more diamond shaped than e l o n g a t e d .  The s t r a -  o f u n g r a f t e d mouse s k i n .  of the epidermis o f an 8 day s k i n  isograft.  I t has the same h i s t o l o g i c a l c h a r a c t e r i s t i c s as t h a t i n the 6 day i s o g r a f t  s  52  Figure  21: This  6 Day A l l o g r a f t i s a s e r i e s of l i g h t microscope p i c t u r e s  of a 6 day s k i n a l l o g r a f t which i n c l u d e s  the 2 l a y e r s of the g r a f t  (epidermis (E) and dermis (D) and the u n d e r l y i n g ( g r a f t bed  of a 1 u s e c t i o n  (B) and panriiculus carnosus (M).  t i s s u e s o f the h o s t  X 95.  53  Figure  22: This  o f an 8 day  8 Day  Skin A l l o g r a f t  i s a s e r i e s o f l i g h t microscope p i c t u r e s o f a 1 y  section  s k i n a l l o g r a f t which i n c l u d e s  graft,  /'epidermis (E) and  dermis (D) and  / g r a f t bed  p a n n i c u l u s adipbsus  (B) and  ;  the  2 l a y e r s of the  the u n d e r l y i n g (A).  X  t i s s u e s of the 95.  host,  54  F i g u r e 23A:  Epidermis o f mouse s k i n  The l a y e r s o f e p i t h e l i a l c e l l s o f the epidermis may be d i v i d e d , morphologically, into various s t r a t a . includes  Note i n normal mouse s k i n t h i s  the stratum germiriativum (SO), s t r a t u m granulosum (SG) and  s t r a t u m corneum  F i g u r e 23B;  (SC).  X 134.  Epidermis o f a s k i n i s o g r a f t removed t r a n s p l a n t a t i o n ( s c o r e : 2)  6 days  following  Note the c l e a r e r d i s t i n c t i o n o f c e l l l a y e r s compared of normal s k i n shown i n f i g u r e 23A.  to that  Above the s t r a t u m germinativum  are s e v e r a l s l a y e r s o f c e l l s which a r e r e f e r r e d t o as the s t r a t u m spinosum (SS).  F i g u r e 23C:  E p i d e r m i s o f a s k i n i s o g r a f t removed t r a n s p l a n t a t i o n ( s c o r e : 2) X 134.  8 days  following  55 (figure 23B) including a prominent stratum spinbsum and granulosum.  stratum  However, o v e r a l l , the epidermis of an 8 day isograft i s  not as thickened as that of a 6 day i s o g r a f t . It should be noted here that the epidermis of neither the 6 day nor 8 day isografts shows any signs of c e l l u l a r necrosis or o v e r a l l tion ( f i gures 23 B and C).  degenera-  Further, the hair f o l l i c l e s seen i n cross sec-  t i o n appear as theyLdo i n the untransplanted pieces of mouse skin.  A skin  graft showing these characteristics (thickened, i d e n t i f i a b l e s t r a t a ; no necrosis, and i n contact with the underlying dermis) i s given a score of 2 (table VIII). (c.)  Epidermis of a l l o g r a f t s removed 4, 6, 7, 8, 9, 10 and 13 days following transplantation  The epidermis of the a l l o g r a f t s shows varying degrees of c e l l u l a r organization and degeneration, depending on the length of time each graft has remained on the host.  Grafts removed 4 days following transplantation  have an epidermis which i s not as thickened as that of the 6 and 8 day i s o grafts (figure 24 A).  There i s , however, a stratum spinosum similar to  that found i n the i s o g r a f t s .  This i s i n contrast to i t s almost  absence from the normal skin of the l a t e r a l thoracic wall. epidermis appears to be detached from the underlying dermis.  complete  In some areas the Some of the  c e l l s present show signs of degeneration including vacuolization of the cytoplasm and pyknosis or karyorrhexis. Furthermore, i n some of the h a i r f o l l i c l e s the epidermal c e l l s appear very disorganized compared to those i n normal skin.  A graft whose epidermis shows these c h a r a c t e r i s t i c s (not  thickened, i d e n t i f i a b l e s t r a t a , l i t t l e necrosis, and occasionally separated  from dermis) i s given a score of 1 (table VIII).  56  Table VIII  Score  3  S c o r i n g system which i n d i c a t e s the appearance o f epidermis•  Appearance o f the Epidermis  the  Illustrated in:  not t h i c k e n e d ; o c c a s i o n a l l y separated from dermis; s t r a t a are i d e n t i f i a b l e ; l i t t l e necrosis  Figure  24A  t h i c k e n e d ; not separated from dermis; s t r a t a a r e i d e n t i f i a b l e ; no n e c r o s i s  F i g u r e s 21A; 23B; 23C; 24B  t h i c k e n e d ; completely separated from dermis; s t r a t a are not i d e n t i f i a b l e ; marked n e c r o s i s  F i g u r e s 22A and 24C  57  F i g u r e 24A:  E p i d e r m i s o f a s k i n a l l o g r a f t removed 4 days t r a n s p l a n t a t i o n ( s c o r e : 1) X 134.  following  F i g u r e 24B:  E p i d e r m i s of a s k i n a l l o g r a f t t r a n s p l a n t a t i o n ( s c o r e : 2)  removed 6 days  following  F i g u r e 24C:  E p i d e r m i s of a s k i n a l l o g r a f t removed 8 days t r a n s p l a n t a t i o n ( s c o r e : 3 ) . X 134  following  N o t i c e the epidermis has (D)  and  the  gap  (G)  separated from the u n d e r l y i n g dermis  i s f i l l e d w i t h some c e l l u l a r  debris.  58  The m a j o r i t y o f the 6 day a l l o g r a f t s have an e p i d e r m i s which structurally,  the same as t h a t of the 6 and 8 day i s o g r a f t s  The e p i d e r m i s i s two  appears,  (score: 2).  to t h r e e times t h i c k e r than normal and, as shown i n  f i g u r e 24 B, c o n s i s t s o f s e v e r a l d i s t i n c t s t r a t a , i n c l u d i n g the s t r a t u m germinativum,  stratum spinosum, stratum granulosum and s t r a t u m corneum.  I n c o n t r a s t to the 4 day a l l o g r a f t epidermis to be f i r m attachment  (score: 1), there  appears  between the e p i d e r m a l c e l l s and the u n d e r l y i n g dermis.  In a d d i t i o n , theaepidermal c e l l s o f the 6 day a l l o g r a f t s do not show s i g n s of d e g e n e r a t i o n . Thus the epidermis o f 6 day a l l o g r a f t s w i t h these c h a r a c t e r i s t i c s i s , as the 6 and 8 day i s o g r a f t s , g i v e n a s c o r e o f 2. The  epidermis o f some of the 6 and 8 day a l l o g r a f t s and a l l o f the  9, 10 and 13 day a l l o g r a f t s showed s i g n s o f p a r t i a l and, complete, mis,  t i s s u e breakdown.  F i g u r e 24C  i s an example o f t h i s .  t h i c k e n e d as i n the 6 and 8 day i s o g r a f t s  ( s c o r e : 2 ) , has  i n some cases The e p i d e r -  ( s c o r e : 2) and 6 day  s e p a r a t e d away from the u n d e r l y i n g dermis.  The gap  p r e s e n t between them i s o f t e n f i l l e d w i t h c e l l u l a r d e b r i s , p r o b a b l y n a t i n g from the breakdown o f the e p i d e r m a l c e l l s .  allografts now origi-  In many areas s e v e r a l l a y -  e r s oof .epidermal • c e l l s are d i s t i n g u i s h a b l e but i d e n t i f i c a t i o n of the except  f o r the stratum corneum, i s not p o s s i b l e due  ( f i g u r e 24 C ) . necrosis.  strata,  t o c e l l breakdown  The h a i r f o l l i c l e s show s i m i l a r s i g n s of d i s o r g a n i z a t i o n  and  A g r a f t whose e p i d e r m i s shows these c h a r a c t e r i s t i c s ( t h i c k e n e d ,  u n i d e n t i f i a b l e s t r a t a , marked c e l l u l a r n e c r o s i s and almost  complete  separa-  t i o n from u n d e r l y i n g dermis) i s g i v e n a s c o r e o f 3 ( t a b l e V I I I ) . In summary, the t i s s u e b l o c k s are s e p a r a t e d i n t o t h r e e c a t e g o r i e s based on the appearance o f the e p i d e r m i s , the c o n d i t i o n o f which i s  59 considered i n d i c a t i v e of the degree of graft breakdown. (2.)  Dermis  Underlying the epidermis i s the dermis (figure 21 and 22). tissue normally  This  consists only of interwoven collagen bundles, a few scat-  tered f i b r o b l a s t s and even fewer macrophages and mast c e l l s .  Just beneath  the epidermis the collagen bundles are less densely packed than elsewhere and blood vessels are frequently present.  In other areas of the dermis the  blood vessels are usually associated with the hair f o l l i c l e s and sebaceous glands. The appearance with the l i g h t microscope of the constituents of the dermis does not a l t e r i n any consistent manner during wound healing or a l l o g r a f t r e j e c t i o n . Frequently the dermis of an a l l o g r a f t or isograft appears the same as that i n ungrafted skin. changes.  However, sometimes there are  Occasionally, red blood c e l l s are found scattered extravascularly  throughout the dermis, suggesting, at least i n older g r a f t s , breakdown of blood vessel walls (figure 22 A). day a l l o g r a f t s .  This i s most frequently seen i n l a t e r  Blood vessel lumerS sometimes appear packed with either red  blood c e l l s or "debris".  The most important change noted occurs i n the  lower dermis of a l l o g r a f t s and i s an increase i n the number of c e l l s there (Figures 21 and 22). into the region.  present  This i s presumably due to movement of host  cells  The extent of the i n f i l t r a t i o n varies between g r a f t s .  It i s somewhat related to the length of time the graft remains on the host and to whether i t i s allogeneic or syngeneic with respect to the host. both the isografts and a l l o g r a f t s there i s an increase i n the number of c e l l s i n the lower dermis.  Many of the early and l a t e r a l l o g r a f t s also  In  60  show an i n c r e a s e d number o f c e l l s i n the middle  r e g i o n of the  dermis,  w h i l e a s m a l l p e r c e n t of the g r a f t s examined shows an i n c r e a s e d p o p u l a t i o n i n the upper most r e g i o n s of the I t s h o u l d be noted  dermis.  t h a t , w i t h the m a t e r i a l viewed h e r e ,  not appear to be a d i r e c t r e l a t i o n s h i p between the degree of and  cell  the degree of i n f i l t r a t i o n of c e l l s i n t o the dermis.  there d i d  degeneration  Sometimes d i r e c t -  l y below an 8 day g r a f t epidermis which shows s i g n s of complete degeneration  ( s c o r e : 3) there are l a r g e numbers of c e l l s .  sent from another  8 day g r a f t  These c e l l s may  be  ab-  (score: 3).  (3.) Beneath the dermis In mouse s k i n a l a y e r o f f a t t y t i s s u e , the p a n n i c u l u s u n d e r l i e s the dermis and lagen bundles  i s f i r m l y united with i t .  o f the dermis,  the f a t c e l l s may  p r e s e n t i n l a r g e groups j u s t beneath.  adiposus,  Around the lower  occur s i n g l y , but are u s u a l l y  F i r m l y u n i t e d to t h i s l a y e r of f a t  t i s s u e i s an u n d e r l y i n g l a y e r o f muscle t i s s u e r e f e r r e d to as the carnosus.  col-  L y i n g between the p a n n i c u l u s  adiposus  are the p r i n c i p a l a r t e r i e s , v e i n s , lymphatics  and  panniculus  the p a n n i c u i u s  and nerves  carnosus  o f mouse s k i n .  The p r e p a r a t i o n of donor s k i n to be used f o r t r a n s p l a n t a t i o n i n c l u d e s removal of these t i s s u e l a y e r s . and dermis.  P r e p a r i n g the host bed  and p a n n i c u l u s panniculus host bed,  Thus, the g r a f t c o n s i s t s o n l y o f  i n v o l v e s removal o f the e p i d e r m i s ,  adiposus w h i l e k e e p i n g  carnosus  intact.  epidermis  the a r t e r i e s , v e i n s and  dermis  underlying  T h e r e f o r e , when donor s k i n i s t r a n s p l a n t e d to  the g r a f t dermis becomes j u x t a p o s e d  and any o v e r l y i n g b l o o d v e s s e l s .  to host p a n n i c u l u s  carnosus  61  D u r i n g the  first  few  days f o l l o w i n g  comes f i r m l y u n i t e d to the h o s t and into  the  tween the as  the  two  layers  g r a f t bed  of t h i s g r a f t bed donor.  I t s appearance and  (a.) The  layers  same as  and  the  p a n n i c u i u s carnosus.  the  f a t and  either  8 day  isografts  electron  fibroblasts  and  m a t e r i a l was  the  g r a f t bed.  I t i s the  host  arises  be-  referred  to  disappearance host  and  depend on  the  host. transplantation  dermis of 6 and The  8 day  isografts  p a n n i c u i u s adiposus  i s o g r a f t i s g r e a t e r than that  or of u n g r a f t e d s k i n  neutrophils.  In one  tissue.  I t should be  show these c e l l s  graft  of  to be  a s m a l l mass o f  noted  mostly  necrotic  dermis.  i n the  t h i n n e s s of the  i s p r e s e n t between the  p a i i n i c u i u s adiposus l a y e r  electron  at such an  6 day  a l l o g r a f t s a "gap"  bundles and  g r a f t bed  cells  Allografts  In the  w i t h the  w i l l be  point also  8 days f o l l o w i n g  microscope i n v e s t i g a t i o n s  a few  be-  However, the number of c e l l s p r e s e n t around  p r e s e n t j u s t below the  (b.)  the  those found i n u n g r a f t e d s k i n :  muscle c e l l s i n the  the  here that  remained on  at any  of t i s s u e u n d e r l y i n g the  the  T h i s gap  g e n e t i c d i s p a r i t y between the  removed 6 and  are  above the  subsequent development or  characteristics  g r a f t has  Isografts  The  the  graft  Thus a narrow gap  f i l l e d with host c e l l s .  depends on  l e n g t h of time the  g r a f t dermis and  p a n n i c u i u s carnosus .  i n t h i s study.  the  t h e r e i s a marked i n f i l t r a t i o n o f  damaged t i s s u e j u s t beneath the  p a n n i c u i u s adiposus and  transplantation  c e l l s of  microscope. 4 day  t h i s a r e a o f the  referred  to as  ( s c o r e : 1).  r e g i o n o r , more l i k e l y ,  the  g r a f t which were examined  D i f f i c u l t y a r i s e s when t r y i n g to l o c a t e  allografts  e a r l y stage i n the  that w i l l be  lower c o l l a g e n  T h i s i s due  either  to  this the  to i t s i n s i g n i f i c a n t development  a l l o g r a f t response.  The  g r a f t beds o f  the  62  6 day ( s c o r e : 2) and 8 and 10 day ( s c o r e : 3) s k i n a l l o g r a f t s a r e e a s i l y located.  There i s some v a r i a t i o n i n t h i c k n e s s ,  and i t i s more prominent  i n some c r o s s s e c t i o n s of the g r a f t than i n o t h e r s . A primary aim o f t h i s t h e s i s i s a comparison between the mutant and non-mutant host  o f the c e l l types comprising  stages o f the a l l o g r a f t response.  