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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 PAT COLLEN B.Sc, University of Brit i s h Columbia A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Master of Science In the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1974 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th i s thes is fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada pate OdbJJMsVnM i i ABSTRACT The populations of c e l l s which i n f i l t r a t e tissue a l l o g r a f t s i n W mutant mice and t h e i r non-mutant littermates were investigated using the l i g h t and electron microscopes. I n i t i a l l y , thyroid a l l o g r a f t s were attempted but t h i s tissue proved unsatisfactory f o r comparative studies and s k i n was used instead. The c e l l s i n f i l t r a t i n g the skin g r a f t s were i s o l a t e d enzymatically and characterized using the l i g h t microscope. In addition, c e l l s i n epon sections of skin grafts were i d e n t i f i e d using the electron miscroscope. The frequency of the various c e l l types i s o l a t e d from grafts 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 that of c e l l s i s o l a t e d from grafts i n non-mutant mice. The electron microscope studies indicated that the c e l l types i n f i l t r a t i n g skin a l l o g r a f t s are the same i n both mutant and non-mutant hosts. i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF FIGURES v LIST OF TABLES v i i i ACKNOWLEDGMENT ix INTRODUCTION 1 TRANSPLANTATION IMMUNITY 1 THE W MUTANT 5 PROPOSAL 8 IMPLANTATION OF THYROID < 11 INTRODUCTION 11 MATERIALS AND METHODS 13 Implantation of thyroid 13 RESULTS 15 Normal thyroid 15 Thyroid grafts implanted subcutaneously 17 Thyroid eisografts 17 Thyroid a l l o g r a f t s 23 Thyroid i s o g r a f t s implanted i n the spleen 25 DISCUSSION 28 TRANSPLANTATION OF SKIN 31 INTRODUCTION 31 MATERIALS AND METHODS 34 Skin g r a f t i n g 34 I s o l a t i o n of the i n f i l t r a t i n g c e l l s 34 Light microscopy 36 Ele c t r o n microscopy 36 RESULTS 39 I s o l a t i o n of the i n f i l t r a t i n g c e l l s 39 C e l l types 39 Data and s t a t i s t i c a l analysis 42 Light microscopy 50 Epidermis 50 Dermis . • 59 Beneath the dermis 60 Ele c t r o n microscope study of the graft bed of 6 and 8 day (score: 2) and 8 and 10 day (score: 3) a l l o g r a f t s 62 i v Table of Contents (cont'd) Page Orientation and i d e n t i f i c a t i o n 62 General c h a r a c t e r i s t i c s of the graf t bed 66 C e l l s of the graft bed 70 DISCUSSION 91 The skin a l l o g r a f t response: General remarks 91 The W mutant I l l BIBLIOGRAPHY 118 V LIST OF FIGURES Page IA. Normal thyroid .. .. 16 IB. Higher magnification of normal thyroid tissue 16 2A. Thyroid graft receiving a score of 1 18 2B? Degenerating f o l l i c l e s 18 3A. Thyroid graft r e c e i v i n g a score of 2 19 3B. Composition of the cen t r a l area of glands r e c e i v i n g a score of 2 or 4 '.. 19 4A. Thyroid graft r e c e i v i n g a score of 5 24 4B. Composition of the c e n t r a l area of glands r e c e i v i n g a score of 5 24 5A. Thyroid i s o g r a f t implanted i n the spleen and removed 1 day following implantation 27 5B. Thyroid i s o g r a f t implanted i n the spleen and removed 33 days following implantation 27 6. General procedure f o r preparation of tissue and i s o l a -t i o n of c e l l s 35 7-20 The c e l l types i s o l a t e d from the skin graft beds on both mutant and non-mutant hosts........ 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. Epidermis of mouse ski n 54 23B. Epidermis of a ski n i s o g r a f t removed 6 days following transplantation (score: 2) 54 23C. Epidermis of a ski n i s o g r a f t removed 8 days following transplantation (score: 2) 54 24A. Epidermis of a s k i n : a l l o g r a f t removed 4 days following transplantation (score: 1) 57 24B. Epidermis of a ski n a l l o g r a f t removed 6 days following transplantation (score: 2) 57 v i L i s t of Figures (cont'd) Page 24C. Epidermis of a s k i n a l l o g r a f t removed 8 days following transplantation (score: 3) 57 25. Tracing of a region of a 6 day skin a l l o g r a f t (score: 2).. 64 26. Tracing of a region of an 8 day skin a l l o g r a f t (score: 3). 65 27. Low power electron micrograph of the graft bed of a 6 day skin a l l o g r a f t (score: 2) 68 28. Low power electron micrograph of the graft bed of a 6 day s k i n a l l o g r a f t (score: 2) ... 69 29. Low power el e c t r o n microphage of the graft bed of an 8 day skin a l l o g r a f t (score: 3) 71 30A. Low power electron micrograph of the graft bed of an 8 day skin a l l o g r a f t (score: 3) 72 30B. Debris 72 31A. Small lymphocyte found i n the g r a f t bed of a 6 day skin a l l o g r a f t (score: 2) 76 31B. Small lymphocyte showing minor changes i n i t s u l t r a -structure 76 32. Immunoblast 77 33A. Activated lymphocyte found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2) 79 33B. Activated lymphocyte found i n the graft bed of a 6 day sk i n a l l o g r a f t (score: 2) 79 34A. Activated lymphocyte found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2)....... 81 34B. Activated lymphocyte found i n the graft bed of a 6 day sk i n a l l o g r a f t (score: 2) 81 35. Monocyte, or "non-activated" form of macrophage, found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2) 82 36A. "Activated" macrophage found i n the graft bed of a 6 day s k i n a l l o g r a f t (score: 2) 84 36B. "Activated" macrophage found i n the graft bed of a 6 day s k i n a l l o g r a f t (score: 2) ... 84 v i i L i s t of Figures (cont'd) Page 36C. Activated macrophage i n the process of phagocytosis within the graft bed of a 6 day skin a l l o g r a f t (score: 2) 84 37A. Eosinophil found i n the graft bed of a s k i n a l l o g r a f t removed 6 days following transplantation (score: 2) 85 37B. Eosinophil found-in the gr a f t bed of a 6 day skin a l l o g r a f t (score: 2) 85 37C. Neutrophil found i n the graft bed of a 6 day s k i n a l l o g r a f t (score: 2) . 85 37D. Degenerating neutrophil found i n the graft bed of a skin a l l o g r a f t removed 6 days following transplantation (score: 2) 85 38A. F i b r o b l a s t found i n the graft bed of a s k i n a l l o g r a f t removed 6 days following transplantation (score: 2) 87 38B. Stacks of rough endoplasmic reticulum of a f i b r o b l a s t i n the graft bed of a 6 day skin a l l o g r a f t 87 38C. F i b r o b l a s t found i n the g r a f t bed of a skin a l l o g r a f t removed 6 days following transplantation (score: 2) 87 v i i i LIST OF TABLES Page I. Scoring system r e l a t i n g the degree of degeneration of the syngeneic and allogeneic thyroid grafts 21 I I . Scores of degeneration of i s o g r a f t s and a l l o g r a f t s of thyroids removed on d i f f e r e n t days following transplantation 22 I I I . 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 37 IV. Number of grafts prepared for electron microscopy 37 V. Summary of the c h a r a c t e r i s t i c s of 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 with the l i g h t microscope 43 VI. Number and percent of the t o t a l number of c e l l s counted of each c e l l type on mutant and non-mutant hosts on given days following transplantation.... 44 VII. Chi-square values and p r o b a b i l i t y that the f r a c t i o n of each category of leukocyte i s the same i n skin a l l o g r a f t s on mutant and non-mutant hosts on given days following transplantation ... 48 VIII. Scoring system which indicates the appearance of the epidermis 56 IX. E s s e n t i a l features of the u l t r a s t r u c t u r e of each c e l l type found within the s k i n a l l o g r a f t s i t e on both mu-tant and non-mutant hosts 89 X. C e l l types i d e n t i f i e d w i t h i n the skin a l l o g r a f t s i t e i n a number of electron microscope studies 98 ACKNOWLEDGMENT I would l i k e to thank Dr. A.B. Acton for h i s help and encouragement during the course of t h i s study. INTRODUCTION Despite intensive research i n the f i e l d of transplantation immunity, our understanding of the mechanism of homograft r e j e c t i o n i s s t i l l incom-pl e t e . Always associated with the necrosis of transplanted t i s s u e i s the i n f i l t r a t i o n of mononuclear c e l l s into the g r a f t . However, uncertainty a r i s e s as 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 action and actual r o l e i n the destruction of the grafted t i s s u e . The approach taken i n t h i s i n v e s t i g a t i o n of the phenomenon involves a comparison of the Dominants-spotting mutant and the corresponding non-mutant of WBB6 mice. TRANSPLANTATION IMMUNITY The transfer of t i s s u e between g e n e t i c a l l y incompatible i n d i v i d u a l s e l i c i t s a number of immunological responses within the host and w i l l nor-mally r e s u l t i n the destruction by the host of the grafted t i s s u e . The immune responses e l i c i t e d are both humoral and c e l l u l a r . The degree to which either system contributes to a l l o g r a f t r e j e c t i o n has been a matter of intense i n v e s t i g a t i o n and considerable controversy. One point i s be-coming c l e a r : In many cases the destruction of transplanted t i s s u e i s mediated s o l e l y by the c e l l u l a r immune system (Najarian and Simmons, 1972; Winn, 1970). The f i r s t i n d i c a t i o n s of the importance of the c e l l u l a r immune re-sponse i n transplantation immunity arose from two separate observations. The f i r s t i s that necrosis of the graft i s always associated with the i n -f i l t r a t i o n of host mononuclear c e l l s into the transplanted t i s s u e (Billingham and S i l v e r s , 1971). Secondly, adoptive t r a n s f e r of homograft immunity can only be effected by v i a b l e lymphocytes (Billingham et a l . , 1954; Mitchison, 1954). Neither serum nor non-viable c e l l s from sensi-t i z e d animals w i l l s u ccessfully t r a n s f e r immunity, thus suggesting that t h i s t r a n s f e r i s dependent on the continued a c t i v i t y of the immunized c e l l s within the second host and i s not due simply to passive transfer of preformed antibody. Other observations now demonstrate that the c e l l u l a r immune response alone can bring about the destruction of some grafted t i s s u e s . In ins t a n -ces where the host i s unable (or has a reduced capacity) to produce humoral antibody, but the c e l l u l a r immune response i s " i n t a c t " , as i n the case of a bursectomized chicken or f e t a l lamb, the r e j e c t i o n of the a l l o g r a f t pro-ceeds normally ( S i l v e r s t e i n et a l . , 1964; Niederhuber et a l . , 1971). Fur-thermore, i t may be noted that during the r e j e c t i o n of grafted tissues the c e l l u l a r immune response, unlike the humoral immune response, can always be demonstrated as active and capable only of the destruction of the trans-planted t i s s u e . Often, depending on the type of tiss u e grafted, the produc-t i o n of humoral antibodies i n response to the a l l o g r a f t cannot be demonstra-ted, or else i t does not coincide with graft destruction (Burger et a l . , 1973). Further, while transfer of s e n s i t i z e d lymphocytes r e s u l t s i n acceler ated a l l o g r a f t r e j e c t i o n (Billingham et_ al_. , 1954) or the breakdown of i t s " p r i v i l e g e d " status i n a tole r a n t host (Billingham et a l . , 1963) , t r a n s f e r of a s p e c i f i c antibody does not always have the same e f f e c t on the r e j e c t i o n response (Najarian and Simmons, 1972). The transferred antibody may acceler ate r e j e c t i o n (Ting et a l . , 1971; Stetson, 1963; Hasek et a l . , 1969), or 3 have no e f f e c t (Steinmuller, 1962; H a r r i s , e t " a l . , 1972), or r e s u l t i n enhancement (Cohen et a l . , 1971). Thus, since the tempo of graf t des-t r u c t i o n proceeds unaltered, (Harris, et a l . , 1971, 1972) while the occurrence and a c t i v i t y of humoral antibodies can vary considerably, i t i s suggested that the humoral antibodies are not obligatory p a r t i c i p a n t s i n the destruction of grafted tissues by the host. On the other hand, i t has been demonstrated that the presence of an i n t a c t c e l l u l a r immune sys-tem i s e s s e n t i a l for the a l l o g r a f t response to proceed normally, blockage of i t s a c t i v i t y r e s u l t i n g i n prolonged graft s u r v i v a l (Billingham and S i l v e r s , 1971). The 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 of the grafted tissue i s not c l e a r l y understood. Among other questions uncertainty e x i s t s as to the i d e n t i t y of the c e l l s i n f i l t r a t i n g the g r a f t , t h e i r mechanism of action and actual r o l e i n the destruction of the grafted t i s s u e . The population of i n f i l t r a t i n g c e l l s i s quite hetero-genous. Further, the c e l l types and t h e i r numbers vary depending on, among other things, the time following transplantation at which the population i s examined, the type of tiss u e which i s grafted and the species involved. Furthermore, the exact i d e n t i f i c a t i o n of the c e l l s i s d i f f i c u l t and remains incomplete. Small lymphocytes, macrophages, plasma c e l l s , b a s o p h i l i c c e l l s , eosinophils and'neutrophils have been c i t e d as part of the c e l l population entering renal (Feldman and Lee, 1967; Kountz et a l . , 1963; L i n q u i s t et a l . , 1971), skin (Titus and Shorter, 1962; Simar and Betz, 1970; Weiner, et a l . , 1969; A l l e g r a et a l . , 1968) and tumour (Journey and Amos, 1962), a l l o g r a f t s , but these may not be present i n a l l types of g r a f t s , and other categories of c e l l s w i l l almost c e r t a i n l y be added to th i s l i s t . 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 population i n f i l t r a -t i n g the graft has been separated into two subpopulations. A s p e c i f i c a l l y s e n s i t i z e d lymphocyte population, which a r i s e s i n response to the presence of the graft antigens; and, a non-specific e f f e c t o r c e l l population, pro-bably p r i m a r i l y of monocyte-macrophage lineage (Prendergast, 1964; Kongshavin and Lapp, 1973). Information as to the r o l e i n the a l l o g r a f t response and mechanism of action of each c e l l 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 with other c e l l s at the graft s i t e a r ises from both i n vivo and i n v i t r o studies. However, the experimental techniques u t i l i z e d have inherent problems which prevent a complete understanding of the e f f e c t o r phase of the a l l o g r a f t response. Many of the i n vivo studies attempt to a l t e r the pool of c e r t a i n c e l l types within an animal and then note the e f f e c t of t h i s change on the a l l o g r a f t response. However, some of the techniques which are u t i l i z e d a l t e r the "background" physiology of the animal as well as the pool of the c e l l type under i n v e s t i g a t i o n . Thus, for example, techniques commonly employed to investigate the r o l e and a c t i v i t y of the lymphocyte i n the a l l o g r a f t r e -sponse include t o t a l body x - i r r a d i a t i o n , thymectomy, thoracic duct drainage and administration of anti-lymphocyte sera, many of which r e s u l t i n animal death due to runt disease (Najarian and Simmons, 1972; Billingham and S i l v e r s , 1971). Caution must be exercised when attempting to c o r r e l a t e the e f f e c t of such treatments on a p a r t i c u l a r c e l l type with t h e i r e f f e c t on the a l l o g r a f t response. Information as to the mechanism by which the i n f i l t r a t i n g c e l l s at the gr a f t s i t e i n t e r a c t with each other and the donor t i s s u e , and mediate 5 the destruction of the grafted t i s s u e , has been d i f f i c u l t to obtain by i n vivo studies, since the experimental systems av a i l a b l e do not provide a means of studying the c e l l a c t i v i t y i n vivo. 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 the e f f e c t o r phase of cell-mediated immunity at the l e v e l of separate i n d i v i d u a l c e l l types. Since the c e l l s under i n v e s t i g a t i o n are removed from the animal and manipulated i n tissue c u l t u r e , these studies are only possible examples of what the c e l l s are capable of doing and do not i n d i c a t e what a c t i v i t y they are performing within the graft s i t e . I t would be informative i f i t were possible to manipulate or a l t e r the property of one of the c e l l s i n f i l t r a t i n g the graft s i t e without a l t e r -ing the "background" physiology of the surrounding t i s s u e s . One would then be able to follow the e f f e c t t h i s a l t e r a t i o n has on the cell-mediated immune response to transplanted t i s s u e . Just such an a l t e r a t i o n may e x i s t i n a mutation that a f f e c t s the blood c e l l s without changing the "background" physiology of the surrounding t i s s u e . I f the phenotypic c h a r a c t e r i s t i c s of the c e l l s a r i s i n g from a mutation resulted i n an a l t e r a t i o n i n the c e l l -mediated immune response and i f the manner of t h i s a l t e r a t i o n could be deter-mined, this would provide information as to the c o n t r i b u t i o n the effected c e l l normally makes toward the destruction of the grafted t i s s u e and, p o s s i -b l y , i t s mechanism of action. THE W MUTANT An i n t e r e s t i n the homograft response of the W mutant i s aroused be-cause of the known phenotypic c h a r a c t e r i s t i c s of the W gene and the p o s s i -b i l i t y that the underlying defect may also a f f e c t the immune system. Muta-6 tions of the W locus i n mice are common (Green, 1966). There are a number of mutant a l l e l e s which e x i s t , two of which are the W, dominant spotting, a l l e l e and the WV, v i a b l e dominant spotting, a l l e l e . The presence i n mice of these a l l e l e s at the dominant-spotting locus i n the homozygous, WV/WV or W/W, or heterozygous, W/WV, condition r e s u l t s i n a "black-eyed, white coat" pigmentation, severe macrocytic anemia and s t e r i l i t y . Although the exact s t r u c t u r a l or enzymatic defect caused by the W gene i s unknown, analy-s i s of the experimental data c o l l e c t e d thus f a r suggests an a f f e c t on the migration or p r o l i f e r a t i o n , or both, of the p r i m i t i v e c e l l types of the affected t i s s u e s . I t has been shown by transplantation experiments that the cause of the black-eyed, white coat c h a r a c t e r i s t i c of the W mutant resides i n a defect i n the stem melanoblast. An i n t r i n s i c property of t h i s c e l l retards or i n -h i b i t s e i t h e r i t s migration from the neural 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 into a melanocyte, or a combination of these (Mayer and Green, 1968; Mayer, 1970). H i s t o l o g i c a l studies in 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 deleterious e f f e c t of the W gene on ei t h e r the migration or p r o l i f e r a -t i o n , or both, of the primordial germ c e l l s . Most of these c e l l s do not migrate from the yolk sac to the g e n i t a l ridge and, any that do, f a i l to p r o l i f e r a t e en route (Mintz and R u s s e l l , 1957). The W mutant also shows severe macrocytic anemia and i s extremely r a d i o s e n s i t i v e (Russell and Bernstein, 1966, 1967). Both of these charac-t e r i s t i c s have been a t t r i b u t e d to a genetic defect i n hematopoiesis, 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 hematopoietic stem c e l l s . Examina-t i o n of the blood-forming tissues of the mutant by transplantation experiments 7 and h i s t o l o g i c a l l y with radioactive tracers reveals a retardation i n p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i n hematopoiesis, including both ery-thropoiesis and leukopoiesis (Borghese, 1959; Russell et a l . , 1953; Altmann and Ru s s e l l , 1964; Rus s e l l and Bernstein, 1967; McCulloch et a l . , 1964<, Bennett et a l . , 1968; and Lewis'et a l . , 1967). Thus, the e f f e c t of the W gene appears early i n mouse development i n several d i f f e r e n t i a t i n g 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 to migrate or p r o l i f e r a t e , or both, during development, compared to those of the +/+ li t t e r m a t e s , i s retarded or i n h i b i t e d . There i s some i n d i c a t i o n that the W gene also a l t e r s the a c t i v i t y of some of the more d i f f e r e n t i a t e d hematopoietic c e l l types i n the adult W/WV mouse. For example, the W/WV mouse is: d e f i c i e n t i n i t s response to erythropoeitin, i n d i c a t i n g a defect i n the erythropoeitin-responsive c e l l l i n e (Russell and Bernstein, 1966). Further, Shearer and Cudkowicz (1967) found the mutant had a reduced number of precursor c e l l s to the antibody-forming c e l l s , suggesting reduced pro-l i f e r a t i o n i n the leukocytic precursor pool. Wong (1969) found that i n v i t r o the W/WV lymphocyte migrates s i g n i f i c a n t l y more slowly on a background of W/WV c e l l s than the +/+ lymphocytes on a background of +/+ 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 reduction or 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 or migration of blood c e l l s at d i f f e r e n t stages of t h e i r d i f f e r e n t i a t i o n . The host 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 are blood c e l l s which are both migratory and p r o l i f e r a t i v e . The question arises as to whether or not