the g r a f t bed a t d i f f e r e n t  I t was a p p r e c i a t e d  e a r l y i n the work  t h a t improper o r l i m i t e d sampling c o u l d l e a d to an e r r o r i n i n t e r p r e t a t i o n of the d a t a when comparing the c e l l and non-mutant h o s t s . day  types found i n the g r a f t beds o f mutant  F o r t h i s r e a s o n examination o f the g r a f t bed o f 4  a l l o g r a f t s ( s c o r e : 1) was d i s c o n t i n u e d ,  c o u l d be done.  since only very  l i m i t e d sampling  An e l e c t r o n microscope i n v e s t i g a t i o n o f the g r a f t beds o f  6 day ( s c o r e : 2) and 8 and 10 day ( s c o r e : 3) a l l o g r a f t s on mutant and nonmutant h o s t s  follows.  E l e c t r o n microscope study o f the g r a f t bed o f 6 and 8 day ( s c o r e : 2) and 8 and 10 day ( s c o r e : 3) a l l o g r a f t s (1.)  O r i e n t a t i o n and i d e n t i f i c a t i o n  There are two i n s t a n c e s when o r i e n t a t i o n becomes extremely i n t h i s s t u d y : (1) When p r e p a r i n g  critical  the epon-blocks f o r t h i n s e c t i o n i n g ; and  (2) when v i e w i n g the image of t h e s e c t i o n s on the f l u o r e s c e n t s c r e e n . i s important when t h i n s e c t i o n i n g t h a t  (a) the o n l y m a t e r i a l s e c t i o n e d i s  g r a f t bed and (b) the c o n d i t i o n o f the e p i d e r m i s d i r e c t l y above t h i s be known. amined.  area  To a c h i e v e t h i s 1 y s e c t i o n s cut f o r l i g h t microscopy a r e ex-  As i n f i g u r e s 21 and 22, when p o s s i b l e , the s e c t i o n s i n c l u d e a l l  the t i s s u e l a y e r s from the e p i d e r m i s t o the p a n n i c u l u s allows  It  f o r (1) c o n f i r m a t i o n  determination  carnosus.  This  t h a t the g r a f t bed i s , i n f a c t , p r e s e n t ,  (2)  of the c o n d i t i o n of the e p i d e r m i s based on the c r i t e r i a o u t -  63  l i n e d i n t a b l e V I I I , and (3) p r o p e r o r i e n t a t i o n w i t h i n the b l o c k , so t h a t it  can be trimmed down to areas o n l y o f the g r a f t bed.  makes i t p o s s i b l e to r e s t r i c t  the comparison between the g r a f t bed c e l l s  o f t h e mutant and non-mutant host the same degree o f  the s e c t i o n s i n the e l e c t r o n microscope, o r i e n t a t i o n critical.  g i o n o f the g r a f t i s present region i s being  t o r e g i o n s under the e p i d e r m i s showing  degeneration.  When viewing a g a i n becomes v e r y  This o r i e n t a t i o n  I t i s obviously  e s s e n t i a l t o know which r e -  i n the s e c t i o n , and whether e x a c t l y the same  compared between one experiment and another.  g r a f t bed l i e s between the dermis and the p a n n i c u l u s  S i n c e the  adiposus and p a n n i c u i u s  carnosus , the presence o r absence o f c o l l a g e n b u n d l e s , f a t and muscle serve  cells  as p o i n t e r s f o r the i d e n t i f i c a t i o n of the p a r t i c u l a r r e g i o n imaged on  the f l u o r e s e n t s c r e e n .  F i g u r e s 25 and 26 i l l u s t r a t e  t h e i r use.  diagram i s a t r a c i n g o f a r e g i o n o f a 6 day s k i n a l l o g r a f t  The f i r s t  ( s c o r e : 2) on  a W/W  h o s t , and d e p i c t s the g r a f t bed as i t can appear a t low m a g n i f i c a t i o n .  Notice  t h a t the c o l l a g e n bundles ( d o t t e d a r e a s ) a r e a t t h e i r h i g h e s t  V  i n the upper r e g i o n of the diagram w h i l e present  the f a t c e l l s  i n the lower edge of the diagram.  (hatched  density  area) a r e  Thus, the c e l l s , d e b r i s and  b l o o d v e s s e l s o f the c e n t r a l a r e a l i e between the dermis and a l a y e r o f fat c e l l s . The  T h i s a r e a i s then the g r a f t bed. importance o f t h i s method o f o r i e n t a t i o n i s most e v i d e n t when  examining areas which a r e d i f f i c u l t i n t e r n a l components.  t o d i s t i n g u i s h as g r a f t bed by t h e i r  T h i s i s i l l u s t r a t e d i n f i g u r e 26 which i s a t r a c i n g  of an a r e a i n an 8 day s k i n a l l o g r a f t  ( s c o r e : 3) on a w/+ h o s t .  l e a s t 50% o f the m a t e r i a l i s l a b e l e d d e b r i s  Here a t  ( f i g u r e 30) and t h e n , even  64  F i g u r e 25: T r a c i n g of a r e g i o n of a 6 day  T h i s diagram d e p i c t s the g r a f t bed cation.  The  g r a f t bed  skin allograft  (score:  as i t appears at low  2)  magnifi-  c o n s i s t s o f a heterogeneous p o p u l a t i o n of  cells,  areas of e x t r a c e l l u l a r m a t e r i a l , i n c l u d i n g c o l l a g e n ( d o t t e d areas) d e b r i s , and having  blood v e s s e l s  (dark a r e a s ) .  N o t i c e i t l i e s between a r e g i o n  a h i g h d e n s i t y o f c o l l a g e n bundles (dermis) and  area). B-  a c t i v a t e d lymphocyte  L-  small  P-  neutrophil  E-  eosinophil  F-  fibroblast  M-  macrophage  H-  immunoblast  lymphocyte  and  fat cells  (hatched  65  F i g u r e 26:  T r a c i n g o f a r e g i o n of an 8 day s k i n a l l o g r a f t  ( s c o r e : 3)  T h i s diagram d e p i c t s the g r a f t bed as i t appears a t low m a g n i f i cation,  Notice  t h a t , i n c o n t r a s t to f i g u r e 25, the major component o f  the g r a f t bed i s the e x t r a c e l l u l a r m a t e r i a l , p r i m a r i l y the d e b r i s . I d e n t i f i c a t i o n o f most of the c e l l s i s i m p o s s i b l e necrosis.  due to marked  cellular  65c  66  though boundaries of s i n g l e c e l l s are o u t l i n e d , o n l y about 20% identified  ( r e f e r to f i g u r e s 26,  29 and  to know i f b l o o d v e s s e l s are p r e s e n t of 3 t h i s s t a t e of the g r a f t bed g i o n may  be  considered  or n o t .  I t i s sometimes  a mass of d e b r i s where c e l l s are d i f f i c u l t  d e p i c t e d i n f i g u r e s 25 and  c l u d i n g c o l l a g e n and  guidecells)  i s , i n f a c t , i n the  26,  bed  the g r a f t bed  between 6 day  d e b r i s , and b l o o d v e s s e l s .  allografts  c o n s i s t s m a i n l y of  c e l l s , areas of e x t r a c e l l u l a r m a t e r i a l , i n The  amount and  o f these components v a r i e s somewhat w i t h i n a g r a f t but ( s c o r e : 2) and  8 and  10 day  condition  d i f f e r s markedly  allografts  (score: 3).  o v e r a l l appearance o f the g r a f t beds i s the same i n g r a f t s w i t h  same s c o r e whether on mutant or non-mutant h o s t s . c r i p t i o n of the g e n e r a l 8 and  10 day  The  cells  to one  to the o t h e r .  allografts comprising  s i g n s of n e c r o s i s  (score:  f o l l o w i n g i s a des-  Figures  25,  (score:  27 and  2)  the g r a f t bed  ( f i g u r e 27 and  another v a r i e s c o n s i d e r a b l y  o f d i s t r i b u t i o n noted. occur  of 6 day  the  allografts.  m a j o r i t y o f the c e l l s  g r a f t do not show any  The  c h a r a c t e r i s t i c s o f the g r a f t beds o f 6 day  ( s c o r e : 3)  (a.) G r a f t bed  o f the  dis-  bed.  a heterogeneous p o p u l a t i o n of  2) and  score  to  (presence or absence o f c o l l a g e n bundles, f a t c e l l s o r muscle  As  bed  a  Under these c i r c u m s t a n c e s the  (2.) G e n e r a l c h a r a c t e r i s t i c s of the g r a f t  The  impossible  In o t h e r g r a f t s w i t h  become v e r y important i n a s s u r i n g o n e s e l f that one graft  be  i s heightened to the p o i n t where the r e -  t i n g u i s h at a l l (as i n f i g u r e 30B). lines  30A).  can  of a 6 day  28).  from one  28 are examples of the two  separated  proximity  a r e a of the g r a f t  In some r e g i o n s , as i n f i g u r e 25 and  s i n g l y or i n groups o f 2 to 4,  The  allo-  by d i s t a n c e s  27, of  main the  kinds cells  .1 u to 4 u.  67  C l o s e r spacing other  regions  o f the c e l l s o f t e n o c c u r s around the b l o o d of the g r a f t bed the p r o x i m i t y  of a c e l l  vessels.  In  to i t s n e i g h b o r s  i s l e s s than .1 .u ( f i g u r e 28), c r e a t i n g masses o f c e l l s w i t h i n the g r a f t bed.  Figure  28 shows a s e c t i o n o f such a c e l l mass.  o f t e n f o l l o w the same contour and, i n some c a s e s , p r o b a b l y to o b l i q u e  sectioning.  Apposing c e l l membranes  appear b l u r r e d , due  With the c e l l s so c l o s e there i s l i t t l e  room f o r e x t r a c e l l u l a r m a t e r i a l a l t h o u g h sometimes a l i t t l e very  s m a l l areas of d e b r i s a r e p r e s e n t . Within  the e x t r a c e l l u l a r spaces of the g r a f t bed there  v e s s e l s , c o l l a g e n , d e b r i s and a few red b l o o d v e s s e l s t r a v e r s e the g r a f t bed.  shows i t to c o n t a i n a few r e d b l o o d cells  ( f i g u r e 25).  the e n d o t h e l i a l c e l l s suggesting  cells  are blood  ( f i g u r e 25).  The b l o o d  T h e i r e n d o t h e l i a l c e l l s do not show any  signs of necrosis or degeneration.  blood  c o l l a g e n and  A c r o s s s e c t i o n of the lumen u s u a l l y cells  Occasionally  and, sometimes, one o r two w h i t e  the w h i t e blood  c e l l s l i e between  and the basement membrane of the b l o o d  vessel wall,  they a r e moving i n t o o r out of t h a t v e s s e l .  The m a t e r i a l i n the e x t r a c e l l u l a r spaces l a b e l e d d e b r i s c o n s i s t s of e i t h e r vacuoles of varying  s i z e and e l e c t r o n d e n s i t y ,  almost c e r t a i n l y de-  r i v e d from broken-down c e l l s , membrane-bound g r a n u l e s (some c h a r a c t e r i s t i c o f e o s i n o p h i l s or n e u t r o p h i l s ) , other amount o f d e b r i s p r e s e n t  c e l l remnants, o r a l l o f these.  The  v a r i e s from one area o f the g r a f t bed to a n o t h e r .  However, s c a t t e r e d among the c e l l s of the g r a f t bed, as shown i n f i g u r e 25, i t s p r e s e n c e i s never v e r y (b.)  extensive.  G r a f t bed of 8 day a l l o g r a f t s ( s c o r e : 3)  The c e l l s o f the 8 day g r a f t bed are d i s t r i b u t e d i n much the same  68  F i g u r e 27:  Low power e l e c t r o n micrograph o f the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  Note the d i s t a n c e between c e l l s and t h e i r d i s t r i b u t i o n to one another.  relative  Only a few o f the c e l l s show any s i g n s o f n e c r o s i s .  69  F i g u r e 28:  Low power e l e c t r o n micrograph o f the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  T h i s i s a s e c t i o n through a mass o f c e l l s bed.  found w i t h i n the g r a f t  N o t i c e t h a t the c e l l s a r e so c l o s e t h e i r apposing c e l l membranes  o f t e n f o l l o w t h e same c o n t o u r . material.  There i s l i t t l e room f o r e x t r a c e l l u l a r  70  was as those found i n t h e 6 day g r a f t beds.  They may o c c u r  i n groups o f 2 to 4, .1 u to 4 u a p a r t , as i l l u s t r a t e d 29. the  s i n g l y , or  i n f i g u r e s 26 and  N e i g h b o r i n g c e l l s may a l s o be v e r y c l o s e to each o t h e r r e s u l t i n g i n formation  o f a s m a l l c e l l mass i n the g r a f t bed (a p o r t i o n o f such a  c e l l mass i s shown i n f i g u r e 30A). A striking  c o n t r a s t which i s i l l u s t r a t e d  i n the f i g u r e s between  the 6 day and 8 day g r a f t beds i s the c o n d i t i o n o f the c e l l s . ty o f the c e l l s  i n the g r a f t bed o f an 8 day a l l o g r a f t  marked s i g n s o f n e c r o s i s i n c l u d i n g p y k n o s i s , i n some cases, and  extrusion of e i t h e r nuclear  swollen  The m a j o r i -  ( s c o r e : 3) show  cytoplasmic  organelles,  c h r o m a t i n o r cytoplasm, o r b o t h ,  absence o f e i t h e r the c e l l membrane o r the n u c l e a r membrane, o r b o t h .  I d e n t i f i c a t i o n o f these c e l l s i s i m p o s s i b l e  ( f i g u r e 26).  Further,  these  s i g n s o f n e c r o s i s a r e seen i n o n l y a s m a l l number o f the c e l l s o f the 6 day allograft  ( f i g u r e s 27 and 28).  A g a i n , i n c o n t r a s t to the g r a f t bed o f the 6 day a l l o g r a f t , the major component i n an 8 day a l l o g r a f t bed i s the e x t r a c e l l u l a r m a t e r i a l , p r i m a r i l y the d e b r i s .  I t c o n s i s t s o f remnants o f c e l l s , v a c u o l e s  ious s i z e s and e l e c t r o n d e n s i t y and granules many c e l l s of the g r a f t bed a r e b r e a k i n g consider  the whole g r a f t bed as d e b r i s  of var-  ( f i g u r e s 29 and 3 0 ) . O f t e n so  down t h a t i t becomes f e a s i b l e to  ( f i g u r e 30B).  The e n d o t h e l i a l c e l l s  of the b l o o d v e s s e l w a l l s a l s o show s i g n s o f breakdown and, i n many a r e a s , no l o n g e r completely  enclose  by masses o f r e d blood  the lumen.  The lumen i s u s u a l l y o b l i t e r a t e d  c e l l s , blood p l a t e l e t s or c e l l u l a r  debris.  (3.) C e l l s o f t h e g r a f t bed (a.) F i x a t i o n procedures and the p r e s e r v a t i o n o f the u l t r a s t r u c t u r e o f the g r a f t bed c e l l s  71  F i g u r e 29:  Low power e l e c t r o n microphage o f the g r a f t bed o f an 8 day s k i n a l l o g r a f t ( s c o r e : 3)  Note the d i s t a n c e between c e l l s and t h e i r d i s t r i b u t i o n r e l a t i v e t o one  another.  The m a j o r i t y o f the c e l l s i n the g r a f t bed show marked s i g n s  of necrosis i n c l u d i n g pyknosis,  swollen cytoplasmic  organelles, extrusion  o f e i t h e r n u c l e a r chromatin o r cytoplasm, o r both, and an absence o f e i t h e r the c e l l membrane o r the n u c l e a r membrane, o r both. o f these c e l l s i s i m p o s s i b l e .  Identification  72  Figure  30A:  Low power e l e c t r o n micrograph of the g r a f t bed o f an 8 day s k i n a l l o g r a f t ( s c o r e : 3)  S i m i l a r to f i g u r e 28, t h i s i s a s e c t i o n through a mass o f c e l l s found w i t h i n the g r a f t bed.  However, n o t i c e t h a t the m a j o r i t y  of the  c e l l s show marked s i g n s o f n e c r o s i s .  Figure  30B:  Debris  Sometimes the c e l l u l a r n e c r o s i s i s so e x t e n s i v e allograft debris. ules .  ( s c o r e : 3) t h a t the whole g r a f t bed may  i n an 8 day s k i n  f e a s i b l y be  Note the remnants of c e l l s , v a c u o l e s o f v a r i o u s  considered  s i z e s and g r a n -  73  Due  to the extent o f c e l l breakdown o c c u r r i n g i n some o f the  beds, i t i s important used.  The  to c o n s i d e r the e f f e c t s o f the f i x a t i o n  method of f i x a t i o n f o l l o w e d was  i n phosphate b u f f e r , pH ide;  or  7.4,  7.4)  procedures  glutaraldehyde  f o l l o w e d by p o s t - f i x a t i o n i n 1% osmium t e t r o x -  (2) Karnovsky's f i x a t i v e  phosphate b u f f e r , pH  e i t h e r : (1) 6%  graft  (paraformaldehyde p l u s g l u t a r a l d e h y d e  f o l l o w e d by p o s t - f i x a t i o n i n 1% osmium t e t r o x i d e .  With the former method o f f i x a t i o n t h e r e i s a h i g h i n c i d e n c e o f breakdown i n the g r a f t bed  in  of  the, 6 d a y . a l l o g r a f t .  cell  These c e l l s show many of  the c h a r a c t e r i s t i c s which can be a t t r i b u t e d to poor f i x a t i o n , i n c l u d i n g marked d i s t e n t i o n of the endoplasmic r e t i c u l u m , g o l g i and broken c i s t e r n a e w i t h i n the m i t o c h o n d r i a , c l e a r membrane, and O f t e n i t was  chromatin clumping a l o n g the  s e p a r a t i o n o f the cytoplasm  found t h a t w i t h two  p r o p e r t i e s i n d i c a t i n g proper p r e s e r v a t i o n . very  nu-  from the n u c l e a r membrane.  c e l l s s i d e by s i d e , the u l t r a s t r u c t u r e o f  one would show the above c h a r a c t e r i s t i c s w h i l e  of c e l l breakdown was  n u c l e a r membrane,  the o t h e r would show a l l t h e  In the 8 day  g r a f t s the  incidence  high.  With such a h i g h and e r r a t i c i n c i d e n c e of c e l l breakdown i n the g r a f t beds, the second method, Karnovsky's f i x a t i v e , was o f c e l l breakdown i n the 6 day a l l o g r a f t s decreased 25,  27 and  and  28  ( n o t i c e the gap  vacuoles  The  amount  s i g n i f i c a n t l y (figures  28), except f o r the n e u t r o p h i l p o p u l a t i o n .  n e u t r o p h i l s always show s i g n s of breakdown.  tried.  F o r some reason  the  T h i s i s e v i d e n t i n f i g u r e s 27  p r e s e n t between the cytoplasm  and broken m i t o c h o n d r i a l c i s t e r n a e ) .  and  nucleus,  empty  Karnovsky's f i x a t i v e d i d not  lower the extremely h i g h i n c i d e n c e of c e l l breakdown i n the g r a f t beds of 8 and  10 day  allografts  ( s c o r e : 3)  ( r e f e r to f i g u r e s 29 and  30).  This  74  breakdown i s p r o b a b l y not due Due  to improper  fixation.  to t h i s c o n d i t i o n o f the c e l l s i n 8 day a l l o g r a f t s  the problem o f sampling o c c u r s a g a i n .  ( s c o r e : 3)  A s e a r c h f o r c e l l s w i t h i n the g r a f t  bed w i t h s u f f i c i e n t p r e s e r v a t i o n o f t h e i r u l t r a s t r u c t u r e f o r i d e n t i f i c a t i o n and comparison  would be f u t i l e .  Thus, any  comparison  of c e l l  types between  the mutant and non-mutant h o s t s on g r a f t s g i v e n a s c o r e o f 3 i s not T h i s a l s o means t h a t the comparisons  of c e l l  possible.  types between beds i n which  the g r a f t s had a s c o r e o f 2 and 3 have been made on the " g e n e r a l appearance" l e v e l o n l y . (b.)  D e s c r i p t i o n o f the c e l l types found i n the g r a f t bed o f 6 day a l l o g r a f t s ( s c o r e : 2) on mutant and non-mutant h o s t s  There i s a wide v a r i e t y o f c e l l c l u d i n g f i b r o b l a s t s , s m a l l lymphocytes,  types p r e s e n t i n the g r a f t bed, i n immunoblasts, a c t i v a t e d  macrophages, e o s i n o p h i l s and n e u t r o p h i l s .  The d i s t r i b u t i o n o f any one  type does not appear to f o l l o w an obvious p a t t e r n ( f i g u r e 25). words, one  cell  specific cell  lymphocytes,  In o t h e r  type does not c o n s i s t e n t l y appear a s s o c i a t e d w i t h  type o r a c e r t a i n k i n d o f e x t r a c e l l u l a r m a t e r i a l .  another The  e x c e p t i o n to t h i s i s the f i b r o b l a s t , which i s u s u a l l y a s s o c i a t e d w i t h gen  cell  only colla-  bundles. There does not appear t o be any d i f f e r e n c e between the mutant  and  non-mutant h o s t s i n e i t h e r the k i n d s of c e l l s which e n t e r the g r a f t bed a 6 day a l l o g r a f t  ( s c o r e : 2) nor i n t h e i r u l t r a s t r u c t u r e .  i s a d e s c r i p t i o n of each c e l l cells  type found i n the g r a f t bed.  The  following  Examples o f the  are taken from g r a f t beds o f a l l o g r a f t s w i t h a s c o r e of 2 on  mutant or non-mutant h o s t s .  of  either  75  1.  Small lymphocyte  Small lymphocytes  a r e p r e s e n t i n the g r a f t beds o f 6 day ( s c o r e : 2)  and 8 day ( s c o r e : 3) a l l o g r a f t s .  I n e l e c t r o n micrographs  i n s i z e from 5 t o 7 u i n diameter  ( f i g u r e 31A).  these c e l l s  range  Many show the u l t r a s t r u c -  t u r a l c h a r a c t e r i s t i c s o f the " r e s t i n g " o r " i n a c t i v e " s t a t e by which they a r e commonly d e s c r i b e d .  The nucleus has a c o n s i d e r a b l e amount o f  t i n and a s m a l l n u c l e o l u s and i s surrounded ( f i g u r e 31A).  by a narrow r i m o f cytoplasm  The contents o f the cytoplasm v a r y .  p r e s e n t and t h e r e are a few, s m a l l m i t o c h o n d r i a . p r e s e n t , i s p o o r l y developed  heterochroma-  and rough endoplasmic  Centrio l e s are u s u a l l y The G o l g i a p p a r a t u s , i f r e t i c u l u m i s sparse.  Ribosomes a r e u s u a l l y f r e e i n the cytoplasm and, sometimes, t h e r e a r e one or two s m a l l e l e c t r o n dense v a c u o l e s p r e s e n t . Some o f the s m a l l lymphocytes ture.  show minor changes i n t h e i r  ultrastruc-  N o t i c e i n f i g u r e 31B the i n c r e a s e i n t h e number o f RER s t r a n d s p r e s e n t  i n the cytoplasm and the number o f ribosomes which a r e r i b o s o m a l aggregates.  and the p r o p o r t i o n o f these  These changes may  (as shown i n f i g u r e 31B)  be accompanied by an i n c r e a s e i n t h e amount o f euchromatin n u c l e u s and by a more prominent  nucleolus.  l a r g e r s u g g e s t i n g they a r e medium o r l a r g e 2.  p r e s e n t i n the  O f t e n times t h e s e c e l l s a r e lymphocytes.  Immunoblas t s  The immunoblast types i n the g r a f t bed.  ( f i g u r e 32) i s one o f the l e a s t f r e q u e n t l y seen  cell  I t i s found i n the g r a f t bed o f 6 ( s c o r e : 2) and  8 ( s c o r e : 3) day a l l o g r a f t s on b o t h mutant and non-mutant h o s t s . to be v e r y s e n s i t i v e to t h e f i x a t i o n procedure  I t appears  s i n c e , more o f t e n than n o t ,  the m i t o c h o n d r i a l c i s t e r n a e appear broken down w i t h i n the m a t r i x .  These  76'.  F i g u r e 31A:  Small lymphocyte found i n the g r a f t bed o f a .6 day a l l o g r a f t ( s c o r e : 2 ) . The bar r e p r e s e n t s 1 u .  F i g u r e 31B:  Small lymphocyte showing minor changes i n i t s u l t r a s t r u c t u r e  This  c e l l was  also  moved 6 days f o l l o w i n g  skin  found i n the g r a f t bed o f a s k i n a l l o g r a f t r e -  transplantation.  The bar r e p r e s e n t s 1 u .  7C*.  F i g u r e 32:  Immunoblast  The most prominent c h a r a c t e r i s t i c o f t h i s c e l l i s t h e abundance o f p o l y r i b o s o m e s w i t h i n the cytoplasm. s i t e o f 6 day s k i n a l l o g r a f t s  This c e l l i s present  (score: 2).  The b a r r e p r e s e n t s  at the gra 1 y.  78  c e l l s range i n s i z e from 9 to 11 u i n diameter i n e l e c t r o n  micrographs.  The n u c l e u s c o n s i s t s p r i m a r i l y of f i n e l y s c a t t e r e d chromatin w i t h a t h i n e l e c t r o n - d e n s e band o f clumped chromatin along the n u c l e a r membrane. a l s o has a prominent  nucleolus.  T h i s nucleus i s l a r g e and round,  It  often  w i t h an i n d e n t a t i o n at one end, and i s surrounded by a somewhat narrow r i m of  cytoplasm.  Only a few c y t o p l a s m i c o r g a n e l l e s are u s u a l l y p r e s e n t , m i t o -  c h o n d r i a and a s t r a n d o r two o f RER.  The most prominent  characteristic of  the immunoblast i s the abundance of polyribosomes w i t h i n the cytoplasm ( r e f e r to f i g u r e 3.  32).  Activated  Another  cell  lymphocyte type p r e s e n t i n the g r a f t bed i s the a c t i v a t e d  c y t e d e s c r i b e d by H a l l et^ al_. (1967).  These c e l l s v a r y markedly  s i z e but are u s u a l l y l a r g e r than the s m a l l lymphocytes g r e a t e r frequency than any o t h e r type of lymphocyte  i n their  and appear w i t h  identified.  H a l l et^ a l .  (1967) judge the v a r i o u s u l t r a s t r u c t u r a l s t a t e s as h a v i n g a " l e a s t e n t i a t e d " or a "more d i f f e r e n t i a t e d " appearance.  lympho-  differ-  Such s t a t e s were a l s o  found i n the p o p u l a t i o n o f c e l l s c o m p r i s i n g the g r a f t  bed.  F i g u r e 33A i s an example o f a " l e a s t d i f f e r e n t i a t e d " b a s o p h i l i c as d e s c r i b e d by H a l l shape.  (1967).  The n u c l e u s i s l a r g e and has an  cell  irregular  There i s a moderate amount o f chromatin clumping a l o n g the n u c l e a r  membrane and a n u c l e o l u s i s u s u a l l y p r e s e n t . developed than a n y t h i n g d e s c r i b e d thus f a r .  The cytoplasm i s much more Ribosomes, abundant i n the  cytoplasm, u s u a l l y o c c u r s i n g l y , a l t h o u g h a few polyribosomes a r e sometimes present.  A few s c a t t e r e d  s t r a n d s o f RER  are present.  L o c a t e d i n the  79  F i g u r e 33A:  The c e l l type.  F i g u r e 33B:  A c t i v a t e d lymphocyte found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  i n s e r t shows the w e l l developed G o l g i apparatus t y p i c a l of The b a r r e p r e s e n t s 1 y.  A c t i v a t e d lymphocyte found i n the g r a f t bed o f a 6 day skin a l l o g r a f t (score: 2).  this  80  c e n t r a l region of the c e l l developed,  i s the G o l g i a p p a r a t u s .  I t i s usually well  c o n s i s t i n g o f 2 o r 3 l a m e l l a r s t a c k s and a s s o c i a t e d v e s i c l e s ,  v a c u o l e s and some s m a l l e l e c t r o n dense v a c u o l e s  (as i n f i g u r e 33B).  F i g u r e s 34A and 34B a r e examples o f a c t i v a t e d lymphocytes w i t h a more " d i f f e r e n t i a t e d appearance".  There i s an abundance o f ribosomes  which m o s t l y o c c u r i n r o s e t t e s and, sometimes, i n s h o r t c h a i n s . becomes more abundant. out the cytoplasm ranged  I n some c e l l s i t i s g e n e r a l l y d i s t r i b u t e d  number o f m i t o c h o n d r i a may a l s o be up. a c t i v a t e d lymphocytes,  (as i n f i g u r e 34B). The  As i n the " l e s s  differentiated"  t h e g o l g i zone i s w e l l developed w i t h a number o f  v a c u o l e s p r e s e n t , some c o n t a i n i n g e l e c t r o n dense g r a n u l e s M i c r o t u b u l e s and m i c r o f i b r i l s a r e o f t e n p r e s e n t  ( f i g u r e 34A).  also.  Macrophage  Another c e l l of  through-  (as i n f i g u r e 34A) w h i l e i n o t h e r s the s t r a n d s a r e a r -  i n s h o r t s t a c k s i n one area o f t h e c e l l  4.  RER a l s o  type p r e s e n t i n t h e g r a f t bed o f a l l o g r a f t s w i t h s c o r e s  2 and 3 on b o t h mutant and non-mutant h o s t s i s the macrophage, which  make up a s u b s t a n t i a l p o r t i o n o f the c e l l p o p u l a t i o n .  The c e l l appears i n  v a r i o u s u l t r a s t r u c t u r a l s t a t e s which have been s e p a r a t e d i n t o " n o n - a c t i v a ted"  and an " a c t i v a t e d " form. F i g u r e 35 i s an example o f the " n o n - a c t i v a t e d " form.  The diameter  of  these c e l l s i s 10 to 14 u .  of  d i s p e r s e d chromatin w i t h a t h i n e l e c t r o n dense band o f clumped  a l o n g the n u c l e a r membrane. high.  The nucleus i s horseshoe-shaped and c o n s i s t s  The volume r a t i o o f cytoplasm  chromatin  to nucleus i s  The cytoplasm c o n t a i n s s c a t t e r e d s t r a n d s o f RER, a g o l g i zone,  s e v e r a l m i t o c h o n d r i a and e l e c t r o n dense v a c u o l e s o f v a r y i n g s i z e .  There  81  Figure  34A:  A c t i v a t e d lymphocyte found i n t h e g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  T h i s i s an example o f an a c t i v a t e d lymphocyte showing a more " d i f f e r e n t i a t e d appearance". cytoplasm.  F i g u r e 34B:  Note the abundance  o f ribosomes i n the  The b a r r e p r e s e n t s l ' u .  A c t i v a t e d lymphocyte found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  T h i s i s an example o f an a c t i v a t e d lymphocyte showing a more " d i f f e r e n t i a t e d appearance". reticulum.  The b a r r e p r e s e n t s  Note the s h o r t s t a c k s o f rough endoplasmic 1 p.  Bio,  82  F i g u r e 35:  Monocyte, o r " n o n - a c t i v a t e d " form o f macrophage, found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2) The b a r r e p r e s e n t s 1 u .  83  are a l a r g e number o f s m a l l v e s i c l e s a l s o .  The c e l l membrane has a v e r y  uneven contour due to the number o f pseudopods p r e s e n t . Examples o f the more " a c t i v a t e d " forms a r e shown i n f i g u r e s 36A, 36B and 36C.  These c e l l s a l s o have diameters o f 12 t o 15 u i n e l e c t r o n  micrographs.  