t h e i r pattern of response to an a l l o g r a f t of tiss u e a l t e r s i n the pre-sence of the W gene. McNay (1974) has demonstrated that the skin homograft response of the W/WV mutant d i f f e r s s i g n i f i c a n t l y from that of i t s W/+, +/+ 8 and WV/+ littermates. Histological observations of skin allografts indicate that the mutant rejects i t s grafts significantly 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 cells during the allograft response, indicates that in the W/W mouse a larger number of activated lymphocytes are present in the blood at an earlier stage i n graft rejec-tion than in the blood of the W/+, +/+ and WV/+ mice at the same stage. It appears, then, that in the W/WV mouse the homograft response i s significantly altered relative to the response in normal mice. The ques-tion arises as to what the basis of this difference in responsiveness i s . An answer could provide information as to the cellular mechanisms respon-sible for the rejection of transplanted tissue in a l l higher mammals. PROPOSAL It appears, then, that the W gene affects either the migratory or proliferative properties of some blood c e l l s , including the lymphocytes. Further, i n the presence of the W gene the skin allograft response, of which the lymphocyte i s an important component, i s altered sufficiently to result i n reduced graft survival time. A preliminary approach to the investigation of the cause of the altered cellular immune response of the mutant w i l l involve a study of cells at the graft site during the allograft 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 in the c e l l populations i n f i l t r a t i n g the transplanted tissue. It i s proposed therefore to investigate the homograft response at the histologi-cal and ultrastructural levels. 9 Such an i n v e s t i g a t i o n has two inherent problems. F i r s t , the target material to be transplanted should have a "background" c e l l population which w i l l allow f o r the easy i d e n t i f i c a t i o n of the various host c e l l types that i n f i l t r a t e the implant i n response to antigenic stimulation. I t should also allow for the determination of t h e i r k i n e t i c s . Secondly, the system used must be 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 exi s t between the mutant and non-mutant i n the types of c e l l s i n f i l t r a t i n g the g r a f t , i n t h e i r movement or i n t h e i r number, i t would be detected. An i d e a l experiment would be to implant an a c e l l u l a r , very antigenic sub-stance into an animal. Upon removal of the material from the host any c e l l s found within i t could e a s i l y be i d e n t i f i e d as having i n f i l t r a t e d the implant. An amorphous substance which was considered i s collagen. However, a l i t e r a t u r e review indicated i t would be unsuitable for several reasons. To obtain collagen suitable for transplantation studies i s a tedious procedure req 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 r e c o n s t i t u t i o n ( G r i l l o and Gross, 1962). Then, whether or not t h i s p u r i f i e d collagen i s antigeneic i s a matter of considerable controversy. Subcutaneous implantation e l i c i t s a v a r i e t y of host responses including encapsulation of the implant by host c e l l s , followed by replacement or breakdown of the material within 45 days. However, the hosts are not s e n s i t i z e d to the collagen, i n d i c a t i n g the c o l -lagen i s not antigenic and therefore not responsible for the response e l i c i -ted. In contrast, other studies i n d i c a t e i t stimulates the c e l l u l a r immune system while others suggest the stimulation of the humoral (Lindsley et a l . , 1971; Kirrane and Glynn, 1968). 10 Thus, two other target tissues were considered whose presence i n allogeneic hosts are known to e l i c i t a c e l l u l a r immune response: Thyroid and skin. This thesis deals with two experimental systems set up to i n -vestigate the cause of the difference i n the response to a l l o g r a f t s be-tween the W mutant and the non-mutant. The f i r s t involves the implanta-t i o n of thyroid, both subcutaneously and into the spleen. I t had been hoped to demonstrate a difference between the c e l l u l a r immune responses of the mutant and non-mutant i n a system d i f f e r e n t from the ski n a l l o g r a f t . However, as w i l l be explained i n the appropriate s e c t i o n , there were many problems with t h i s system and i t was subsequently abandoned. The second system involves transplantation of skin, i n c l u d i n g enzymatic i s o l a t i o n of the c e l l s i n f i l t r a t i n g the grafts and an electron microscope study of the graft beds. 11 IMPLANTATION OF THYROID INTRODUCTION The r e s u l t s of some early studies on the transplantation of the thyroid suggested that i t might serve as the basis for a microscopic comparison between the c e l l mediated immune responses e l i c i t e d by the mutant and non-mutant hosts. I t had been found i n rats and mice that thyroid i s o g r a f t s could be transplanted to any part of the body and r e -t a i n the physiology and morphological c h a r a c t e r i s t i c s found i n the non-transplanted tissues (Bennett and Gordman, 1951; Dempster and Doniach, 1955). Further, contrary to general b e l i e f , a thyroid d e f i c i e n c y i n the host i s not e s s e n t i a l f or the take and s u r v i v a l of the thyroid tissue implant (Dempster and Doniach, 1955; Gibbs and F i e l d , 1971). On the other hand, a l l o g r a f t s had been found to "take, regenerate and function, only to succumb l a t e r to an obscure r e a c t i o n " (Dempster and Doniach, 1955). Concerning t h i s reaction of the host to the allogeneic thyroid t i s s u e , i t i s stated that "lymphocytic i n f i l t r a t i o n i s the r u l e . " More recent work done by Gibbs and F i e l d (1971) involved implantation of the thyroid subcutaneously i n the thigh of both syngeneic and allogeneic host rats and determination of the v i a b i l i t y of the graft by i t s a b i l i t y to concentrate radioiodine. Ninety percent of the i s o g r a f t s transplanted by the i n c i s i o n method (used here) were functioning a f t e r 29 days i n the host. This i s i n contrast to the method involving the use of a trochar and cannula which showed only f i f t y - t h r e e percent success. None of the a l l o g r a f t s were functioning a f t e r 19 days i n the host. Thus, a method of transplanting the thyroid with reasonable s u r v i v a l of the i s o g r a f t s i s 12 a v a i l a b l e . Further, the homograft response occurs within a period of 19 days which i s s u f f i c i e n t time to view the various stages of i n f i l t r a -t i o n . The c e l l u l a r composition of the thyroid provides good background material f o r studying i n f i l t r a t i o n . The s t r u c t u r a l units of the thyroid are f o l l i c l e s which are packed f a i r l y c l o s e l y together and surrounded by an outer and inner capsule. The inner capsule consists of f i b r o e l a s t i c connective tissue and forms the septae of the gland, carrying blood and lymphatic vessels and nerves throughout the organ. The e p i t h e l i a l walls of the f o l l i c l e s are composed p r i m a r i l y of low cuboidal e p i t h e l i a l c e l l s involved i n c o l l o i d s ecretion and a few p a r a f o l l i c u l a r c e l l s (Ham, 1969). The types of c e l l s believed to be involved i n immune responses are not normally present i n the thyroid, and any i n f i l t r a t i n g c e l l s moving into 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 foreign to the gland t i s s u e . However, as well be shown i n the re s u l t s section, there were so many pro-blems with t h i s system that i t was abandoned. 13 MATERIALS AND METHODS Implantation of thyroid Male and female mice of the st r a i n s DBA/2 and WBB6, which d i f f e r i n the a l l e l e s at the H-2 locus, were used i n these experiments. The re c i p i e n t s , always of the same sex as the donors, were 14 to 16 weeks of age at the time of the experiment. While the donor animal was under Nembutal anaesthesia, the larynx and part of the trachea, with the thyroid attached, were removed through an i n c i s i o n along the ventral midline i n the neck region and placed i n Ringers s o l u t i o n . Then, under a di s s e c t i n g microscope, the thyroid to be used for transplantation was dissected away from the trachea. Two se r i e s of experiments were c a r r i e d out. I. The f i r s t series of experiments involved implantation of DBA/2 thyroid lobes subcutaneously i n the thigh of (1) DBA/2 (syngeneic grafts) or (2) WBB6 (allogeneic grafts) mice. A thyroid lobe was placed i n a sub-cutaneous pocket made by placing sharply pointed s c i s s o r s into a small i n -c i s i o n i n the skin and opening the blades to separate the skin from the underlying body w a l l . The wound was closed with Michel c l i p s . The grafts were harvested, f o r (1) the syngeneic g r a f t s , at 4, 6, 8 and 12 days f o l -lowing transplantation and, for (2) the allogeneic g r a f t s , at 3, 4, 6, 8, 10, 12, 13, 14 and 17 days following transplantation. When removed, the grafts were fi x e d i n Bouin's f l u i d , embedded i n p a r a f f i n wax, sectioned, and stained with haematoxylin and eosin. I I . The second s e r i e s of experiments involved implantation of thyroid lobes into the spleens (see page 25) of r e c i p i e n t mice of the same s t r a i n 14 as the donor (syngeneic g r a f t s ) . Small i n c i s i o n s were made i n the skin and i n the underlying body wall to allow f o r the exposure of the spleen. The surface of the spleen was cut and the thyroid lobe was inserted into the opening. The body wall was s t i t c h e d closed, while the more s u p e r f i -c i a l s k i n wound was closed with Michel c l i p s . The grafts were removed on days 1-6, 10, 18-21, 33 and 35, fixed i n Bouin's f l u i d , embedded i n paraf-f i n wax, sectioned and stained with haematoxylin and eosin. For comparison, a thyroid gland was removed from a DBA donor and pre-pared i n the same manner as the thyroid g r a f t s . 15 RESULTS Normal thyroid A b r i e f d e s c r i p t i o n i s given here of the microscopic structure of the normal thyroid gland removed from a DBA donor and prepared for l i g h t microscopy. The thyroid gland consists of i r r e g u l a r lobules, c a l l e d f o l l i c l e s , which are packed close together and are surrounded by a fibrous capsule (figure 1A). Generally, the ce n t r a l f o l l i c l e s are smaller i n s i z e than the f o l l i c l e s i n the peripheral area of the gland. Each f o l l i -c l e consists of a single layer of 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 with c o l l o i d ( f i g u r e IB). The e p i t h e l i a l w a l l i s composed of 2 c e l l types; f o l l i c u l a r (dark) c e l l s 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 . The f o l l i c l e c e l l s are e i t h e r cubodial or columnar. 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 region toward the lumen and basal region next to the basement membrane. Their n u c l e i , situated c e n t r a l l y or within the basal region and occupying two thirds of the c e l l , are sp h e r i c a l and contain f i n e l y dispersed chromatin. There are one to two c l e a r l y delineated n u c l e o l i . The cytoplasm i s homo-geneous and i s s l i g h t l y b a s o p h i l i c . There are only a few p a r a f o l l i c u l a r c e l l s located along the basement membrane between the f o l l i c l e c e l l s . They show no p o l a r i t y : At no point i s the c e l l surface open to the lumen of the f o l l i c l e s . These c e l l s are larger than the f o l l i c l e c e l l s and have a cl e a r e r cytoplasm. The c o l l o i d , within the lumen of 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 . C e l l u l a r i n c l u s i o n s within the c o l l o i d 16 Figure LA: Normal thyroid This gland was removed from a DBA donor and prepared f o r l i g h t microscopy. I t i s considered to show the "normal" appearance of thyroid t i s s u e , which i s designated by a score of 0 (table I ) . X 51. Figure IB: Higher magnification of normal thyroid t i s s u e Notice the organization of the f o l l i c l e s (F). Each consists of a s i n g l e layer of 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). Note also the d e f i n i t e p o l a r i t y of the c e l l s and t h e i r s p h e r i c a l n u c l e i containing f i n e l y dispersed chromatin and 1 or 2 c l e a r l y delineated n u c l e o l i . X 138. 17 are sometimes present but are probably due only to f o l l i c u l a r damage. The separation of the c o l l o i d from the f o l l i c l e c e l l s , g i v i n g the c o l l o i d a serrated edge, i s an a r t i f a c t produced by the preparation procedure. Often the c o l l o i d of the c e n t r a l f o l l i c l e s s tains more darkly than the c o l l o i d of the peripheral f o l l i c l e s . The f o l l i c l e s are surrounded by an inner capsule, or septum, which consists of f i b r o e l a s t i c connective t i s s u e , carrying blood and lymphatic vessels and nerves throughout the organ. In tissue sections of normal thyroid the components of the septum are d i f f i c u l t to see. Thyroid grafts implanted subcutaneously The f i r s t series of transplants made included both syngeneic g r a f t s , DBA thyroid to DBA mice, and allogeneic g r a f t s , DBA thyroid to WBB6 mice. These graft s were implanted subcutaneously. (1.) Thyroid i s o g r a f t s There i s a marked contrast between the microscopic appearance of the DBA thyroid i s o g r a f t and that of the 'normal untransplanted thyroid gland. The syngeneic grafts show varying degrees of necrosis. Generally, only a few of the f o l l i c l e s at the periphery of the gland appear s t r u c t u r a l l y normal, occurring there s i n g l y or i n groups of 6 to 8 (figure 2A and 3A). The inner f o l l i c l e s found i n a normal thyroid gland are u s u a l l y absent from the g r a f t s , replaced by e i t h e r degenerating f o l l i c l e s (figure 2B) or an amorphous material and a few scattered c e l l s (figure 3B). That some of these inner f o l l i c l e s appear to be degenerating i s sug-gested by t h e i r s t r u c t u r a l appearance. The endothelial w a l l no longer 18 Figure 2A: Thyroid graft r e c e i v i n g a score of 1 Note that only the f o l l i c l e s at the periphery of the gland appear s t r u c t u r a l l y normal, occurring there s i n g l y or i n groups of 6 to 8. The inner f o l l i c l e s found i n normal thyroid (figure 1A) are absent, replaced by degenerating f o l l i c l e s , X 51. Figure 2B: Degenerating f o l l i c l e s Notice that the endothelial w a l l no longer encloses the c o l l o i d and i s disrupted at various points. Most of the f o l l i c l e c e l l s are f l a t -tened, with very l i t t l e cytoplasm, and contain i r r e g u l a r l y shaped n u c l e i . X 138. 19 Figure 3A: Thyroid graft receiving a score of 2 Only the f o l l i c l e s at the periphery of the gland appear s t r u c t u r a l l y normal, occurring there s i n g l y or i n groups of 6 to 8. The inner f o l -l i c l e s found i n normal thyroid are absent, replaced by an amorphous ma-t e r i a l and a few scattered c e l l s . X 51. Figure 3B: Composition of the c e n t r a l area of glands r e c e i v i n g a score of 2 or 4 Here the central region of the gland consists of an amorphous ma-t e r i a l and a few scattered c e l l s . X 138. 20 encloses the c o l l o i d , and i s disrupted at various points. The f o l l i c l e c e l l s are f l a t t e n e d , with very l i t t l e v i s i b l e cytoplasm and contain i r -r e g u l a r l y shaped, fla t t e n e d n u c l e i (figure 2B), a marked contrast to the dispersed, round n u c l e i of the normal f o l l i c l e c e l l s ( f i g u r e 1A). Further, the c o l l o i d normally contained within the lumen i s e i t h e r grainy and very weakly stained (figure 2B) or absent (figure 3A and 3B). In other i s o g r a f t s the c e n t r a l region consists of an amorphous, very weakly stained material. Located within i t are a few scattered c e l l s and, sometimes, c e l l u l a r debris (figure 3B). Areas of connective t i s s u e , not e a s i l y v i s i b l e i n the normal thyroid, are now e a s i l y distinguished from the r e s t of the tissue present. Based on these various 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 scoring the degree of degeneration of the syngeneic and allogeneic thy-r o i d grafts was established (table I ) . The two main aspects considered were (1) the number of s t r u c t u r a l l y normal f o l l i c l e s present i n the p e r i -pheral region of the gland as a percentage of the t o t a l region and (2) the composition of the c e n t r a l region of the gland. The scores range from 0 to 5. Based on this system the thyroid i s o g r a f t s were scored and the r e s u l t s recorded i n table I I . Note that a l l of the i s o g r a f t s show some degree of degeneration. In almost every gland the c e n t r a l region, which i n a normal, untransplanted gland consists of f o l l i c l e s (score: 0), now contains only an amorphous material and a few scattered c e l l s (scores: 2 or 4). Another c h a r a c t e r i s t i c of these thyroid i s o g r a f t s i s the presence of a c e l l u l a r capsule probably a r i s i n g from the host which surrounds the 21 Table I Scoring System Relating the Degree of Degeneration of the Syngeneic and Allogeneic Thyroid Grafts. Score I l l u s t r a t e d i n : % normal f o l l i c l e s present i n peripher-a l area Composition of the ce n t r a l area of the gland 0 Figure 1A and 5B 100% normal f o l l i c l e s 1 Figure 2 and 5A ->50% degenerating f o l l i c l e s 2 Figure 3 >50% amorphous material with scattered c e l l s 3 <50% degenerating f o l l i c l e s 4 <50% amorphous material with scattered c e l l s 5 Figure 4 <50% (usually 410%) Table II Scores of degeneration of i s o g r a f t s and a l l o g r a f t s of thyroids removed on d i f f e r e n t days following transplantation Days Grafts were Removed Following Transplantation  Day Thyroid graft 1 2 3 4 5 6 8 10 12 13 14 17 18 19 20 21 33 Implanted sub-cutaneous l y (1.) DBA to DBA 2 1,2,4 2,4 2 (2.) DBA to WBB6 1 1,2 2,2,2 2,2 2,2 2,5 1 2,5 2.2 4 Implanted i n the spleen (1.) DBA to DBA 3 1 2,3 1 2 4,4 4,2 2,3 3 3 0 0 0.1 or WBB6 to WBB6 ro 23 subcutaneously implanted gland. The c e l l s are elongated and orientated such that t h e i r long axes are p a r a l l e l to the surface of the gland. The capsule i s not present around the grafts removed soon a f t e r trans-p l a n t a t i o n but appear^ r e g u l a r l y around the grafts removed on days 8 and 12. (2.) Thyroid a l l o g r a f t s The thyroid a l l o g r a f t s 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 the thyroid i s o g r a f t s discussed i n the previous section. Therefore, these grafts were scored as outlined i n table I and the values obtained are recorded i n s t a b l e I I . Examination of table IT reveals two important points about the degree of degeneration of the a l l o g r a f t s . The f i r s t i s that i t i s not r e l a t e d to the length of time the g r a f t has remained i n the host. Thus, a spec-trum of increasing c e l l u l a r i n f i l t r a t i o n and f o l l i c u l a r c e l l death, from the day 4 graft through to the day 17 g r a f t , was not found. Some of the gra f t s consisted p r i m a r i l y of an amorphous material with scattered c e l l s (score: 4 or 5) while others, for example the 17 day g r a f t , contained 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 second important point about the degree of degeneration of the a l l o g r a f t s i s that the range i s almost comparable to that of 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 include a stage of complete thyroid degeneration (score: 5) ( r e f e r to figures 4A and 4B). The peripheral and c e n t r a l region' of two of the a l l o g r a f t s consist of a disorganized mass of c e l l s and debris ( f i g u r e 4B). This never occurred with the i s o g r a f t s . The scores received by the rest of the allogeneic grafts were on a par with 24 Figure 4A: Thyroid g r a f t receiving a score of 5 This degree of thyroid t i s s u e degeneration was only found i n the a l l o g r a f t s . There i s almost a complete absence of normal thyroid f o l -l i c l e s . Both the peripheral and c e n t r a l region consist of a disorgan-ized mass of c e l l s and debris. Notice the c e l l u l a r capsule (CAP) surrounding the gland. X 51. Figure 4B: Composition of the ce n t r a l area of glands r e c e i v i n g a score of 5. The c e n t r a l region consists of a disorganized mass of c e l l s and debris. 