The nucleus shows an i r r e g u l a r i t y o f i t s s u r f a c e  t a i n s more clumped chromatin than the " n o n - a c t i v a t e d "  form.  and con-  I t a l s o has  a w e l l developed g o l g i zone, s e v e r a l m i t o c h o n d r i a , s c a t t e r e d s t r a n d s o f RER and l a r g e numbers o f s m a l l v e s i c l e s i n i t s cytoplasm. the  cytoplasm o f the " n o n - a c t i v a t e d "  and  number o f e l e c t r o n dense v a c u o l e s and t h e reduced number o f s u r f a c e  processes.  Figures  density,  Figure  36C shows a macrophage e n g u l f i n g  Such a s s o c i a t i o n w i t h c e l l s o r d e b r i s 5.  Eosinophil  The  e o s i n o p h i l i s another c e l l  another  i s not uncommon.  type found i n the g r a f t bed.  regions  i t makes up a s u b s t a n t i a l p o r t i o n of the c e l l p o p u l a t i o n  others,  as i n f i g u r e 25, there  a r e o n l y one or two c e l l s .  n u c l e u s o f an e o s i n o p h i l i s l o b e d , u s u a l l y 2 o r 3 lobes  The c e l l i s 9  s i z e and s e v e r a l m i t o c h o n d r i a .  characterize  the e o s i n o p h i l .  vesicles varying  The  are p r e s e n t i n a  s e c t i o n , and the chromatin i s d e n s e l y packed a l o n g the n u c l e a r W i t h i n the cytoplasm a r e a few s t r a n d s  I n some  while i n  t o 13 u i n diameter i n e l e c t r o n micrographs ( f i g u r e s 37A and 37B).  varying  size  36A and 36B a r e examples o f two u l t r a s t r u c t u r a l s t a t e s  of t h i s a c t i v a t e d form. cell.  form a r e the i n c r e a s e d  In c o n t r a s t t o  membrane.  o f RER, a few empty v e s i c l e s o f A l s o p r e s e n t a r e the g r a n u l e s which  These a r e membrane-bound, e l e c t r o n - d e n s e  from .5 t o 1 u i n diameter.  Most c o n t a i n a s t i l l  denser  e l e c t r o n band w i t h i n t h e i r c e n t r a l area which appears i n s e c t i o n s as a  84  Figure  36A:  " A c t i v a t e d " macrophage found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2 ) . The b a r r e p r e s e n t s l u ,  Figure  36B:  " A c t i v a t e d " macrophage found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  Notice  the i n c r e a s e d number o f e l e c t r o n dense v a c u o l e s compared  to f i g u r e 36A.  Figure  36C:  The b a r r e p r e s e n t s l u .  " A c t i v a t e d " macrophage i n the p r o c e s s o f p h a g o c y t o s i s w i t h i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2). The b a r r e p r e s e n t s l u .  85  Figure  37A:  E o s i n o p h i l found i n the g r a f t bed of a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2)  Note t h a t the e l e c t r o n dense g r a n u l e s c o n t a i n a l e s s dense e l e c t r o n band w i t h i n them.  Figure  37B:  The b a r r e p r e s e n t s l y  E o s i n o p h i l found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  Note t h a t the e l e c t r o n dense g r a n u l e s c o n t a i n a s t i l l  denser e l e c -  t r o n band w i t h i n t h e i r c e n t r a l area which appears i n s e c t i o n s as a r e c tangle.  Figure  The b a r r e p r e s e n t s l y  37C:  F i g u r e 37D:  N e u t r o p h i l found i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t ( s c o r e : 2)  Degenerating n e u t r o p h i l found i n the g r a f t bed o f a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2). The b a r r e p r e s e n t s l y  86  square o r r e c t a n g l e ( f i g u r e 37B). the same p o s i t i o n 6.  I n some, a l e s s dense band o c c u p i e s  ( f i g u r e 37A) i n the g r a n u l e .  Neutrophil  Another  c e l l type found i n the g r a f t bed o f a l l o g r a f t s on the  mutant and non-mutant h o s t s i s the n e u t r o p h i l .  This c e l l  i s always p r e s e n t ,  and sometimes, i t makes up a s u b s t a n t i a l p o r t i o n o f the c e l l p o p u l a t i o n ( f i g u r e 25). The n u c l e u s i s m u l t i - l o b e d and c o n s i s t s almost e n t i r e l y o f clumped chromatin  ( f i g u r e s 37C and 37D).  W i t h i n the cytoplasm a r e a few  m i t o c h o n d r i a and some c l e a r v a c u o l e s a p p r o x i m a t e l y  .01 y i n diameter.  A  g o l g i i s u s u a l l y l o c a t e d c e n t r a l l y i n the c e l l , as seen i n f i g u r e 37C. A l s o i n the cytoplasm a r e a number o f e l e c t r o n dense g r a n u l e s  approximately  .1 t o .3 y i n diameter which are a c h a r a c t e r i s t i c o f the n e u t r o p h i l . the n e u t r o p h i l s appear as i n f i g u r e 37D. tion.  This c e l l  Often  shows s i g n s o f degenera-  The n u c l e a r membrane i s s w o l l e n and w i t h i n the c y t o p l a s m t h e r e a r e a  l a r g e number o f empty v e s i c l e s as w e l l as e l e c t r o n dense v a c u o l e s o f v a r y i n g size. in  The background m a t r i x o f the cytoplasm appears  the l i m i t s o f the c e l l 7.  membrane.  Fibroblast  Fibroblasts of  to have clumped w i t h -  the g r a f t bed.  ( f i g u r e 38B and 38C) a r e a l s o p a r t o f t h e c e l l p o p u l a t i o n I n e l e c t r o n micrographs  t h e s e c e l l s a r e 11 t o 13 y i n  diameter and a r e o f t e n a s s o c i a t e d w i t h c o l l a g e n b u n d l e s .  They have a l a r g e  o v a l n u c l e u s which c o n t a i n s f i n e l y d i s p e r s e d chromatin and one o r two p r o minent n u c l e o l i . of  The prominent  RER as shown i n the i n s e r t  f e a t u r e o f the cytoplasm i s the abundance  ( f i g u r e 38A).  There a r e a l s o a number o f  87  F i g u r e 38A:  F i b r o b l a s t found i n the g r a f t bed of a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2) The bar r e p r e s e n t s l y .  Figure  38B:  Stacks of rough endoplasmic r e t i c u l u m o f a f i b r o b l a s t i n the g r a f t bed o f a 6 day s k i n a l l o g r a f t . The b a r r e p r e sents l y .  Figure  38C:  F i b r o b l a s t found i n the g r a f t bed o f a s k i n a l l o g r a f t removed 6 days f o l l o w i n g t r a n s p l a n t a t i o n ( s c o r e : 2) The b a r r e p r e s e n t s l y .  88 mitochondria present, and a w e l l developed golgi i s often c e n t r a l l y l o cated.  Sometimes there are a few electron dense granules i n the cytoplasm.  The shape of the fibroblasts i s very i r r e g u l a r , depending on t h e i r assoc i a t i o n with other c e l l s or collagen bundles. The essential features of the ultrastructure of each c e l l type found i n the graft bed o f a a l l o g r a f t s on both mutant and non-mutant host are summarized i n table IX.  Table I X Cell  E s s e n t i a l f e a t u r e s o f t h e u M t r a s t r u c t u r e o f each c e l l type found w i t h i n t h e s k i n a l l o g r a f t on b o t h mutant and non-mutant h o s t s  type  approximate  Nuclei  Nucleoli  Ribosomes  RER  Golgi  v e r y z: sparse  'G~ p o o r l y developed  size  site  Vesicles & Vacuoles  MitoOther chondria  S m a l l Lymphocyte a. " r e s t i n g " state  5 t o 7y i n diameter  round and i n dented; l a r g e amount o f heterochromatin  small  free  b. w i t h "minor change  5 t o 8y i n diameter  increase i n the amount o f euchromatin  more prominent  increase i n increase # & % aggre-in # of gates strands  Immunoblas t  9 t o l l y i n round  1 or 2  diameter  strands  & finely scattered chro. matin;thin electron-dense band a t n u c l e a r membrane  r  prominent  abundance of p o l y ribosomes  few e l e c t r o n dense v a c u oles present  p r e s e n t same as above  few, small  centrio l e usually present  same as above  few e l e c t r o n dense v e s i cles  few  well developed  few e l e c t r o n dense v a c u oles; l o t of vesicles  present  well developed  l o t of both; some e l e c t r o n dense granules  Activated Lymphocyte a. l e s s d i f ferentiated  more d i f f e r entiated  8 t o lOu i n diameter  large & irreg u l a r shape; moderate amount o f clumping  8 t o l l y i n same as diameter above  present  abundantoccur singly  few s c a t tered strands  present  abundantmore abunmost o c c u r dant i n rosettes  increased i n numbers  (continued) 00  T a b l e IX  Cell  (continued)  type  Approximate size  Nuclei  Nucleoli  Ribosomes  RER  Golgi  Vesicles & Vacuoles  Mitochondria  Other  Macrophages a. n o n - a c t i v a ted  10 to  14u  Horse-shoeshaped; d i s persed chromatin  present  few  scattered strands  present  electron dense v a cuoles o f varying size; large & small vesicles  several  cell mem brane un even con tour  b. a c t i v a t e d  12 to  15u  irregular shape more clumped chromatin  present  few  scattered strands  well developed  increased #, d e n s i t y and s i z e of e l e c t r o n dense vacuoles  several  reduced # at surf a c e processes  Neutrophil  10 to  12u  l o b e d ; chrom a t i n densel y packed  absent  few  scattered short strands  centrally located  clear vacuoles; # of • dense granuoles  Eosinophil  10 to  13u  l o b e d ; chrom a t i n denser l y packed  absent  few  few strands  present  few e l e c several tron vesicles containing electron dense bands  Fibroblast  11 to  14u  large;oval; finely dispersed chromatin  1 or 2 prominent  few  abundant  well developed  few e l e c t r o n dense granules  few  several  91  DISCUSSION  The s k i n a l l o g r a f t  response: g e n e r a l  remarks  With r e g a r d to the s k i n a l l o g r a f t r e s p o n s e , o n l y v e r y few p o i n t s have become c l e a r as to what i s happening at the g r a f t s i t e . (1964) s t u d i e s , a l o n g w i t h the work o f o t h e r s ( H a l l , 1967; Cooper, 1972; Moore and H a l l , 1972;  Prendergast's  Lance and  1973; N a j a r i a n and Feldman,  c l e a r l y demonstrate that o n l y a s m a l l percentage o f the c e l l s  1962),  infiltrating  the g r a f t s i t e a r e s p e c i f i c a l l y s e n s i t i z e d t o t h e s k i n a n t i g e n s .  These  c e l l s have a r i s e n i n the r e g i o n a l lymph node i n response t o the p r e s e n c e o f the g r a f t .  P r e n d e r g a s t e s t i m a t e s , on average, t h a t 5.6%  t r a t i n g lymphocytes are s p e c i f i c a l l y s e n s i t i z e d .  o f the  infil-  The r e s t o f the g r a f t bed  c e l l population c o n s i s t s p r i m a r i l y of n o n - s p e c i f i c e f f e c t o r c e l l s ,  including  those o f monocyte-macrophage l i n e a g e a r i s i n g from the bone marrow (Giroud et S L I . , 1970; Kongshavin and Lapp, 1973; Volkman and Gowans, 1965) s e n s i t i z e d lymphocytes.  and  non-  A c c u m u l a t i o n of b o t h the s p e c i f i c and the "uncom-  m i t t e d " e f f e c t o r c e l l s appears t o be an e s s e n t i a l component o f the s k i n g r a f t response ( B i l l i n g h a m and S i l v e r s , 1971; and Frank, 1970).  Giroud e t a l . ,  1970;  allo-  Lambert  N e i t h e r the cause of t h i s massive a c c u m u l a t i o n o f c e l l s  at the g r a f t s i t e , nor the c e l l u l a r i n t e r a c t i o n and mechanisms o f a c t i o n which r e s u l t i n the d e s t r u c t i o n o f the g r a f t e d t i s s u e are known. The o n l y h i n t as to what may be happening a r i s e s from i n v i t r o dies.  stu-  Of p r i m a r y i n t e r e s t here a r e the t i s s u e c u l t u r e s t u d i e s o f lympho-  c y t e a c t i v i t y which demonstrate t h r e e i m p o r t a n t p r o p e r t i e s o f t h e lymphocyte. The s p e c i f i c a l l y s e n s i t i z e d lymphocyte responds o n l y to t h e s t i m u l a t i n g  92  antigen, thus demonstrating a c a p a b i l i t y for s p e c i f i c recognition.  Second-  l y , upon proper stimulation the lymphocyte i s capable of c y t o t o x i c i t y , damaging the target c e l l by some unknown mechanism.  Further, upon recog-  n i t i o n of s p e c i f i c antigen the lymphocyte, i n v i t r o at l e a s t , i s capable of releasing any one or more of a number of effector molecules, referred to as lymphokines (Dumonde et a l . , 1969).  Included  i n this group of affector  molecules are substances that i n h i b i t macrophage migration 1972;  Lamelin, 1971;  Pick and Turk, 1972;  (David and David,  and Pick, 1974), that are chemo-  taxic for neutrophils (Ward et a l . , 1970), eosinophils (David and David, 1972)  or mononuclear c e l l s (David and David, 1972), that stimulate  tized lymphocytes to transform  to blast c e l l s and divide (Falk et a l . , 1970;  Dumonde et a l . , 1969), that stimulate macrophages, increasing a b i l i t y (Nathan ejt a l . , 1973) (Evans and Alexander, 1972;  unsensi-  phagocytotic  and "arming" them for d i r e c t c y t o t o x i c i t y  Evans and Grant, 1972;  Lohmann-Matthes et a l . ,  1973) , that stimulate the p r o l i f e r a t i o n of "granulocytic and macrophage precursor populations"  (Parker and Metcalf, 197&).  These properties of the lymphocyte suggest that the lymphocyte popul a t i o n , e s p e c i a l l y the s p e c i f i c a l l y sensitized portion, may  play an important  r o l e i n the accumulation and a c t i v a t i o n of the c e l l s at the graft s i t e . Upon i n t e r a c t i o n with and recognition of the s p e c i f i c antigen within the graft s i t e the few sensitized lymphocytes present produce and release e f f e c tor molecules capable of either a t t r a c t i n g or a c t i v a t i n g , or both, macrophages, eosinophils, neutrophils and non-sensitized lymphocytes.  Such ac-  t i v i t y would, i n fact, place these sensitized lymphocytes i n an i n i t i a t i n g role i n the mediation of graft destruction.  93  The  lymphocyte:  L i g h t microscope  A l i g h t microscope  study  s t u d i e s of the s k i n a l l o g r a f t  response  commonly  c i t e the lymphocyte as a major component of the i n f i l t r a t i n g c e l l t i o n (Medawar, 1944;  Simar and B e t z , 1970;  B i l l i n g h a m and S i l v e r s ,  The o b s e r v a t i o n s and d a t a on c e l l counts p r e s e n t e d i n t h i s sistent with this finding.  popula1971).  