25 those received by the syngeneic g r a f t s . Thus, f o r example, the two 17 day a l l o g r a f t s show the same degree of necrosis as the 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 with the syngeneic g r a f t s , encapsulation of the a l l o g r a f t s occurs. However, i t i s most frequently present i n , and thi c k e s t around, the grafts removed a f t e r longer periods of transplantation (figure 4A). Thyroid i s o g r a f t s implanted i n the spleen Because the scores obtained and recorded i n table II in d i c a t e that subcutaneous implantation of the thyroid lobes involves marked degenera-t i o n of the glands another procedure was followed. I t was hoped that implantation of the glands into the spleen, because of i t s r i c h blood supply, would eliminate the marked degrees of degeneration and the en-capsulation of the g r a f t s . The thyroid i s o g r a f t s , implanted i n the host's spleen and removed 1-6, 10, 18-21 and 33 days following transplantation, 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 allogeneic grafts im-planted subcutaneously, except that encapsulation of the lobes does not occur. Thus, the system ou t l i n e d i n table I i s used to score these syn-geneic grafts and the r e s u l t s are recorded i n table I I . A l l of the i s o g r a f t s implanted i n the spleen and removed from 1 to 14 days following transplantation show the same degree of degeneration as occurred when the thyroid lobes were implanted subcutaneously. Again, there i s no c o r r e l a t i o n between the degree of degeneration and the length of time the graft remained i n the host. Indeed, 3 of the grafts removed 26 on days 20, 21 and 33 received scores of 0, since they were s t r u c t u r a l l y normal (figure 5B). Figure 5A: Thyroid i s o g r a f t implanted i n the spleen and removed 1 day following implantation. X 51 Figure 5B; Thyroid i s o g r a f t implanted i n the spleen and removed 33 days following implantation. X 49 28 DISCUSSION Generally, the thyroid i s o g r a f t s and a l l o g r a f t s , which were im-planted subcutaneously, showed, with a few exceptions, the same degree and range of f o l l i c l e t i s s u e degeneration. Although some degree of f o l -l i c l e degeneration was expected with the e a r l i e r i s o g r a f t s , i t was not expected to be so extensive nor to be present at a l l the stages examined following transplantation. Neither did the a l l o g r a f t s show the expected spectrum of increase i n degeneration with time. Much of the f o l l i c u l a r degeneration occurring i n the syngeneic and early allogeneic g r a f t s implanted subcutaneously was probably ischemic necrosis. This i s suggested since the ce n t r a l f o l l i c l e s were always the most s e r i o u s l y affected. The connective ti s s u e of the subcutaneous space i s poorly vascularized and, according to Billingham and S i l v e r s (1971) does not f a c i l i t a t e very prompt v a s c u l a r i z a t i o n of an endocrine organ. Some of the tissue damage may be due also to the handling of the t i s s u e . The apparently good condition of the long-standing thyroid a l l o g r a f t s may be accounted for i n two ways. F i r s t , i t appears that a capsule of c e l l s , probably of host o r i g i n (Billingham and S i l v e r s , 1971), surrounds the implanted t i s s u e . This host response would cover the antigens and thereby hinder the host's r e j e c t i o n response. Secondly, i t has been proposed by Woodruffs (Billingham and S i l v e r s , 1971) that regeneration of f o l l i c l e s by the surviving graft c e l l s i s accompanied by an induction of the surrounding host tissues to form the stroma of the gland. If t h i s i s the case t h i s would further reduce the magnitude of antigenic stimula-t i o n and thus prolong graft s u r v i v a l . 29 In order to minimize ischemic necrosis and, hopefully, eliminate the problem of encapsulation the thyroid i s o g r a f t s were implanted i n the spleen. C e l l u l a r encapsulation of the grafts d i d not occur. However, except for the much longer s u r v i v a l , the i s o g r a f t s showed the same degree and range of f o l l i c u l a r degeneration as the a l l o g r a f t s and i s o g r a f t s im-planted subcutaneously. Since r e j e c t i o n occurs within the f i r s t 19 days (Gibbs and F i e l d , 1970) a f t e r g r a f t i n g , i t would not be possible to separate immunological c e l l death from "background" necrosis due to ischemia and the handling of the t i s s u e . It i s very i n t e r e s t i n g to note that three thyroid i s o g r a f t s which remained i n the host for 20, 21 and 33 days received a score (table II) which indicates 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 of a normal thyroid. The explanation of t h i s could be e i t h e r that the t i s s u e was not damaged dur-ing the transplantation procedure, or r e v a s c u l a r i z a t i o n occurred quickly or, most l i k e l y , the grafts had enough time to r e - e s t a b l i s h themselves. This s u r p r i s i n g "return to normal" suggests that, given enough time, the thyroid i s o g r a f t s w i l l r e - e s t a b l i s h themselves. That the thyroid i s o g r a f t s can r e - e s t a b l i s h themselves suggests that the c e l l u l a r immune response of a host to thyroid a l l o g r a f t s might be studied by using the a v a i l a b l e methods to induce tolerance and adoptively transfer immunity (Billingham, et a l . , 1963). The procedure followed would involve the following steps: (1.) Tolerance to donor antigens i s induced i n host mice at b i r t h (Billingham and Brent, 1958; S e l l e r , 1968); (2.) At the proper age these mice receive a t h y r o i d a l l o g r a f t and the 30 g r a f t i s allowed to " r e - e s t a b l i s h " i t s e l f ; (3.) The tolerant mice bearing the "normal appearing" thyroid a l l o g r a f t s are i n j e c t e d with syngeneic lymphocytes, normal or s p e c i f i c a l l y s e n s i t i z e d to the donor antigens (Billingham et a l . , 1963); (4.) The r e j e c t i o n procedure i s followed. At the time t h i s was considered i t was not known i f tolerance could be induced i n WBB6 mice to DBA antigens. Sometimes, i f the mice d i f f e r markedly at 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 , i t i s not p o s s i -ble to induce tolerance. Since a great deal of time would be required to determine proper c e l l dosages and procedure to induce tolerance, and with the p o s s i b i l i t y that i t s induction between these 2 s t r a i n s may be impossible, i t was decided not to attempt the implantation of thyroid as a means of i n v e s t i g a t i n g the c e l l u l a r immune response of the W mutant. 31 TRANSPLANTATION OF SKIN INTRODUCTION Several factors suggested that the s k i n a l l o g r a f t response could serve as the basis of a comparison of the cell-mediated immune responses e l i c i t e d by the mutant and non-mutant hosts. F i r s t , i t has been c l e a r l y demonstrated with adoptive transfer studies and lymphocyte depletion ex-periments that the host c e l l s which migrate into the s k i n a l l o g r a f t are somehow responsible for i t s subsequent destruction (Billingham et_ a l . , 1963; Najarian and Simmons, 1972). Humoral antibodies are not obligatory p a r t i c i p a n t s and thus the skin a l l o g r a f t response i s , b a s i c a l l y , a c e l l -mediated one (Winn, 1970). Secondly, preliminary work by McNay (1974) has established that there 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 skin 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 . The mutant r e -j e c t s i t s grafts s i g n i f i c a n t l y f a s t e r and there are larger numbers of ac-t i v a t e d lymphocytes present i n i t s blood at an e a r l i e r stage i n the a l l o -graft response. Another factor favoring the s k i n a l l o g r a f t response i s that some background information on i t b i s already provided by other microscopic i n -v e s t i g a t i o n s . Such information aids i n s e t t i n g up experiments and carry-ing out the preliminary studies. Light microscope observations reveal that the s k i n a l l o g r a f t response develops i n three successive stages (Medawar, 1944). The f i r s t stage involves would repair and healing phenomena, 32 similar to the response seen in isografts. It i s characterized by proliferation of fibroblasts and graft epithelium, formation of new capillaries i n the graft bed and a large i n f i l t r a t i o n of polymorpho-nuclear leukocytes. The second stage is marked by the i n f i l t r a t i o n of mononuclear cells into the graft site. These cells 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 cells spread from the graft bed through the dermis toward the epidermis (Weiner, et a l . , 1964). During this second stage there are no signs of epidermal degeneration. The third and f i n a l stage is 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 in mice (Simar and Betz, 1970). Another favorable factor i n skin transplantation i s that the cellular "background" composition of the dermis into which the i n f i l t r a -ting cells migrate consists only of fibroblasts, macrophages and blood cell s , none of them numerous (Montagne, 1956; Ham, 1969). Although d i f -f i c u l t y in identification can arise due to the problem of clearly identifying any c e l l i n a section of skin graft prepared for light microscopy (often the cytoplasm of cells i n the dermis cannot be dis-tinguished from the surrounding tissue components and i t i s impossible to identify or describe a c e l l by i t s nuclear structure alone) proper identification of these cells can be made with the electron microscope. Furthermore, the histological appearance of the epidermis provides a means by which the degree of graft degeneration may be scored. This \ 33 allows a c o r r e l a t i o n to be made between the stage of 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 comprising 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 that provides a means of quantita-t i n g the i n f i l t r a t i n g c e l l population and of determining the c e l l types comprising i t . The c e l l s move f i r s t i nto the region between the tissues of the host and those of the sk i n g r a f t . For lack of a better term, th i s region w i l l be re f e r r e d to as the graft bed. A method, using enzyme dig e s t i o n , has been developed by Jakobisiak et^ a l . (1971) i n which the c e l l s from the graft bed and po s s i b l y the lower dermis can be separated from the surrounding t i s s u e . This then makes i t possible to study the d i f f e r e n t c e l l types that i n f i l t r a t e the sk i n a l l o g r a f t s on both the mutant and non-mutant host. 34 MATERIALS AND METHODS Skin g r a f t i n g Male and female mice of the s t r a i n s WBB6 and DBA/2 were used i n these experiments. The WBB6 r e c i p i e n t s were 14 to 24 weeks of age at the time of the experiment and were always of the same sex as the donor. The skin g r a f t i n g technique employed was as outlined i n Billingham and Medawar (1951). Pieces of donor skin, 10 mm i n diameter, were pre-pared by scraping away both the f a t t y layer and the periniculus carnosus beneath the dermis. The grafts were then placed i n an i n t a c t bed of penniculus carnosus i n the l a t e r a l thoracic w a l l of the host. Grafts were covered with a square of gauze covered i n vaseline and the thorax was encased i n a p l a s t e r cast. These experiments included two types of skin 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 the transplantation of ski n from DBA/2 mice to WBB6 mice; and, (2) i s o g r a