t h e s i s are  con-  Depending on the time e l a p s e d s i n c e t r a n s p l a n -  t a t i o n , the lymphocyte can account t i o n i n f i l t r a t i n g the g r a f t s i t e  f o r up to 60% o f the h o s t c e l l  (table VIII).  Since l i g h t  popula-  microscope  s t u d i e s of the g r a f t bed have been l i m i t e d , i n the m a j o r i t y o f c a s e s , to paraffin-embedded  t i s s u e s e c t i o n s i n which the s u r r o u n d i n g t i s s u e compon-  ents i n t e r f e r e w i t h attempts  to i d e n t i f y c e l l types p r e c i s e l y ,  further  c l a s s i f i c a t i o n of the lymphocyte s u b - p o p u l a t i o n s has not been p o s s i b l e . By e x t r a c t i n g the c e l l s from the g r a f t bed, as i n t h i s s t u d y , i t becomes p a s s i b l e to i d e n t i f y d i f f e r e n t types o f lymphocytes,  based  on the morpho-  l o g i c a l d e s c r i p t i o n s from s t u d i e s o f .blood smears ( E l v e s , 1966; and o f b l o o d and lymphoid  c e l l s i n tissue culture.  The  Ling,  1968)  lymphocyte p o p u l a -  t i o n i s o l a t e d here has been d i v i d e d i n t o the f o l l o w i n g c a t e g o r i e s ; s m a l l lymphocyte,  l a r g e lymphocyte;  I t was  transformed lymphocyte and b a s o p h i l i c  hoped t h a t t h i s more p r e c i s e i d e n t i f i c a t i o n of  cell.  lymphocytes  would p r o v i d e some i n s i g h t as to the k i n d s of lymphocyte a c t i v i t y o c c u r r i n g w i t h i n the g r a f t s i t e .  However, accumulative i n f o r m a t i o n i n d i c a t e s  that  p r e c i s e f u n c t i o n a l c l a s s i f i c a t i o n o f the lymphocyte i s not p o s s i b l e by s t r i c t l y m o r p h o l o g i c a l c r i t e r i a s i n c e a group o f m o r p h o l o g i c a l l y s i m i l a r c e l l s may  be f u n c t i o n a l l y q u i t e d i v e r s e .  to base c o n c l u s i o n s about  T h e r e f o r e , i t may  lymphocyte a c t i v i t y on s t r i c t l y  be m i s l e a d i n g  morphological  94  criteria. In t h i s s t u d y , s m a l l and as  those of mouse b l o o d and  l a r g e lymphocytes, s t r u c t u r a l l y the same  lymph (Schermer, 1967), appear to peak i n con-  c e n t r a t i o n , r e l a t i v e to the o t h e r c e l l t y p e s , transplantation Jaboisiak  (chart I ) .  (1971) who,  t i n g a s k i n g r a f t and days f o l l o w i n g The  following  T h i s i s c o n s i s t e n t w i t h the f i n d i n g s  u s i n g mice, s i m i l a r l y i s o l a t e d the  cells  of  infiltra-  found t h a t the number o f lymphocytes i s h i g h e s t  s m a l l lymphocyte, i d e n t i f i e d w i t h the l i g h t m i c r o s c o p e ,  authors c o n s i d e r  d i v e r s e number o f m e t a b o l i c a c t i v i t i e s .  the s m a l l lymphocyte to be  to i t s undeveloped cytoplasm and  (Zucker-Franklin, stimulated"  1969).  state.  stimulation.  a " r e s t i n g " or  They can  or s p e c i f i c r e c o g n i t i o n o f the  "un-  strictly  sensiti-  to d i f f e r e n t i a t e f u r t h e r f o l l o w i n g  "morphologically"  " i n a c t i v e " as  states that small  proper  lymphocytes  s m a l l lymphocyte as seen w i t h  the  f a r as o n l y r e t a i n i n g a  p o t e n t i a l f o r l a t e r a l t e r a t i o n of morphology and W i l s o n (1965, 1967)  "inactive" cell  l a r g e amount of h e t e r o c h r o m a t i n  Data from i n v i t r o t i s s u e c u l t u r e s t u d i e s o f  l i g h t microscope i s not  Many  then be viewed as c e l l s w i t h the p o t e n t i a l  and w i t h the a b i l i t y  suggests t h a t the  has  I t i s suggested t h a t t h e s e c e l l s are i n an  for i n i t i a l r e c o g n i t i o n of antigen zing antigen  6  transplantation.  been a t t r i b u t e d a r a t h e r  due  on the s i x t h day  activity.  In p a r t i c u l a r ,  lymphocytes, removed from the  regional  lymph node or t h o r a c i c duct lymph of mice which have p r e v i o u s l y  rejected a  s k i n a l l o g r a f t , are capable o f d e s t r o y i n g  target  c e l l s containing  monolayer c u l t u r e s o f  the a n t i g e n i c determinants o f the s k i n donor.  s e a r c h groups have a l s o c i t e d the  s m a l l or l a r g e lymphocyte as  Other r e responsible  95 in vitro  f o r s p e c i f i c target c e l l l y s i s  sometimes,  (Able and Rosenau, 1970)  and,  f o r r e l e a s e of e f f e c t o r m o l e c u l e s (Lawerence and Landy,  i 1969).  Another c a t e g o r y o f lymphocytes noted i n t h i s l i g h t microscope study i s the transformed lymphocyte.  I t has not been d e s c r i b e d i n o t h e r  l i g h t microscope o b s e r v a t i o n s as p a r t o f the c e l l p o p u l a t i o n the g r a f t bed.  T h i s i s p r o b a b l y due to the t e c h n i q u e o f i n v e s t i g a t i o n .  C e l l s of the same morphology  have been noted i n a number o f i n s t a n c e s i n  both i n v i t r o and i n v i v o s t u d i e s .  They have been d e s c r i b e d as a r i s i n g  from s m a l l or l a r g e lymphocytes upon s t i m u l a t i o n w i t h PHA studies  infiltrating  ( B i b e r f e l d , 1971; E l v e s , et^ al.,  1964).  i n tissue  culture  H a l l and co-workers demon-  s t r a t e t h e i r appearance i n the e f f e r e n t lymph o f a lymph node s t i m u l a t e d by e i t h e r s o l u b l e or t r a n s p l a n t a t i o n a n t i g e n s , and suggest t h a t the c e l l s have a r i s e n from s m a l l lymphocytes s t i m u l a t e d w i t h i n the lymph node.  In  v i t r o s t u d i e s of lymphocyte c y t o t o x i c i t y d e s c r i b e c e l l s o f s i m i l a r morphol o g y as a r i s i n g from s m a l l lymphocytes phocytes (mixed lymphocyte  culture)  i n the presence o f h e t e r o g e n e i c lym-  (Andersson and Hayry, 1973; Hayry e t a l . ,  1972) , or from s p e c i f i c a l l y s e n s i t i z e d s m a l l lymphocytes i n the presence o f the s e n s i t i z i n g a n t i g e n ( H e i s e and Weiser, 1969;  Granger and K l o b , 1968).  In t h e s e i n s t a n c e s the c e l l s have been termed b l a s t c e l l s .  I t a l s o appears  they a r e capable o f s p e c i f i c o r n o n - s p e c i f i c t a r g e t c e l l l y s i s and the r e l e a s e of e f f e c t o r m o l e c u l e s (Granger and K l o b , 1968; Andersson and Hayry, 1973;  Shacks e t a l . ,  1973).  Another lymphocyte i d e n t i f i e d i n t h i s l i g h t microscope study i s r e f e r r e d to as a b a s o p h i l i c c e l l due to i t s v e r y b a s o p h i l i c c y t o p l a s m . comparable  c e l l type i s mentioned  No  i n o t h e r l i g h t microscope i n v e s t i g a t i o n s  96  o f the s k i n a l l o g r a f t response or t i s s u e c u l t u r e s t u d i e s o f activity.  A g a i n , t h i s i s p r o b a b l y due  Jakobisiak  (1971) d e s c r i b e s  c e l l population  lymphocyte  to the t e c h n i q u e of i n v e s t i g a t i o n .  a " b a s o p h i l i c c e l l " as p a r t of  the  infiltrating  at the s k i n g r a f t s i t e , but h i s d e s c r i p t i o n i s not  and  the p r o p o r t i o n  and k i n e t i c s of t h i s b a s o p h i l i c c e l l  ent  from those o f the b a s o p h i l i c c e l l d e s c r i b e d  b a s o p h i l i c s t a i n i n g property  clear  are q u i t e  i n t h i s study.  differ-  The  very  of these c e l l s suggests they are t a k i n g  a c t i v e p a r t i n the a c t i v i t y o c c u r r i n g w i t h i n the g r a f t s i t e .  an  Some of  the  i n v i t r o t i s s u e c u l t u r e s t u d i e s o f lymphocyte c y t o t o x i c i t y have demonstrated that " k i l l e r " c e l l s a r i s i n g from s m a l l lymphocytes when newly formed blast cells  ( i . e . transformed lymphocytes).  these b l a s t c e l l s become s m a l l e r i n s i z e and as k i l l e r  cells  basophilic cell  (Andersson and  Hayry, 1973;  However, i n the o l d e r simultaneously,  Hayry et a l . , 1972).  of--'the. " " o l d e r " c e l l s o f Andersson and  a c t i v e l y s y n t h e s i z i n g m a t e r i a l as p a r t of the c e l l - m e d i a t e d to s k i n a l l o g r a f t  f i n d i n g s from the e l e c t r o n microscope study c o n c e r n i n g  An  (1973),  e l e c t r o n microscope study  c e l l s are d i s c u s s e d  i n a s e p a r a t e s e c t i o n from the  scope study, s i n c e combining the i n f o r m a t i o n  would r e q u i r e an attempt  types observed i n each study to one  another.  d i f f e r e n t means by which the t i s s u e s were t r e a t e d and they were c l a s s i f i e d  and  the i n -  l i g h t micro-  the  to  T h i s I do  t h i n k would be v a l i d w i t h o u t a s p e c i a l study o f t h i s p o i n t due  population.  perhaps  antigens.  The  cell  the  immune response  lymphocyte:  r e l a t e the  Since  Hayry  The  filtrating  cultures  more e f f e c t i v e  forms such a l a r g e p o r t i o n of the c e l l p o p u l a t i o n  they are the e q u i v a l e n t  are  to the  not  grossly  c r i t e r i a by which  i d e n t i f i e d , p a r t i c u l a r l y concerning  the  lymphocyte  97  Lymphocytes have been noted as a major p a r t o f the  cellular  t r a t e i n a l l e l e c t r o n microscope s t u d i e s of the s k i n a l l o g r a f t Some d i s c r e p a n c y  does appear between r e s e a r c h  lymphocytes which are p r e s e n t lymphocytes t h a t c o u l d be  (table X).  infil-  response.  groups as to the k i n d s  of  In t h i s t h e s i s the types o f  i d e n t i f i e d a t the u l t r a s t r u c t u r a l l e v e l  included  the s m a l l lymphocyte, some showing minor changes i n t h e i r u l t r a s t r u c t u r e , the immunoblast and The as b e i n g  the a c t i v a t e d lymphocyte.  s m a l l lymphocyte i s commonly seen w i t h the e l e c t r o n microscope  one  B e t z , 1970;  o f the c e l l s i n f i l t r a t i n g the s k i n a l l o g r a f t Weiner, L a t t e s and  are e a s i l y i d e n t i f i e d by cytoplasmic 1966;  P e a r l , 1969;  A l l e g r a e t a l . , 1968).  by  considered  by  Simar and  as c o n t r i b u t i n g  stimulated.  the s m a l l lymphocytes "with minor changes". i n the g r a f t bed  tissue. attaching PHA  little  described  Betz (1970) i n v e s t i g a t i n g u l t r a In v i t r o s t u d i e s i n d i c a t e  t a k i n g an a c t i v e p a r t i n the d e s t r u c t i o n of the  B i b e r f e l d (1971, 1973)  describes  grafted  c e l l s o f s i m i l a r morphology as  to Chang c e l l s and b r i n g i n g about t h e i r l y s i s i n the presence of  or a n t i b o d y i n t i s s u e c u l t u r e .  Ax  e t a l . (1968) d e s c r i b e c e l l s o f  morphology as capable o f c y t o t o x i c i t y by d i r e c t c e l l c o n t a c t . has  (Elves,  Of i n t e r e s t here  These were a l s o  s t r u c t u r a l l y the s k i n a l l o g r a f t response i n mice. these c e l l s c o u l d be  of  some authors to be " i n a c t i v e "  to the s k i n a l l o g r a f t response u n t i l p r o p e r l y  as p r e s e n t  and  They  abundance o f h e t e r o c h r o m a t i n  They are c o n s i d e r e d  such t h a t , i n t h i s case, they c o u l d be  are  (Simar  t h e i r s i z e , high nucleus/cytoplasm r a t i o , l a c k  o r g a n e l l e development and  L i n g , 1968).  site  a l s o been shown to be  ( H a r r i s e t a l . , 1966).  This  this  cell  capable of p r o d u c i n g hemolysis i n plaque t e s t s  B i b e r f e l d (1971) d e s c r i b e s  c e l l s o f t h i s morphology  as a p p e a r i n g i n c u l t u r e s o f " t y p i c a l " s m a l l lymphocytes, 2 hours  following  Table X  C e l l types i d e n t i f i e d studies  w i t h i n the s k i n a l l o g r a f t  s i t e i n a number o f e l e c t r o n microscope  Simar and Betz (1970) (between 2 to-. 11 days) ( i n mice)  (day  s m a l l lymphocytes  s m a l l lymphocytes  s m a l l lymphocytes  b l a s t c e l l s (as desc r i b e d by H a l l , 1967)  transformed cytes  macrophages  macrophages  immunoblast  histiocytes  monocytes  immature plasma cells  macrophage  monocytes  polymorphonuclear leukocytes  plasma c e l l s  Weiner e t a l . (1964)  A l l e g r a e_t a l . (1968)  (to day 11)  (days 5 and 7)  (in  ( i n rhesus monkeys)  rabbits)  graft rejection c e l l  reticulum  plasma c e l l s ( a f t e r r e j e c t i o n f a r advanced)  lymphocytes  D i d not see:  eosinophils  cells  Weiner e t a l . (1969) ( b e f o r e 7 o r 8 days) ( i n mice)  Collen  (1974)  6 and 8)  ( i n mice)  lympho- a c t i v a t e d cytes  lympho-  neutrophil eosinophil  D i d not see: plasma c e l l s ( s m a l l number)  hemocytoblasts  Did not see: plasma  cells  VO  CO  99  t h e i r i n c u b a t i o n w i t h PHA.  I t i s i n t e r e s t i n g to note that i n c u l t u r e s  of PHA-stimulated lymphocytes, lymphotoxin ( W i l l i a m s and Granger, and macrophage m i g r a t i o n i n h i b i t o r y f a c t o r c o u l d be i s o l a t e d o f PHA,  (Lawerence and Landy,  from the c u l t u r e o n l y 3 to 6 hours f o l l o w i n g  1969) 1969)  introduction  even though, w i t h the l i g h t m i c r o s c o p e , t h e r e d i d not appear to be  any n o t i c e a b l e changes i n the morphology  of the s m a l l lymphocyte.  Another c e l l type found at the g r a f t s i t e i n t h i s study i s the immunoblast,  though Simar and Betz (1971) c l e a r l y s t a t e t h a t a " t y p i c a l  immunoblast"  i s not p a r t o f the c e l l u l a r i n f i l t r a t e , d u r i n g t h e i r s t u d y of  the s k i n a l l o g r a f t  response i n mice.  However, i n my  investigation,  c h a r a c t e r i z e d by the presence of a l a r g e n u c l e u s , a l a r g e n u c l e o l u s , tively l i t t l e  cells rela-  rough endoplasmic r e t i c u l u m and many f r e e ribosomes a r e p a r t  o f the c e l l u l a r  infiltrate.  The immunoblast  ( a l s o r e f e r r e d to as the hemocytoblast or p y r o n i n o -  p h i l i c c e l l ) i s d e f i n e d w i t h t h e s e c h a r a c t e r i s t i c s by s e v e r a l r e s e a r c h groups. Andre-Schwartz  (1964), u s i n g r a b b i t s , and B i n e t and Hathe  (1962), u s i n g mice,  f i n d and d e s c r i b e t h e s e c e l l s which a r i s e w i t h i n the r e g i o n a l lymph node i n response to the presence of a s k i n a l l o g r a f t .  