f t s , i n v o l v i n g the transplantation of skin from a WBB6 mouse to i t s WBB6 li t t e r m a t e s . In order to compare the c e l l u l a r response of the mutant with that of the non-mutant, the gr a f t i n g was done i n pairs such that the ski n from DBA/2 donor was trans-planted to a p a i r c o n s i s t i n g of a W/WV mouse and one of i t s +/+, +/W or +/WV l i t t e r m a t e s . When the graft was l a t e r removed from the host, h a l f of i t was used for the extraction of c e l l s i n f i l t r a t i n g the g r a f t while the remaining h a l f was prepared for electron microscopy. The procedure i s summarized i n figure 6. Is o l a t i o n of the i n f i l t r a t i n g c e l l s The procedure for enzymatic i s o l a t i o n of the i n f i l t r a t i n g c e l l s from the g r a f t bed and dermis i s outlined by Jakobisiak et_ a l . (1971). The 35 Figure 6: General procedure for preparation of tissue and i s o l a t i o n of c e l l s . D O N O R (DBA or W B B 6 ) , Q HOST ( W B B 6 ) COMPARED TO o wv/w y + l w/ + or + G R A F T lisp™5* EPIDERMIS "^oo O o C2J FATTY LAYER P. C A R N O S U S F I X A T I V E E P O N E M E N Z Y M A T I C MIXTURE ISOLATED C E L L S IDENTIFICATION a n d CELL C O U N T 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 (crystal-li n e ) , in Hanks balanced salt solution)) at 37°C for 30 minutes. The pellet of cells obtained was smeared on a microscope slide and stained with Wright's stain. In some instances the pellet was resuspended in 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 cells were isolated are shown i n table III. The cells 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 distribution in the nucleus. These were later re-grouped on the basis of blood c e l l classification and light microscope studies of cells in tissue culture. Light microscopy lu sections of the skin grafts prepared for electron microscopy were cut and stained with toluidine blue for examination with the light 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 for 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 cells were isolated. GENOTYPE DAYS AFTER TRANSPLANTATION Host Donor 0 4 6 8 10 WBB6 (W/+, wv/+, +/+) DBA/2 5 5 5 3 WBB6 (Wv/W) DBA/2 - 5 5 5 3 no host DBA/2 5 - - -Table IV Number of grafts prepared for electron microscopy HOST GENOTYPE DAYS AFTER TRANSPLANTATION Allografts 4 6 4 7 8 9 10 13 WBB6 (WV/w) 5 6 2 6 2 3 1 WBB6 (WV/+, +/+, W/+) 5 6 2 6 2 4 1 Isografts WBB6 3 3 (W v/+, +/+, W/+) 38 of the graf t and include only the pieces from the c e n t r a l region. Two f i x a t i o n procedures were t r i e d : (1) For some, the g r a f t material was fi x e d i n 6.25% glutaraldehyde i n 0.1M phosphate buffer at pH 7.2 for 2 hours, rinsed i n b u f f e r f o r 15 minutes and then post-fixed i n 1% osmium tetroxide f o r 1 hour; or, (2) the graft was f i x e d i n Karnovsky's f i x a -t i v e (Karnovsky, 1965) for 2 to 5 hours, rinsed i n 0.1M phosphate buffer for 3 to 7 hours and then post-fixed i n 1% osmium tetroxide for 1 hour. D i r e c t l y following e i t h e r f i x a t i o n procedure, the material was stained with 2% aqueous uranyl acetate at 60°C overnight (Lock, e_t al_. , 1971) . The tissues were then dehydrated i n alcohols and embedded i n epon from propylene oxide. Sections were cut on an LKB ultratone and examined with a H i t a c h i HS7S and AEI 801. 39 RESULTS I s o l a t i o n of the i n f i l t r a t i n g c e l l s (1.) C e l l types Based on the c r i t e r i a outlined i n Materials and Methods (see page 34) the c e l l s i s o l a t e d from the graft bed were separated into the following categories: (a.) Small lymphocyte The nucleus of t h i s c e l l has a diameter of 5 to 6 u and, with Wright's s t a i n , appears dark purple. The chromatin i s clumped and i s surrounded by a narrow rim of cytoplasm which either lacks color or stains very pale blue or pink. The diameter of the e n t i r e c e l l i s us-u a l l y 6 to 7 u (figure 7). (b.) Large lymphocyte The nucleus of this c e l l has a diameter of 8 to 10 u and s t a i n s dark purple. The chromatin i s clumped, but not as markedly as that of the small lymphocyte, and i s surrounded by a narrow rim of cytoplasm which ei t h e r lacks color or stains very pale blue or pink. The diameter of the en t i r e c e l l i s 9 to 11 u (figure 8). (c.) "Transformed" lymphocyte These c e l l s may be separated into two groups based on t h e i r s i z e . In one the nuclear diameter i s 8 to 10 u while i n the other i t i s 10 to 15 u. The chromatin i s evenly dispersed and i s surrounded by varying amounts of cytoplasm, appearing as a wide rim as shown i n fig u r e 10 or i n an amount approximately twice the width of the nucleus. S i m i l a r l y , 40 although the color of the cytoplasm i s always blue, i t v a r i e s markedly i n depth of color. The color ranges from a pale sky blue to a very dark, almost black, blue as i n figures 9 and 10 r e s p e c t i v e l y , (d.) Macrophage This category includes a rather morphologically diverse population of c e l l s . C h a r a c t e r i s t i c s common among these c e l l s include a large amount of cytoplasm r e l a t i v e to the s i z e of the nucleus, and cytoplasmic color of pale pink, gray or deep pink. The appearance of the nucleus varies somewhat between c e l l s . The nuclear chromatin sometimes appears loo s e l y clumped while i n other instances i t i s evenly dispersed. As with the transformed lymphocyte, two groups of c e l l s can be distinguished based on t h e i r nuclear s i z e . Some f a l l i nto a nuclear s i z e range from 8 to 10 y while others f a l l i n a range from 10 to 12 y (figures 15, 16 and 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 are separated into two categories based on the amount of cytoplasm surrounding the nucleus: B l which has a rim of cytoplasm, and B2 which has a large amount of cytoplasm. In both ca-tegories the nuclear diameter ranges from 8 to 10 y. The chromatin i s clumped but i s somewhat open as i n the medium lymphocyte. The color of the cytoplasm varies from blue to very dark blue or dark purple, as demon-strated i n figures 12, 13 and 14. (f . ) Polymorphonuclear leukocyte Both eosinophils and neutrophils were present and counted. The n u c l e i are lobed and stained dark purple. The eosinophil i s characterized by red cytoplasmic granules (figures 19 and 20). 41 The c e l l types Isolated from the skin graft beds on both mutant and non-mutant hosts. X 270. Figure 7: Small lymphocyte Figure 8: Large lymphocyte Figures 9, 10 and 11: Transformed lymphocytes. Note the varying degrees of cytoplasmic b a s o p h i l i a . Figures 12, 13 and 14: Basophilic c e l l s Figures 15, 16 and 17: Macrophages Figure 18: F i b r o b l a s t Figures 19 and 20: Neutrophils 4 42 (g.) Fibroblast The f i b r o b l a s t nucleus has a diameter of 8 to 12 y and i s surround-ed by a large amount of cytoplasm. The c e l l appears elongated, the cytoplasm extending as strands from the area of the nucleus as demon-strated i n figure 18. The color of the cytoplasm i s usually pale blue, though sometimes i t i s deeper blue. The number of m i t o t i c f i g u r e s , basophils and plasma c e l l s i n the c e l l counts were extremely small. The e s s e n t i a l features of each c e l l type i s o l a t e d from the graf t-siteaar-essummarizediinltable V. (2.) Data and s t a t i s t i c a l analysis (a.) Donor s k i n The c e l l s of normal skin were i s o l a t e d i n order to get an idea as to the contribution the donor sk i n might make to the o v e r a l l c e l l counts i n the a l l o g r a f t s and to see the types of c e l l s normally present p r i o r to the i n f i l t r a t i o n by host c e l l s . Only a few c e l l s could be i s o l a t e d from ungrafted s k i n , r e l a t i v e to the number of c e l l s i s o l a t e d from the sk i n a l l o g r a f t s . Of those i s o l a t e d , the majority were f i b r o b l a s t s and macrophages, with a few blood c e l l s . (b.) Skin a l l o g r a f t s removed following transplantation. Five a l l o g r a f t s were removed from mutant hosts and f i v e from non-mutant hosts at 4, 6, 8 and 10 days following transplantation. The c e l l s below the graft dermis were extracted and 500 of these c e l l s per g r a f t were i d e n t i f i e d and counted. The values are recorded i n table VI. In order to determine i f the frequencies of the 9 c e l l types comprising the i n f i l t r a t i n g population were the same for both the mutant and non-mutant Table V Summary of the c h a r a c t e r i s t i c s of the c e l l types i s o l a t e d from the skin a l l o g r a f t s and viewed with the l i g h t microscope Nucleus Cytoplasm Fig: ures Cell-. Types Size Color (Wright's stain) Chromatin Color (Wright's stain) Amount I l l u s t r a t e d i n : Small Lymphocyte 5y-6y dark purple clumped (often notched) no color or pale pink or blue rim around the nucleus 7 Large 1 Lymphocyte 8y-10y dark purple clumped, but somewhat open no color or pale pink or blue rim around the nucleus 8 Transformed Lymphocyte 8y-10y or 10y-15y dark blue dispersed blue to very dark blue wide rim to area of the cleus twice nu-9,10,11 Macrophage 8y-10y or 10y-15y dark purple dispersed or some clumping gray, pale or deep pink At least twice the area of the nucleus 15,16,17 Basophilic C e l l I 8y-10y dark purple clumped, but open blue, deep blue or deep purple rim around the nucleus 12,14 Basophilic 8 y - l l y dark purple clumped, but blue, deep blue at least twice 13 C e l l II open or deep purple the area of the nucleus Polymorpho-nuclear Leukocytes dark purple lobed gray (eosinophil rim of cytoplasm 19,20 have red granules Table VI Number and percent of the t o t a l number of c e l l s counted of each c e l l type on mutant and non-mutant hosts on given days following transplantation. Chi-square and p r o b a b i l i t y values that the frequencies of the various c e l l types within the graft bed on mutant and non-mutant hosts are the same are included Day Small Large Transformed Baso. I Baso II Macro- Neutro- Eosino- Fi b r o - T o t a l Following Lymphocyte Lymphocyte Lymphocyte phage p h i l p h i l b l a s t # Transplan- # % # % # % # % # % # % # % # % # % ta t i o n 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 2497 b. Non-mu- 411 16.5 280 11.7? 377 15.1 379 15.2 149 5.9 592 23.7 283 11.3 17 7 „ 2495 tant 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 2499 b. Non-mu- 597 23.9 225 9.0 269 10.8 325 13.0 184 7.4 520 20.8 320 12.8 44 14 2498 x = 28.56 tant tant 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 2500 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 2 2494 x = 79.04 P< .005 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-mu- 239 16.0 90 6.0 81 8.3 107 7.2 52 3.5 345 23.1 539 36.2 28 9 1490 tant x = 22.4 P< .005 45 hosts a chi-square test i s used. Recorded i n table VI are the chi-square values obtained and the p r o b a b i l i t i e s that the frequencies of the leuko-cytes w i t h i n the skin grafts on mutant and non-mutant hosts on given days following transplantation are the same. I t i s clear that the d i s t r i b u t i o n of the various leukocytes within the mutant graft 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 that of the non-mutant on a l l days following transplantation ( i n a l l cases, P < .005). In order to obtain some idea as to the k i n e t i c s of c e l l movement at the gra f t s i t e a percentage of each c e l l type within the t o t a l number of c e l l s were calculated and are included i n table VI. These values have been plotted against days following transplantation i n chart I and chart I I . Some trends which are i l l u s t r a t e d include the following: The small and large lymphocytes and b a s o p h i l i c c e l l s peak i n concentration on day 6 while the highest percent of macrophages occurs on day 8 while the per-centage of polymorphonuclear leukocytes increases from day 6 to day 8 and day 8 to day 10. In order to get some idea 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 popu-l a t i o n s a r i s e s , i n d i v i d u a l chi-square tests were done f o r each c e l l cate-gory. In table VII are recorded the chi-square values and p r o b a b i l i t y that the f r a c t i o n of each category of leukocyte i s the same i n skin grafts on mutant and non-mutant hosts on given days following transplantation. These c a l c u l a t i o n s suggest where the difference i n the frequencies of the leukocytes of mutant and non-mutant hosts may l i e and may be summarized as follows: 46 Chart I: Percentage of lymphocytes ("normal" and basophilic) out of the t o t a l number of c e l l s counted (per 5 grafts) on days 4, 6, 8 and 10 following transplantation. For the explana-t i o n of I and I I (basophilic c e l l s ) see page 40). 4 6 8 1 0 D a y s F o l l o w i n g T r a n s p l a n t a t i o n 47 Chart I I : Percent of macrophages and neutrophils out of the t o t a l number of c e l l s counted (per 5 grafts) on days 4, 6, 8 and 10 following transplantation. 40 48 Table VII Chi-square values and p r o b a b i l i t y that the f r a c t i o n of each category of leukocyte i s the same i n ski n a l l o g r a f t s on mutant and non-mutant hosts on given days following transplan-t a t i o n . Days Following Transplantation C e l l Types 4 2 X values P 6 2 X values P 8 2 X values P 10 x 2 P values Transformed Lymphocyte 7.49 < .01 6.80 < .01 10.95 < .005 11.38 <.005 Large Lymphocyte 22.72 < .005 6.198 < .05 0.093 0.857 Small Lymphocyte 12.41 < .005 0.23 35.8 < .005 0.240 Basophilic C e l l I 2.34 0.572 7.128 < .01 2.42 Basophilic C e l l II 2.40 3.46 4.02 < .05 0.09 Macrophage 0.03 0.28 11.88 < .005 0.28 Neutrophil 29.2 <.005 0.039 0.309 4.170 < .05 49 1. Transformed lymphocyte Their number i s s i g n i f i c a n t l y higher (P <.01 or P < .005) i n the skin gr a f t on the mutant host on a l l of the days following transplantation. 2. Large lymphocyte The number of these c e l l s i s s i g n i f i c a n t l y lower on day 4 (P <.005) and s i g n i f i c a n t l y higher (P < .05) on day 6 i n the ski n a l l o g r a f t on the mutant host. 3. Small lymphocyte The number of small lymphocytes i s s i g n i f i c a n t l y lower (P < .005) i n the s k i n graft on the mutant host on days 4 and 8 following transplanta-t i o n . 4. Basophilic c e l l The number of these c e l l s i s s i g n i f i c a n t l y d i f f e r e n t on day 8 be-tween the mutant host and non-mutant host:. The B l c e l l s are s i g n i f i c a n t l y higher (P < .01) while the B2 c e l l s are s i g n i f i c a n t l y lower (P < .05). 5. Macrophage There are a higher number i n the mutant (P < .005) on day 8. 6. Polymorphonuclear leukocytes A s i g n i f i c a n t d i f f e r e n c e i n the number of neutrophils between mutant and non-mutant hosts occurs on days 4 (P < .005) and 10 (P < .05). On day 4 the numbers are higher i n the mutant while on day 10 they are lower than the number for the non-mutant host. 50 Light Microscopy Whenever pos s i b l e the 1 u sections of the epon-embedded material included the two layers of the skin graft (epidermis and dermis) and the underlying tissues of the host (graft bed, panniculus adiposus and pannicu- lus carnosus) (refer to figures 21 and 22). This made i t possible to characterize each layer and to orient the t i s s u e block i n preparation for t h i n sectioning. The general appearance of the grafted tissue varied depending on the degree of genetic d i s p a r i t y between donor and host and the length of time the graft had remained on the host. The various components of the graft and underlying tissues w i l l be considered separately. (1.) 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 graft epidermis i s important since a comparison of the graft'febed c e l l s between the mutant and non-mutant hosts must be l i m i t e d to tissue between mutant and non-mutant blocks which show the same degree of graft degeneration, allowing for comparison of c e l l populations present at s i m i l a r stages of the a l l o g r a f t response, and i t i s the condition of the epidermis, rather than that of the dermis, that i s most i n -d i c a t i v e of the degree of t h i s degeneration. Thus, the c h a r a c t e r i s t i c s of the epidermis of ungrafted mouse skin, skin i s o g r a f t s and a l l o g r a f t s were c a r e f u l l y noted, and c r i t e r i a established for separating the a l l o g r a f t s into d i f f e r e n t categories of degeneration. (a.) Epidermis of mouse ski n The epidermis of normal mouse skin removed from the l a t e r a l thoracic w a l l consists of 3 to 6 i l l - d e f i n e d layers of 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 i n t o various s t r a t a (figure 23A). The most con-51 spicuous of these i s the stratum germiriativum separating the epidermis from the dermis; t h i s has a sing l e layer of cubodial or columnar c e l l s which rest on the basement membrane. There are from one to three layers of rounded c e l l s above t h i s which do not have any other d i s t i n g u i s h i n g 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 . The c e l l s of the outermost layer, the stratum granulosum, contain basophil granules and are elongated with t h e i r long axies p a r a l l e l to the surface of the skin . The c e l l s of the stratum corneum above t h i s layer are dead. Numerous hair f o l l i c l e s are present and, less frequently, sebaceous glands. (b.) Epidermis of i s o g r a f t s removed 6 days and 8 days following transplantation The epidermis of the 6 day i s o g r a f t i s two to three times thicker than the epidermis of ungrafted mouse skin removed from the same region (figure 23B). Furthermore, there i s a marked increase i n the kind and number of c e l l l a y e r s . Above the stratum germinativum there are several layers of c e l l s which are ref e r r e d to as the stratum spinosum. This layer i s normally found only i n mammalian thick skin. The c e l l s are polyhedral but, except for narrow cytoplasmic connections, are separated from one another by a wide space. With the connections t h i s gives 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 figure 23B. The stratum granulosum i s &.more d i s t i n c t layer than the stratum granulosum previously described. I t . • i s c l e a r l y 2 to 3 c e l l s thick and many of the c e l l s of the lower l a y e r s , a l l of which contain granules, are more diamond shaped than elongated. The s t r a -tum corneum i s the same as that of the epidermis of ungrafted mouse s k i n . Figure 23C i s a micrograph of the epidermis of an 8 day skin i s o g r a f t . 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 that i n the 6 day i s o g r a f t 52 Figure 21: 6 Day A l l o g r a f t This i s a seri e s of l i g h t microscope pictures of a 1 u section of a 6 day skin a l l o g r a f t which includes the 2 layers of the gra f t (epidermis (E) and dermis (D) and the underlying tissues of the host (graft bed (B) and panriiculus carnosus (M). X 95. 53 Figure 22: 8 Day Skin A l l o g r a f t This i s a series of l i g h t microscope pictures of a 1 y section of an 8 day s k i n a l l o g r a f t which includes the 2 layers of the g r a f t , /'epidermis (E) and dermis (D) ; and the underlying tissues of the host, / g r a f t bed (B) and panniculus adipbsus (A). X 95. 54 Figure 23A: Epidermis of mouse skin The layers of e p i t h e l i a l c e l l s of the epidermis may be divided, morphologically, i n t o various s t r a t a . Note i n normal mouse skin t h i s includes the stratum germiriativum (SO), stratum granulosum (SG) and stratum corneum (SC). X 134. Figure 23B; Epidermis of a skin i s o g r a f t removed 6 days following transplantation (score: 2) Note the clearer d i s t i n c t i o n of c e l l layers compared to that of normal sk i n shown i n figure 23A. Above the stratum germinativum are severalslayers of c e l l s which are referred to as the stratum spinosum (SS). Figure 23C: Epidermis of a ski n i s o g r a f t removed 8 days following transplantation (score: 2) X 134. 55 (figure 23B) including a prominent stratum spinbsum and stratum  granulosum. However, overall, the epidermis of an 8 day isograft i s not as thickened as that of a 6 day isograft. It should be noted here that the epidermis of neither the 6 day nor 8 day isografts shows any signs of cellular necrosis or overall degenera-tion ( f i gures 23 B and C). Further, the hair f o l l i c l e s seen in cross sec-tion appear as theyLdo in the untransplanted pieces of mouse skin. A skin graft showing these characteristics (thickened, identifiable strata; no necrosis, and i n contact with the underlying dermis) is given a score of 2 (table VIII). (c.) Epidermis of allografts removed 4, 6, 7, 8, 9, 10 and 13 days following transplantation The epidermis of the allografts shows varying degrees of cellular 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 is not as thickened as that of the 6 and 8 day iso-grafts (figure 24 A). There i s , however, a stratum spinosum similar to that found i n the isografts. This is in contrast to i t s almost complete absence from the normal skin of the lateral thoracic wall. In some areas the epidermis appears to be detached from the underlying dermis. Some of the cells present show signs of degeneration including vacuolization of the cy-toplasm and pyknosis or karyorrhexis. Furthermore, i n some of the hair f o l l i c l e s the epidermal cells appear very disorganized compared to those in normal skin. A graft whose epidermis shows these characteristics (not thickened, identifiable strata, l i t t l e necrosis, and occasionally separa-ted from dermis) is given a score of 1 (table VIII). 