Subsequent work on t h e i r  g i n and p o s s i b l e f a t e show t h a t they a r i s e from s m a l l lymphocytes  ori-  (Gowans,  1962)  and w i l l s u b s e q u e n t l y d i v i d e to g i v e r i s e to medium and s m a l l lympho-  cytes  (Gowans et a l . ,  1962; Andre-Schwartz, 1964), which a r e p o s s i b l y the  e f f e c t o r c e l l s o f the s k i n a l l o g r a f t response ( P r e n d e r g a s t , 1964; Andre et^ al.,  1962).  A c e l l of t h i s morphology  i s a l s o d e s c r i b e d as p r e s e n t w i t h i n  the r e g i o n a l lymph node r e s p o n d i n g to the presence o f s o l u b l e a n t i g e n (Leduc et a l . ,  1968; Avrameas and Leduc, 1970).  I t i s suggested t h a t t h e s e c e l l s  a r e the most " p r i m i t i v e " c e l l type of the plasmacyte o r  lymphoplasmacyte  100  line.  Further,  i t i s demonstrated t h a t these " e a r l i e s t " c e l l s  a n t i b o d y s p e c i f i c f o r the s t i m u l a t i n g a n t i g e n If  contain  ( B o u t e i l l e , 1971).  these c e l l s are the d i r e c t r e s u l t o f a n t i g e n e i c  s t i m u l a t i o n of  s m a l l lymphocytes, the p r e s e n c e of these c e l l s a t the g r a f t s i t e t h a t there may  be  some primary r e c o g n i t i o n ^  i n the g r a f t s i t e d u r i n g would be e q u i v a l e n t  of g r a f t antigen  suggests  occurring  with-  the e f f e c t o r phase o f the a l l o g r a f t response.  This  to t h a t a n t i g e n i c s t i m u l a t i o n o f s m a l l lymphocytes which  o c c u r s w i t h i n the r e g i o n a l lymph node d u r i n g i s sheer s p e c u l a t i o n .  I t i s impossible  the  s e n s i t i z a t i o n phase.  to a t t r i b u t e a p o s s i b l e r o l e to  immunoblast s i n c e i t appears t h a t , a l t h o u g h m o r p h o l o g i c a l l y i s c h a r a c t e r i s t i c o f b o t h the B and  This  T c e l l l i n e s which are  the  identical, i t functionally dis-  tinct. Another type of lymphocyte found at the g r a f t s i t e i s the lymphocyte which makes up t i n g the g r a f t s i t e . and  Pearl  activated  a major p o r t i o n of the mononuclear c e l l s  infiltra-  T h i s i s c o n s i s t e n t w i t h the f i n d i n g s o f Weiner, L a t t e s  (1964) s t u d y i n g  rabbit r e j e c t i o n of s k i n a l l o g r a f t s .  c e l l s o f t h i s morphology as p r e s e n t  They  and p a r t of the c e l l p o p u l a t i o n  through the venule endothelium i n t o the s k i n g r a f t bed. a l s o f i n d these c e l l s i n the g r a f t bed  during  Simar and  describe moving  B e t z (1971)  the s k i n a l l o g r a f t response i n  mice. T h i s c e l l has  been r e f e r r e d to as a b a s o p h i l i c c e l l  1967), transformed lymphocyte (Simar and (Weiner, S p i r o and R u s s e l l , 1964) 1969).  I n every i n s t a n c e  ( H a l l et^ a l _ . ,  B e t z , 1971), g r a f t r e j e c t i o n c e l l  and b l a s t c e l l  (Wiener, L a t t e s and  Pearl,  t h i s c e l l i s c h a r a c t e r i z e d by a l a r g e number o f  f r e e ribosomes, a w e l l developed G o l g i apparatus and  a rudimentary endoplasmic  101  reticulum.  Lymphocytes w i t h t h i s morphology have been noted i n a number  of d i f f e r e n t instances  by  a number of r e s e a r c h  groups.  This  description  i s c h a r a c t e r i s t i c of the  i n v i t r o PHA-transformed lymphocyte ( B i b e r f e l d ,  1971,  Douglas_et a l . , 1967).  1973;  E l v e s , 1966;  t h i s morphology which appear i n the ted by  "soluble proteins,  Further,  i t is cells  e f f e r e n t lymph o f lymph nodes  soluble extracts  of b a c t e r i a and  of  stimula-  helminths,  k i l l e d b a c t e r i a , v i r u s e s , h e t e r o l o g o u s r e d c e l l s , homologous lymphocytes, h e t e r o l o g o u s tumour implants and 1967) .  skin grafts"  ( H a l l et a l . , 1967;  These c e l l s are v e r y a c t i v e m i t o t i c a l l y and  Hall,  have been shown to  be  i m m u n o l o g i c a l l y s p e c i f i c f o r the a n t i g e n which i n d u c e s t h e i r appearance ( H a l l and  Smith, 1971;  H a l l et a l . , 1967;  A g a i n , i t appears t h a t c a l l y to be  small  classified  morphologi-  a m p l i f i c a t i o n of the and,  A number of experiments have demonactivated  Smith, 1970;  c e l l s are r e s p o n s i b l e  f u r t h e r , that  they may  f o r the  Moore and  immune response throughout the  reach t h e i r d e s t i n a t i o n .  lymphocytes i n the  lymph node are d e s t i n e d  i n t e s t i n e ( H a l l and  a l s o been suggested t h a t the  et a l . , 1967)  c e l l types.  "many, perhaps most" o f the  f e r e n t lymph of a s t i m u l a t e d o f the  this c e l l population  composed o f a c t i v a t e d lymphocytes i s a c t u a l l y a composite o f  functionally quite d i s t i n c t s t r a t e d that  Cunningham, et a l . , 1966).  lamina  ef-  propria  H a l l , 1972).  f o r the  It  propagation  lymphoid t i s s u e s  and  (Hall  become plasma c e l l s when they  These c e l l s have been demonstrated as  capable of  p r o d u c i n g a n t i b o d y (Cunningham et a l . , 1966;  P e t r i s and K a r l s b a d , 1971),  possibly  and  lymphotoxin ( F o r e s t e r  e t a l . , 1969)  Tissue  culture studies  target  c e l l c y t o x i c i t y (Weiss, 1968;  has  other e f f e c t o r molecules.  show c e l l s o f t h i s morphology to be Koren and  Ax,  1973).  capable o f d i r e c t  102  The is  f i n a l c e l l type to be  the plasma c e l l .  considered  i n the  lymphocyte  " T y p i c a l " plasma c e l l s as d e s c r i b e d  Leduc (1970) w i t h the " c a r t w h e e l " nucleus and lum were not observed i n the g r a f t bed  (1964) who  Pearl  by Avrameas  of s k i n a l l o g r a f t s 6 to 8 days This  complete.  as an important p a r t of the g r a f t bed  Simar  "young plasma  c e l l population  the  Russell  infiltrate  In c o n t r a s t ,  Betz (1971) d e s c r i b e both " t y p i c a l " plasma c e l l s and  fol-  i s s i m i l a r to  (1969) and Wiener, S p i r o and  found t h a t plasma c e l l s d i d appear i n the c e l l  o n l y a f t e r the r e j e c t i o n response was  and  abundant endoplasmic r e t i c u -  lowing t r a n s p l a n t a t i o n p r i o r to epidermal n e c r o s i s . f i n d i n g s o f Wiener, L a t t e s , and  section  but and  cells"  a l t h o u g h they do  not  s t i p u l a t e at what stage f o l l o w i n g t r a n s p l a n t a t i o n of the s k i n they are  pre-  sent . When viewed w i t h b o t h the e l e c t r o n microscope and scope t h e r e graft site.  appear t o be Further,  each type appears, i n i n v i t r o  synthesis  and  r e l e a s e of lymphokines  graft occurs,  d i v e r s e , i t does not by  the  micro-  r u l e out  and  either  (or a n t i b o d y ) ,  lymphocyte i n the m e d i a t i o n of g r a f t d e s t r u c t i o n  through  research  and  too  specific  d i r e c t target c e l l  controversy.  how  played  lysis.  Attempts to e l u c i d a t e the mechanisms of these lymphocyte a c t i v i t i e s themselves f i e l d s of i n t e n s e  lysis  reveal p o s i t i v e l y  the p o s s i b i l i t y o f an important r o l e  be  o r both.  s i n c e t h e i r p o s s i b l e a c t i v i t i e s are  r e c o g n i t i o n , r e l e a s e of e f f e c t o r m o l e c u l e s and  the  s t u d i e s anyway, to  s e n s i t i z i n g antigen  Thus, a l t h o u g h the presence o f these c e l l s does not d e s t r u c t i o n of the  light  a l a r g e number o f k i n d s of lymphocytes w i t h i n  c a p a b l e o f s p e c i f i c r e c o g n i t i o n of the of t a r g e t c e l l s ,  the  are  103  The bearing  c l a s s o f lymphocytes which mediate g r a f t d e s t r u c t i o n a r e 0  T ( o f thymus o r i g i n ) c e l l s .  c a l l y recognize  a n t i g e n i c determinant s i t e s i s n o t c l e a r .  studies, intimate  contact  between the e f f e c t o r T c e l l  appears t o be n e c e s s a r y b e f o r e et  The means by which T c e l l s  In i n v i t r o  and the t a r g e t  l y s i s of the t a r g e t c e l l w i l l  cell  occur (Ax  a l . , 1968; Hayry e t a l . , 1972; W i l s o n , 1967; Cohen and Feldman, 1971).  I t i s suggested t h a t i n t i m a t e  contact  occurs v i a a s p e c i f i c a n t i g e n i c  n i t i o n u n i t on the lymphoid c e l l  surface.  has  although i t i s f e l t  be  specifi-  proven to be v e r y  difficult,  I d e n t i f i c a t i o n of this  recog-  receptor  t h a t the r e c e p t o r  an immunoglobulin (Crone e t a l . , 1972; Feldman, e t a l . , 1972).  must  It i s  suggested t h a t e a r l y membrane i n t e r a c t i o n which occurs between the immune lymphocyte and the s e n s i t i z i n g a n t i g e n  or n o n - s p e c i f i c i n d u c e r may " t r i g g e r "  the lymphocyte to l y s e the t a r g e t c e l l o r r e l e a s e e f f e c t o r m o l e c u l e s . i s suspected t h a t cyclic-AMP a c t s as a modulator o f lymphocyte  It  activity  (Hadden e t a l . , 1970; Henney, 1973; Strom e t a l . , 1973). From the v a r i o u s  i n vitro  systems used t o i n v e s t i g a t e lymphocyte  c y t o t o x i c i t y , s e v e r a l mechanisms have been proposed by which lymphocyte a c t i v i t y could  d i r e c t l y r e s u l t i n t a r g e t c e l l l y s i s f o l l o w i n g an i n i t i a l  s p e c i f i c r e c o g n i t i o n o f the s e n s i t i z i n g a n t i g e n ; d i a t e d by a c e l l  n o n - s p e c i f i c c y t o l y s i s me-  t o x i n (Granger and K l o b , 1968; Granger and W i l l i a m s , 1971;  H e i s e and Weiser, 1969); s p e c i f i c c y t o l y s i s p o s s i b l y r e q u i r i n g cell Ax,  contact  (Andersgon and Hayry, 1973; Forman and M o l l e r ,  continued  1973; Koren and  1973) : and, n o n - s p e c i f i c l y s i s i n v o l v i n g antibody bound t o the t a r g e t  cells 1974) .  (Perlmann and Holm, 1969; Perlmann and Perlmann, 1970; Perlmann e t a l . , An important p o i n t t o note i s the s u g g e s t i o n  t h a t l y s i s may be  104  n o n - s p e c i f i c , as suggested by as suggested by  the involvement of c e l l  i n both i n v i t r o and  matter o f c o n s i d e r a b l e  controversy  Singh et a l . , 1973; With r e g a r d  portant  i n vivo studies.  ( E i f e and  August, 1973;  t a i n two H-2  This i s a  K l e i n and  Klein,  Svedmyr and Hodes, 1970).  to the s k i n a l l o g r a f t response there has  been one  i n v i v o study which s t r o n g l y suggests t h a t the l y t i c  lymphocytes w i t h i n the s k i n g r a f t i s h i g h l y s p e c i f i c 1970).  specific,  the demonstration of o n l y s p e c i f i c l y s i s o f the s e n s i t i z i n g  membrane-bound a n t i g e n  1972;  t o x i n s , or  A l l o p h e n i c mice are used as s k i n donors.  (Mintz  im-  a c t i o n of and  Silvers,  Thus, a g r a f t w i l l  con-  homozygous s u b - p o p u l a t i o n s o f c e l l s w i t h d i f f e r e n t a l l e l e s at  locus.  When p l a c e d on a member of one  found t h a t the c e l l s  c a r r y i n g the same H-2  parental isogeneic antigens  s t r a i n , i t was  as the host  survived  w h i l e c e l l s w i t h f o r e i g n H-2  antigens  s u r v i v a l of m e l a n o b l a s t s and  h a i r f o l l i c l e c e l l s w i t h i n an a r e a o f lympho-  cyte c y t o l y s i s against In v i t r o o f a n t i g e n by  f o r e i g n H-2  were d e s t r o y e d .  the  There was  antigens.  t i s s u e culture studies i n d i c a t e that s p e c i f i c  lymphocytes may  selective  recognition  a l s o r e s u l t i n the r e l e a s e o f s o l u b l e sub-  stances which have v a r y i n g b i o l o g i c a l e f f e c t s ( d i s c u s s e d p r e v i o u s l y ) be  the means by which a few  s e n s i t i z e d lymphocytes a t t r a c t and  numbers o f n o n - s p e c i f i c c e l l s c u l e s are s y n t h e s i z e d 1972;  P i c k and  subsequent to lymphocyte s t i m u l a t i o n  Turk, 1972).  can be produced by  i n the homograft r e s p o n s e .  I t i s not known how  The  and  involve  may  large  e f f e c t o r mole-  (David  and  David,  many d i f f e r e n t molecules  the same immune lymphocyte.  The macrophage:  A l i g h t microscope study  Another major component of the mononuclear c e l l p o p u l a t i o n  found at  105  the g r a f t s i t e and d e s c r i b e d i n t h i s study a r e the macrophages.  A number  of l i g h t microscope s t u d i e s have c i t e d the macrophage as b e i n g a major p a r t o f t h e h o s t c e l l p o p u l a t i o n i n f i l t r a t i n g the g r a f t s i t e .  Radiotracer  s t u d i e s i n d i c a t e t h a t , as i n d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n s  (Volkman  and Gowans, 1965), these c e l l s a r e n o n - s e n s i t i z e d e f f e c t o r c e l l s o f bonemarrow o r i g i n  (Kongshavin and Lapp, 1973; Giroud e t a l . ,  study the c e l l s c l a s s i f i e d t e r i s t i c o f macrophages  1970).  In t h i s  as macrophages, a l t h o u g h showing t r a i t s  charac-  ( i n c l u d i n g low n u c l e a r / c y t o p l a s m i c r a t i o and a non-  b a s o p h i l i c cytoplasm) d i d v a r y somewhat i n t h e i r morphology.  T h i s i s con-  s i s t e n t w i t h the f i n d i n g s o f J a k o b i s i a k (1971) who d e s i g n a t e d t h i s o f c e l l s macrophage-like.  category  The v a r i a t i o n s i n morphology w i t h i n the macro-  phage p o p u l a t i o n are not s u r p r i s i n g .  The macrophages have been i s o l a t e d  from a s i t e i n which t h e r e i s an abundant s u p p l y o f s t i m u l i ,  including  t i s s u e d e b r i s and a c t i v a t e d lymphocytes, which as demonstrated by i n v i t r o s t u d i e s , can t r i g g e r a l t e r a t i o n s i n macrophage morphology, as w e l l as a c tivity  (Nathan et_ a l . ,  1973; N e l s o n , 196 ; W i l l i a m s and Mayhew, 1973).  I t has been c l e a r l y demonstrated t h a t the p r o p o r t i o n o f h i s t o c h e m i c a l l y a c t i v e macrophages i n the c e l l u l a r i n f i l t r a t e o f mouse s k i n  allografts  i n c r e a s e s markedly d u r i n g the homograft response, as demonstrated by quant i t a t i o n o f a c i d phosphatase and l y s o s m a l p r o t e a s e a c t i v i t y o f the i n f i l trate c e l l s  ( P o u l t e r e_t a l . , 1971) .  L i g h t microscope s t u d i e s show t h a t  macrophage a c t i v a t i o n i n the presence o f proper s t i m u l i i s expressed by i n creased c e l l s i z e , i n c r e a s e d n o n - s p e c i f i c p h a g o c y t i c a c t i v i t y and i n c r e a s e d proliferation  (Nathan e t a l . ,  1973).  These c h a r a c t e r i s t i c s a r e expressed by  macrophages removed from animals d u r i n g d e l a y e d h y p e r s e n s i t i v i t y by macrophages i n v o l v e d i n i n v i t r o  reactions,  c e l l mediated immunity and by macrophages  106  a c t i v a t e d i n v i t r o by  a lymphokine, macrophage a c t i v a t i n g f a c t o r  (Evans and A l e x a n d e r , 1972;  Nathan e_t a l . , 1971,  1973;  (MAF)  Godal et a l . , 1971).  Thus, depending on the f u n c t i o n a l s t a t e of the c e l l , e n t r a n c e i n t o g r a f t s i t e could be  f o l l o w e d by  such c e l l u l a r changes as monocyte d i f f e r -  e n t i a t i o n i n t o macrophage; m i t o t i c d i v i s i o n o f macrophage or the o f the " a c t i v a t e d " c h a r a c t e r i s t i c s . These f a c t o r s e x p l a i n the a morphologically The  d i v e r s e macrophage  d a t a gathered on c e l l  macrophages may c e l l types,  groups.  C57B1 mice, t h a t the h i g h e s t  f i n d i n g of  population.  concentration,  8 days f o l l o w i n g t r a n s p l a n t a t i o n . research  acquiring  counts i n t h i s study suggest t h a t  be a t t h e i r h i g h e s t  findings of other  the  with respect  the  to the  This i s consistent with  G i l l e t t e and Lance (1971) f i n d ,  using  transplantation.  S i m i l a r l y , P o u l t e r e t a l . (1971), a g a i n u s i n g HALB/c mice, f i n d t h a t 10 f o l l o w i n g t r a n s p l a n t a t i o n the h i g h e s t s i t e i s formed by h i s t o c h e m i c a l l y (1971), i s o l a t i n g the g r a f t bed t h e i r peak i n number occurs on  In c o n t r a s t ,  from s k i n g r a f t s on A/PI  the s i x t h day  Jakobisiak  mice, noted  following transplantation.  l i e i n the techniques and  The  c r i t e r i a used  i d e n t i f y the macrophages at the g r a f t s i t e .  macrophage:  An  e l e c t r o n microscope study  Some f e a t u r e s which are c h a r a c t e r i s t i c o f macrophages at the s t r u c t u r a l l e v e l are low n u c l e u s / c y t o p l a s m r a t i o and of phagocytic  days  p r o p o r t i o n of the c e l l s at the g r a f t  a c t i v e macrophages.  cells  s l i g h t d i f f e r e n c e s i n r e s u l t s may  The  the  accumulation o f ^"'"Cr-labelled macrophages at  the s k i n a l l o g r a f t s i t e occurs around 9 to 10 days f o l l o w i n g  to l o c a t e and  other  v a c u o l e s and  lysosomes.  ultra-  the p r e s e n c e of a number  These c r i t e r i a f o r i d e n t i f y i n g macro-  phages are most r e l i a b l e i n s i t u a t i o n s where they are l i k e l y p h a g o c y t o s i n g , o r are o t h e r w i s e s t i m u l a t e d .  to be a c t i v e l y  Such an environment i s s u p p l i e d  107  by  the s k i n g r a f t s i t e .  The  macrophages were found to be  o f a c t i v a t i o n , as o u t l i n e d by Nathan et a l . (1971). macrophages contained  at varying  stages  The more a c t i v a t e d  a l a r g e r number of m i t o c h o n d r i a and  lysosomes i n t h e i r  cytoplasm (Williams  and Mayhew, 1973).  suggesting  f u n c t i o n a l s t a t e o f the macrophage can vary w i t h i n  t h a t the  Some monocytes were a l s o noted,  graft s i t e , r e s u l t i n g i n a morphologically The  d i v e r s e macrophage  population.  r o l e o f the macrophage i n the s k i n a l l o g r a f t r e s p o n s e as a  s e n s i t i z e d e f f e c t o r c e l l i s not absence of the n o n - s e n s i t i z e d  population  of c e l l s at the g r a f t s i t e  Frank, 1970).  The  t o s i s i s w e l l accepted as an a c t i v i t y o f the macrophage.  will  I t has  Phagocya l s o been  i n v i t r o s t u d i e s , t h a t the macrophages, when a p p r o p r i a t e l y  "armed", are capable o f c e l l to c e l l c o n t a c t  lysis.  T h i s "arming" p r o b a b l y  o c c u r s i n a s s o c i a t i o n w i t h the immune lymphoid system (Hersey, 1973; Weiser, 1964  Since  and  1966;  Evans and A l e x a n d e r , 1972;  a c t i v a t e d lymphocytes are p r e s e n t  a c t i v a t i o n c o u l d occur and, The  Grant, 1972).  t h e r e f o r e , p o s s i b l y macrophage t a r g e t c e l l I t i s not  the macrophage s p e c i f i c a l l y l y s e s the t a r g e t  The polymorphonuclear l e u k o c y t e s : study  there  Evans and  Granger  w i t h i n the g r a f t s i t e macrophage  arming i s i m m u n o l o g i c a l l y s p e c i f i c .  present  since  macrophages were  o f t e n observed i n the a c t o f e n g u l f i n g c e l l s o r c e l l u l a r d e b r i s .  suggested by  non-  c l e a r , a l t h o u g h i t i s an e s s e n t i a l one  r e s u l t i n g r a f t s u r v i v a l (Lambert and  and  the  c l e a r l y understood  lysis. how  cell.  A l i g h t and  e l e c t r o n microscope  Polymorphonuclear l e u k o c y t e s ,  both n e u t r o p h i l s and  at the g r a f t s i t e .  transplantation of allogeneic skin,  are two  Following  eosinophils,  i n s t a n c e s when the primary c e l l type i n f i l t r a t i n g the  t i s s u e i s the n e u t r o p h i l ; d u r i n g  the p e r i o d o f primary wound h e a l i n g  are  grafted and  \  108  d u r i n g the f i n a l stages of g r a f t r e j e c t i o n .  T h i s was  demonstrated i n t h i s  study and has been shown i n both l i g h t and e l e c t r o n microscopy o f r e s e a r c h groups (Wiener, and B e t z , 1971). inflammatory  Medawar, 1944;  The n e u t r o p h i l i s the c h a r a c t e r i s t i c c e l l  exudates of n o n s p e c i f i c inflammation.  e a r l y accumulation, and  L a t t e s and P e a r l , 1969;  by a number Simar  type i n e a r l y  "This c o u l d e x p l a i n  and p o s s i b l y a l s o the l a t e r one,  as due  to t i s s u e damage  trauma r e s u l t i n g from the p r o c e s s o f t r a n s p l a n t a t i o n or t i s s u e d e s t r u c -  tion.  The  cause of i t s accumulation  at inflammation  sites  i s not  clear  ( H a r r i s , 1960). The main f u n c t i o n of the n e u t r o p h i l i s p h a g o c y t o s i s and t h i s  (Cochrane,  i s p r o b a b l y i t s main o c c u p a t i o n w i t h i n the g r a f t s i t e and  The p e r i o d s d u r i n g a l l o g r a f t r e j e c t i o n of i t s h i g h e s t accumulation the p e r i o d s o f the most severe t i s s u e damage and, damaged c e l l s and c e l l u l a r d e b r i s .  tissues. are  thus, accumulation  also  of  The n e u t r o p h i l e n g u l f s and, when p o s s i -  b l e , d i g e s t s the i n j e s t e d p a r t i c l e s .  D e s t r u c t i o n of the i n j e s t e d m a t e r i a l  i s mediated by i n t r a c e l l u l a r enzymes c o n t a i n e d w i t h i n the g r a n u l e s o f the n e u t r o p h i l .  1968)  characteristic  The enzymes c o n t a i n e d w i t h i n t h e s e lysosomes  i n c l u d e a c i d phosphatase, a l k a l i n e - p h o s p h a t a s e , c o l l a g e n a s e , r i b o n u c l e a s e , d e o x y r i b o n u c l e a s e , l i p a s e , ^ - g l u c u r o n i d a s e and p r o t e o l y t i c enzymes.  Also  i n c l u d e d are b a s i c p r o t e i n s (one e f f e c t s d e g r a n u l a t i o n o f mast c e l l s  while  the o t h e r 3 i n c r e a s e v a s c u l a r p e r m e a b i l i t y ) and a n t i b a c t e r i a l lipozyme  and p h a g o c y t i n  (Cochrane,  substances,  1968).  P h a g o c y t o s i s o f the t i s s u e d e b r i s and immune complexes, i f p r e s e n t , r e s u l t s i n degrees  o f d e g r a n u l a t i o n of the n e u t r o p h i l .  s t r a t e d w i t h the e l e c t r o n microscope  I t has been demon-  t h a t there i s f u s i o n of the lysosome  g r a n u l e w i t h the p h a g o c y t i c v a c u o l e which r e s u l t s i n the emptying of the  109  enzyme contents i n t o the v a c u o l e c o n t a i n i n g complex.  i n s t a b i l i t y may o c c u r t o the extent  may  o f the l y s o s o m a l membranes.  Such  t h a t t h e enzyme c o n s t i t u e n t s a r e r e -  i n , g r e a t e r amounts i n t o the s u r r o u n d i n g t i s s u e s . The  be  p a r t i c l e o r immune  P h a g o c y t o s i s o f p a r t i c l e s and t h e i r subsequent d e g r a n u l a t i o n  l e a d t o c o n d i t i o n s which f a v o r i n s t a b i l i t y  leased  the e n g u l f e d  r e l e a s e o f these m a t e r i a l s  instrumental  i n t o surrounding g r a f t t i s s u e s could  i n the f i n a l stages o f g r a f t d e s t r u c t i o n .  c l e a r l y demonstrated i n v a r i o u s  I t has been  forms o f immune r e n a l d i s o r d e r s t h a t a t t r a c -  t i o n o f n e u t r o p h i l s by the complement system f o l l o w e d by engulfment o f immune complexes can r e s u l t i n the r e l e a s e of the lysosome c o n s t i t u e n t s surrounding t i s s u e structures  (Cochrane, 1968).  i n t o the  I t seems as though, w h i l e  c a r r y i n g out t h e i r j o b o f r i d d i n g the t i s s u e s o f b a c t e r i a o r immune complexes, the v e r y tures.  c o n s t i t u e n t s o f these c e l l s d e s t r o y  the s u r r o u n d i n g t i s s u e s t r u c -  The p o s s i b i l i t y e x i s t s t h a t the c o n d i t i o n s a t the g r a f t s i t e may be  such that i n s t a b i l i t y of the l y s o s o m a l membrane r e s u l t s and o f the n e u t r o p h i l s o c c u r s ,  degranulation  r e l e a s i n g enzyme c o n t e n t s i n t o g r a f t t i s s u e s .  T h i s c o u l d f e a s i b l y a i d i n the f i n a l d e s t r u c t i o n of the g r a f t e d t i s s u e . Another c e l l type noted i n t h i s study a t p r e s e n t filtrate  during  s k i n g r a f t d e s t r u c t i o n i s the e o s i n o p h i l .  i n the c e l l u l a r i n Neither  the k i n e -  t i c s n o r the means by which i t accumulates a t the s i t e , a r e c l e a r . An a c c u m u l a t i o n o f e o s i n o p h i l s i s u s u a l l y a s s o c i a t e d w i t h a n a p h y l a x i s o r p a r a s i t i c i n f e s t a t i o n , a l t h o u g h e o s i n o p h i l s have a l s o been noted around tumours, i n h e a l i n g wounds, i n b u r n t t i s s u e s and i n the p r e s e n c e o f immune complexes (Archer,  1963).  The f u n c t i o n of the e o s i n o p h i l and i t s r o l e i n  immunological r e a c t i o n s remains unknown ( G l e i c h e t a l . , 1973; al.,  1973).  Mahmoud e_t  The e o s i n o p h i l s phagocytose m a t e r i a l , a l t h o u g h t h e i r  efficiency  110  i s l e s s than that o f the n e u t r o p h i l  (Cotran  and L i t t ,  1969).  c o n t a i n c h a r a c t e r i s t i c granules which may be d e s i g n a t e d to n e u t r o p h i l s  These  lysosomes.  cells Similar  and macrophages, engulfment o f p a r t i c l e s by e o s i n o p h i l s i s  f o l l o w e d by membrane f u s i o n o f enzyme-containing v a c u o l e s w i t h t h e phagocyt i c v a c u o l e and, subsequently, enzyme d e g r a d a t i o n o f the e n g u l f e d (Cotran  and L i t t ,  1969).  material  I n f o r m a t i o n gathered thus f a r i n d i c a t e s the con-  t e n t s o f the e o s i n o p h i l lysosome i s s i m i l a r to t h a t o f t h e n e u t r o p h i l  except  t h a t the e o s i n o p h i l s l a c k a n t i b a c t e r i a l enzymes, lysozyme and p h a g o c y t i n , and  have much h i g h e r  concentrations  o f p e r o x i d a s e ( G l e i c h ej: a l . , 1973).  Much o f the problem i n the e l u c i d a t i o n o f e o s i n o p h i l a c t i v i t i e s l i e s i n t e c h n i c a l problems ( G l e i c h , e t a l . , 1973 ; Mahmoud e t a l . , 1973). t h a t can be s a i d about t h e e o s i n o p h i l and the s k i n a l l o g r a f t that the e o s i n o p h i l i s present site.  i n the c e l l population  Thus, a l l  response i s  infiltrating  the g r a f t  Ill The  W mutant  I n f o r m a t i o n gathered i n t h i s study about the s k i n a l l o g r a f t r e sponse o f the W mutant i s o f two k i n d s .  First,  i t was found t h a t , when  viewed u l t r a s t r u c t u r a l l y , the c e l l types i n f i l t r a t i n g the g r a f t bed on the mutant do not d i f f e r mutant h o s t .  from those i n f i l t r a t i n g the g r a f t bed on the non-  Second, the d a t a o b t a i n e d from t h e c e l l counts i n d i c a t e  there  i s a s i g n i f i c a n t d i f f e r e n c e between t h e mutant and non-mutant h o s t s i n the frequencies  o f these c e l l types a t the g r a f t s i t e .  s i o n i s concerned w i t h the p o s s i b l e  The f o l l o w i n g  discus-  cause o f t h i s s i g n i f i c a n t d i f f e r e n c e  and what i t s u g g e s t s , i f a n y t h i n g , about (1) the mechanism o f the s k i n  allo-  g r a f t response and (2) the e f f e c t o f the W gene as r e l a t e d t o a l l t h e phenotypic characteristics. on  The emphasis o f the d i s c u s s i o n  the lymphocyte p o p u l a t i o n .  i s placed  primarily  There a r e two reasons f o r t h i s .  F i r s t , i t i s r e a s o n a b l y c l e a r from i n v i v o r a d i o t r a c e r ( P r e n d e r g a s t , 1964; Gowans, 1962), lymphocyte d e p l e t i o n  experiments  experiments ( M i l l e r  e t a l . , 1971) and experiments i n v o l v i n g t r a n s f e r o f a d o p t i v e immunity (Billingham  e t a l . , 1954; Mitchenson, 1954), and from i n v i t r o  ture s t u d i e s ,  that  the lymphocyte p o p u l a t i o n  t h r e e phases: Available  ability  into  s e n s i t i z a t i o n ; a c e n t r a l phase; and, an e f f e c t o r phase. suggests t h a t each phase i s dependent on lymphocyte  S e n s i t i z a t i o n involves  recognition  t a b l i s h m e n t o f s e n s i t i v i t y to them. the  allo-  t h a t i t s r o l e may be  A h o s t ' s response t o a s k i n a l l o g r a f t may be d i v i d e d  information  activity.  cul-  i s a component i n the s k i n  g r a f t r e s p o n s e , and, f u r t h e r , as suggested p r e v i o u s l y , a c e n t r a l one.  tissue  of the g r a f t a n t i g e n s and e s -  The immuno-competent c e l l ,  retaining  to r e c o g n i z e and respond t o t h e presence o f a n t i g e n i s t h e  112  lymphocyte.  T h i s phase Is f o l l o w e d by a c e n t r a l phase d u r i n g which a m p l i -  f i c a t i o n of the f i r s t  component occurs along w i t h p r o d u c t i o n of  sensitized effector cells. to  the presence  The  the a l l o g r a f t response  g r a f t and  the c e l l undergoing  o f a n t i g e n and becoming s p e c i f i c a l l y  gen i s the lymphocyte. of  Again,  their activity  division in  response  s e n s i t i z e d to t h a t a n t i -  f i n a l phase i s r e f e r r e d to as the e f f e c t o r  limb  d u r i n g which host c e l l s accumulate w i t h i n the results i n graft destruction.  t i s s u e c u l t u r e s t u d i e s , i t i s suggested i s dependent on the s p e c i f i c a l l y ing  specifically  Based on  lymphocyte  t h a t the i n i t i a t i o n o f t h i s phase  s e n s i t i z e d lymphocytes.  These c e l l s  enter-  the g r a f t s i t e r e c o g n i z e the s k i n a n t i g e n s there to which they have been  specifically  sensitized.  T h i s r e c o g n i t i o n can t r i g g e r t a r g e t c e l l l y s i s o r  r e l e a s e of e f f e c t o r m o l e c u l e s , of o t h e r c e l l t y p e s .  r e s u l t i n g i n the a c c u m u l a t i o n  I t i s the a c c u m u l a t i o n  or a c t i v a t i o n  o f t h e s e c e l l s w i t h i n the g r a f t e d  t i s s u e which b r i n g s about i t s d e s t r u c t i o n . Secondly,  the lymphocyte p o p u l a t i o n has  e f f e c t e d by the presence  a l r e a d y been shown to be  of the W gene by o t h e r r e s e a r c h groups.  Wong (1967)  showed t h a t the W lymphocyte migrates more s l o w l y than non-mutant lymphocytes on normal f i b r o b l a s t c u l t u r e c e l l s .  Work by Shearer  demonstrated t h a t the W mutant has a reduced the a n t i b o d y - f o r m i n g B cell line.  cell,  and  Cudcowitz (1967)  number o f p r e c u r s o r c e l l s  to  s u g g e s t i n g t h e r e i s a d e f i c i e n c y i n the lymphocyte  These o b s e r v a t i o n s f o l l o w from the o t h e r p h e n o t y p i c  character-  i s t i c s of the W gene where e i t h e r the m i g r a t i o n o r p r o l i f e r a t i o n , o r b o t h , of  a c e l l l i n e appears to be reduced  or i n h i b i t e d .  I f p a r t o f the lymphocyte p o p u l a t i o n i n i t i a t e s  the a c t i v i t y  that  r e s u l t s i n the d e s t r u c t i o n of g r a f t e d t i s s u e , then an a l t e r a t i o n i n the  113  p r o p e r t i e s of t h i s c e l l p o p u l a t i o n , say, due  t o the presence- o f the W gene,  would be expected to a l t e r the tempo and c h a r a c t e r i s t i c s o f the s k i n g r a f t response. of  In the p r e s e n t study, i t was  transformed lymphocytes  was  allo-  found t h a t the c o n c e n t r a t i o n  s i g n i f i c a n t l y h i g h e r (P <  .005)  i n the  graft  s i t e o f the mutant than of the non-mutant h o s t on a l l o f the days i n v e s t i gated.  McNay (1974) has demonstrated  t h a t a s k i n a l l o g r a f t on a mutant h o s t  has a s h o r t e r s u r v i v a l time than i t would on a non-mutant. c r e a s e d tempo of the s k i n a l l o g r a f t response may number o f transformed  Thus, t h i s i n -  be due to the i n c r e a s e d  lymphocytes.  As d i s c u s s e d p r e v i o u s l y , s e v e r a l f u n c t i o n a l a b i l i t i e s have been a t t r i b u t e d to t h i s  transformed lymphocyte  as i d e n t i f i e d by the l i g h t  microscope.  These p r o p e r t i e s i n c l u d e a b i l i t y to d i v i d e , g i v i n g r i s e to more of the same c e l l type; a b i l i t y to r e v e r t i n s i z e ; a b i l i t y ability  to k i l l by s e c r e t i o n o f lymphotoxin;  k i n e s ; and a b i l i t y t o produce  antibody.  to k i l l by d i r e c t c o n t a c t ; a b i l i t y to produce o t h e r lympho-  Thus, a l t h o u g h t h i s m o r p h o l o g i c a l l y  d i s t i n c t p o p u l a t i o n of c e l l s i s o b v i o u s l y q u i t e d i v e r s e f u n c t i o n a l l y , a common c h a r a c t e r i s t i c i s an a b i l i t y ing  to s p e c i f i c a l l y r e c o g n i z e the  a n t i g e n a n d , i n some manner, respond  to i t s presence.  sensitiz-  The presence o f a  l a r g e r number o f c e l l s w i t h i n the g r a f t s i t e o f the mutant h o s t , compared to  the non-mutant h o s t , responding to the a v a i l a b l e a n t i g e n c o u l d r e s u l t i n  a s h o r t e r g r a f t s u r v i v a l time on the mutant host, as noted by McNay An a l t e r a t i o n i n the response of the lymphocyte presence of a s k i n a l l o g r a f t , might, is  (1974).  p o p u l a t i o n t o the  i f the i d e a o f i t s r o l e as  initiator  t r u e , a l s o be expected to r e s u l t i n an a l t e r e d tempo o f e i t h e r the a c c u -  m u l a t i o n o r a c t i v a t i o n , or b o t h , of the o t h e r c e l l types a t the g r a f t  site.  114  A n a l y s i s of the c e l l counts o b t a i n e d  i n t h i s study  i n d i c a t e d that  i s a d i f f e r e n c e between the mutant and non-mutant host  there  infiltrating  popu-  l a t i o n s i n the f r e q u e n c i e s of o t h e r c e l l types w i t h i n the g r a f t s i t e ( t a b l e VII) . These f i n d i n g s c o n c e r n i n g  the c e l l - m e d i a t e d immune response of  W mutant suggest that the lymphocyte may  indeed  p l a y an important  r o l e i n the s k i n a l l o g r a f t response, as i n v i t r o it  the use o f the W mutant.  type i n f i l t r a t i n g the s k i n g r a f t  Further, be  -  able with  initiating  s t u d i e s suggest.  suggests an in ; v i v o means of i n v e s t i g a t i n g the phenomenon may  S e p a r a t e i n v e s t i g a t i o n s o f each  availcell  f o r a comparison between mutant and  mutant would s u b s t a n t i a t e or d i s p r o v e  the  non-  the above s p e c u l a t i o n s and p o s s i b l y  g i v e some i n s i g h t i n t o the r o l e and mechanism o f a c t i o n of the v a r i o u s types p r e s e n t w i t h i n the g r a f t bed whose a c t i v i t i e s r e s u l t i n the  cell  destruc-  t i o n of the s k i n a l l o g r a f t . The  next q u e s t i o n i s :  why  the i n c r e a s e d number o f transformed  phocytes w i t h i n the mutant's g r a f t s i t e ? s t i m u l a t i o n of the lymphocyte p o p u l a t i o n . available.  Presumably, i t i s due Two  lym-  to i n c r e a s e d  p o s s i b l e explanations  are  I n the f i r s t , emphasis f a l l s on the f a c t t h a t the W mutant shows  a d e f i c i e n t population of B c e l l s , to form a n t i b o d y .  thereby  i n d i c a t i n g a reduced c a p a c i t y  A l t h o u g h i t i s c l e a r that antibody  production  i s not  an  o b l i g a t o r y p a r t i n the s k i n a l l o g r a f t response, i t has been demonstrated i n a number of cases  t h a t antibody  i s produced.  I t s e f f e c t s vary.  o f t e n i t seems to have the e f f e c t o f enhancement. produced n o r m a l l y  i n the  However,  I f such a n t i b o d i e s  are  WBB6 l i n e d u r i n g a s k i n a l l o g r a f t response (as i n  the non-mutant) then comparison w i t h a mouse l i n e l a c k i n g the a b i l i t y to produce such a n t i b o d y  (such as  could r e s u l t  the W mutant) i n an a c c e l e r a t e d  115  r e j e c t i o n time by the W h o s t .  Presumably t h i s would be due to the i n c r e a s e d  number o f a n t i g e n i c determinant s i t e s a v a i l a b l e t o the lymphocyte p o p u l a t i o n d u r i n g the e n t i r e a l l o g r a f t r e s p o n s e , thereby of the lymphocyte  stimulating a higher  proportion  population.  In the second e x p l a n a t i o n as to t h e cause of t h e i n c r e a s e d number o f s t i m u l a t e d lymphocytes i n the W mutant emphasis f a l l s on the p o s s i b i l i t y t h a t i t i s a d i r e c t r e s u l t o f a s i n g l e e f f e c t o f the W gene. a c o n s i d e r a t i o n o f a l l the phenotypic ing  the "black-eyed  This involves  c h a r a c t e r i s t i c s o f the W gene, i n c l u d -  white coat" pigmentation,  sterility,  anemia and an en-  hanced c e l l u l a r immune response. A common b a s i s which these phenomena share  i s t h a t the a f f e c t e d c e l l s  are a l l a t a p o i n t where e x t e r n a l s t i m u l a t i o n from t h e i r s u r r o u n d i n g ment may p l a y an important or a c t i v i t y .  part i n determining  environ-  t h e i r subsequent development  T h i s i s e s p e c i a l l y apparent i n the lymphocyte c e l l l i n e .  These  c e l l s a r e immunocompetent: a s p e c i a l i z e d c h a r a c t e r i s t i c i n v o l v i n g the i n t e r a c t i o n o f the c e l l w i t h a n t i g e n  ( e x t e r n a l s t i m u l a t i o n ) which i n i t i a t e s a  response by the c e l l o f e i t h e r p r o l i f e r a t i o n , m i g r a t i o n , a c t i v i t y , or a combination o f t h e s e .  increased  metabolic  As mentioned p r e v i o u s l y , t h e r e a r e  three phases o f the s k i n a l l o g r a f t response d u r i n g each of which lymphocyte a c t i v i t y i s an important  component.  The i n c r e a s e d number o f s t i m u l a t e d lym-  phocytesamay.vb.eydue t o the e f f e c t o f the W gene a t any stage a c t i v i t y i n t h i s response.  of lymphocyte  However, k e e p i n g i n mind the o t h e r  phenotypic  c h a r a c t e r i s t i c s of the W mutant where there appears t o be a l a c k o f m i g r a t i o n or p r o l i f e r a t i o n , the i n i t i a l r e c o g n i t i o n response may be c o n s i d e r e d . known t h a t lymphocyte r e c o g n i t i o n i s a s u r f a c e phenomenon, although spect  It is  with r e -  t o the T c e l l p o p u l a t i o n , i d e n t i f i c a t i o n o f the r e c o g n i t i o n u n i t s has  116  not been p o s s i b l e .  The  slower m i g r a t i o n ,  m e l a n o b l a s t s , the germ c e l l s , and t h a t there may  be an a l t e r a t i o n i n s u r f a c e p r o p e r t i e s .  divisions.  sensitivity  of  r e s u l t i n g i n more c e l l s which r e -  o r the T c e l l s which do respond undergo more m i t o t i c  In e i t h e r case the r e s u l t would be  a c t i v a t e d lymphocytes w i t h i n the blood response by  In the case of T  r e s u l t i n increased  to the p r e s e n c e of a n t i g e n ,  spond to the a n t i g e n  the  e s p e c i a l l y the lymphocytes, suggests  c e l l r e c o g n i t i o n , t h i s a l t e r a t i o n may the T c e l l s  or l a c k o f i t , o f p o s s i b l y  the a v a i l a b i l i t y of more  along w i t h ,  p o s s i b l y , an  increased  the u n s e n s i t i z e d immunocompetent c e l l s at the g r a f t s i t e .  c o u l d r e s u l t i n a f a s t e r s k i n a l l o g r a f t ; response.  This idea, that  Both  the  primary e f f e c t of the W gene i n v o l v e s an a l t e r a t i o n i n the response o f to  e x t e r n a l s t i m u l a t i o n , a l s o f i t s as an e x p l a n a t i o n  of the o t h e r  cells  phenotypic  c h a r a c t e r i s t i c s of the W gene. I t i s q u i t e f e a s i b l e t h a t f o r the p r o p e r c o m p l e t i o n of t h e i r d e v e l o p ment, m e l a n o b l a s t s , germ c e l l s and  h e m a t o p o i e t i c c e l l s , at some s t a g e ,  q u i r e s p e c i f i c e x t e r n a l s t i m u l a t i o n from an environmental s o u r c e . gard to the m e l a n o b l a s t and d e f e c t due  to the p r e s e n c e o f the W gene i s an i n t r i n s i c p r o p e r t y  normal development o f non-mutant c e l l l i n e s R u s s e l l and The  Bernstein,  With r e -  hematopoietic c e l l l i n e s i t i s c l e a r that  c e l l s , s i n c e the environment i n which these c e l l s are found w i l l  1968;  re-  (Mayer, 1970;  of  the the  support  Mayer and  the  Green,  1967).  d a t a gathered thus f a r i n d i c a t e s t h a t i t i s p o s s i b l e t h a t  d e f e c t i n the m e l a n o b l a s t , germ c e l l and  hematopoietic c e l l l i e s  a b i l i t y to m i g r a t e or p r o l i f e r a t e p r o p e r l y .  The  a l l o g r a f t response appears to l i e i n an i n c r e a s e d  the  in its in-  cause of the i n c r e a s e d response on the p a r t  the lymphocyte to the presence of the s k i n a n t i g e n s .  skin of  This could involve  pro-  117  l i f e r a t i o n or m i g r a t i o n . o c c u r r i n g i n the  These a p p a r e n t l y c o n t r a d i c t o r y  presence of the W gene are  the p r i m a r y e f f e c t o f the W gene t o be perties  of the  ternal  stimulation.  an  explicable  characteristics  i f one  a l t e r a t i o n i n the  considers  surface pro-  c e l l s which thereby a l t e r s t h e i r a b i l i t y to respond to  ex-  118  BIBLIOGRAPHY  A b l e , M.E., Lee, J.C. and Rosenau, W. 1970. Lymphocyte-target interaction i nvitro. Amer. J . P a t h o l . 60: 421-428.  cell  A l l e g r a , F., B e l l , M. and G i a c o m e t t i , L . 1968. S k i n homografts i n rhesus monkeys (Macaca mtilatta) : f i r s t and second r e j e c t i o n . 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