56 Table VIII Scoring system which indicates the appearance of the epidermis• Score Appearance of the Epidermis I l l u s t r a t e d i n : not thickened; occasionally separated from Figure 24A 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 thickened; not separated from dermis; s t r a t a Figures 21A; are i d e n t i f i a b l e ; no necrosis 23B; 23C; 24B 3 thickened; completely separated from dermis; Figures 22A s t r a t a are not i d e n t i f i a b l e ; marked necrosis and 24C 57 Figure 24A: Epidermis of a s k i n a l l o g r a f t removed 4 days following transplantation (score: 1) X 134. Figure 24B: Epidermis of a s k i n a l l o g r a f t removed 6 days following transplantation (score: 2) Figure 24C: Epidermis of a skin a l l o g r a f t removed 8 days following transplantation (score: 3). X 134 Notice the epidermis has separated from the underlying dermis (D) and the gap (G) i s f i l l e d with some c e l l u l a r debris. 58 The majority of the 6 day a l l o g r a f t s have an epidermis which appears, s t r u c t u r a l l y , the same as that of the 6 and 8 day i s o g r a f t s (score: 2). The epidermis i s two to three times thicker than normal and, as shown i n figure 24 B, consists of several d i s t i n c t s t r a t a , i n c l u d i n g the stratum  germinativum, stratum spinosum, stratum granulosum and stratum corneum. In contrast to the 4 day a l l o g r a f t epidermis (score: 1), there appears to be f i r m attachment between the epidermal c e l l s and the underlying dermis. In addition, theaepidermal c e l l s of the 6 day a l l o g r a f t s do not show signs of degeneration. Thus the epidermis of 6 day a l l o g r a f t s with these charac-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 , given a score of 2. The epidermis of some of the 6 and 8 day a l l o g r a f t s and a l l of the 9, 10 and 13 day a l l o g r a f t s showed signs of p a r t i a l and, i n some cases complete, tissue breakdown. Figure 24C i s an example of t h i s . The epider-mis, thickened as i n the 6 and 8 day i s o g r a f t s (score: 2) and 6 day a l l o g r a f t s (score: 2), has separated away from the underlying dermis. The gap now present between them i s often f i l l e d with c e l l u l a r debris, probably o r i g i -nating from the breakdown of the epidermal c e l l s . In many areas several l a y -ers oof .epidermal • c e l l s are distinguishable but i d e n t i f i c a t i o n of the s t r a t a , except for the stratum corneum, i s not possible due to c e l l breakdown (figu r e 24 C). The h a i r f o l l i c l e s show s i m i l a r signs of diso r g a n i z a t i o n and necrosis. A g r a f t whose epidermis shows these c h a r a c t e r i s t i c s (thickened, 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 necrosis and almost complete separa-t i o n from underlying dermis) i s given a score of 3 (table V I I I ) . In summary, the tissue blocks are separated into three categories based on the appearance of the epidermis, the condition of which i s 59 considered indicative of the degree of graft breakdown. (2.) Dermis Underlying the epidermis is the dermis (figure 21 and 22). This tissue normally consists only of interwoven collagen bundles, a few scat-tered fibroblasts 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 light microscope of the constituents of the dermis does not alter in any consistent manner during wound healing or allograft rejection. Frequently the dermis of an allograft or isograft appears the same as that i n ungrafted skin. However, sometimes there are changes. Occasionally, red blood cells are found scattered extravascularly throughout the dermis, suggesting, at least i n older grafts, breakdown of blood vessel walls (figure 22 A). This is most frequently seen in later day allografts. Blood vessel lumerS sometimes appear packed with either red blood cells or "debris". The most important change noted occurs i n the lower dermis of allografts and is an increase in the number of cells present there (Figures 21 and 22). This is presumably due to movement of host cells into the region. The extent of the i n f i l t r a t i o n varies between grafts. It i s somewhat related to the length of time the graft remains on the host and to whether i t is allogeneic or syngeneic with respect to the host. In both the isografts and allografts there i s an increase i n the number of cells i n the lower dermis. Many of the early and later allografts also 60 show an increased number of c e l l s i n the middle region of the dermis, while a small percent of the grafts examined shows an increased c e l l population i n the upper most regions of the dermis. I t should be noted that, with the material viewed here, there did 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 degeneration and the degree of i n f i l t r a t i o n of c e l l s into the dermis. Sometimes d i r e c t -l y below an 8 day gr a f t epidermis which shows signs of complete degenera-t i o n (score: 3) there are large numbers of c e l l s . These c e l l s may be ab-sent from another 8 day gr a f t (score: 3). (3.) Beneath the dermis In mouse ski n a layer of f a t t y t i s s u e , the panniculus adiposus, underlies the dermis and i s f i r m l y united with i t . Around the lower c o l -lagen bundles of the dermis, the f a t c e l l s may occur s i n g l y , but are usually present i n large groups j u s t beneath. Firmly united to t h i s layer of f a t tissue i s an underlying layer of muscle tissue referred to as the panniculus  carnosus. Lying between the panniculus adiposus and the pannicuius carnosus are the p r i n c i p a l a r t e r i e s , veins, lymphatics and nerves of mouse skin. The preparation of donor skin to be used for transplantation includes removal of these tissue l a y e r s . Thus, the graft consists only of epidermis and dermis. Preparing the host bed involves removal of the epidermis, dermis and panniculus adiposus while keeping the a r t e r i e s , veins and underlying panniculus carnosus i n t a c t . Therefore, when donor skin i s transplanted to host bed, the graft dermis becomes juxtaposed to host panniculus carnosus and any overlying blood vessels. 61 During the f i r s t few days following transplantation the graft be-comes f i r m l y united to the host and there i s a marked i n f i l t r a t i o n of c e l l s into the damaged tissue j u s t beneath the graft dermis and above the host pannicuius adiposus and pannicuius carnosus. Thus a narrow gap arises be-tween the two layers f i l l e d with host c e l l s . This gap w i l l be r e f e r r e d to as the graft bed i n this study. The subsequent development or disappearance of t h i s graft bed depends on the genetic d i s p a r i t y between the host and donor. Its appearance and c h a r a c t e r i s t i c s at any point also depend on the length of time the graft has remained on the host. (a.) Isografts removed 6 and 8 days following transplantation The layers of tissue underlying the dermis of 6 and 8 day i s o g r a f t s are the same as those found i n ungrafted skin: The pannicuius adiposus and the pannicuius carnosus. However, the number of c e l l s present around the fat and muscle c e l l s i n the 6 day i s o g r a f t i s greater than that of e i t h e r the 8 day i s o g r a f t s or of ungrafted skin t i s s u e . I t should be noted here that electron microscope investigations show these c e l l s to be mostly f i b r o b l a s t s and a few neutrophils. In one graft a small mass of n e c r o t i c material was present j u s t below the dermis. (b.) A l l o g r a f t s In the a l l o g r a f t s a "gap" i s present between the lower collagen bundles and the paiinicuius adiposus layer that w i l l be r e f e r r e d to as the g r a f t bed. It i s the c e l l s of t h i s area of the graft which were examined with the electron microscope. D i f f i c u l t y arises when t r y i n g to locate t h i s g r a f t bed i n the 4 day a l l o g r a f t s (score: 1). This i s due e i t h e r to the thinness of the region or, more l i k e l y , to i t s i n s i g n i f i c a n t development at such an early stage i n the a l l o g r a f t response. The g r a f t beds of the 62 6 day (score: 2) and 8 and 10 day (score: 3) skin a l l o g r a f t s are e a s i l y located. There i s some v a r i a t i o n i n thickness, and i t i s more prominent i n some cross sections of the graf t than i n others. A primary aim of t h i s thesis i s a comparison between the mutant and non-mutant host of the c e l l types comprising the graft bed at d i f f e r e n t stages of the a l l o g r a f t response. I t was appreciated e a r l y i n the work that improper or l i m i t e d sampling could lead to an error i n i n t e r p r e t a t i o n of the data when comparing the c e l l types found i n the g r a f t beds of mutant and non-mutant hosts. For th i s reason examination of the graf t bed of 4 day a l l o g r a f t s (score: 1) was discontinued, since only very l i m i t e d sampling could be done. An electron microscope i n v e s t i g a t i o n of the graf t beds of 6 day (score: 2) and 8 and 10 day (score: 3) a l l o g r a f t s on mutant and non-mutant hosts follows. E l e c t r o n microscope study of the graft bed of 6 and 8 day (score: 2)  and 8 and 10 day (score: 3) a l l o g r a f t s (1.) Orientation and i d e n t i f i c a t i o n There are two instances when o r i e n t a t i o n becomes extremely c r i t i c a l i n t h i s study: (1) When preparing the epon-blocks f o r t h i n sectioning; and (2) when viewing the image of the sections on the fluorescent screen. I t i s important when t h i n sectioning that (a) the only material sectioned i s graft bed and (b) the condition of the epidermis d i r e c t l y above t h i s area be known. To achieve t h i s 1 y sections cut for l i g h t microscopy are ex-amined. As i n figures 21 and 22, when possible, the sections include a l l the t i s s u e layers from the epidermis to the panniculus carnosus. This allows f o r (1) confirmation that the graft bed i s , i n f a c t , present, (2) determination of the condition of the epidermis based on the c r i t e r i a out-63 l i n e d i n table VIII, and (3) proper o r i e n t a t i o n within the block, so that i t can be trimmed down to areas only of the graft bed. This o r i e n t a t i o n makes i t possible to r e s t r i c t the comparison between the g r a f t bed c e l l s of the mutant and non-mutant host to regions under the epidermis showing the same degree of degeneration. When viewing the sections i n the electron microscope, o r i e n t a t i o n again becomes very c r i t i c a l . I t i s obviously e s s e n t i a l to know which r e -gion of the graft i s present i n the section, and whether exactly the same region i s being compared between one experiment and another. Since the graft bed l i e s between the dermis and the panniculus adiposus and pannicuius  carnosus, the presence or absence of collagen bundles, f a t and muscle c e l l s serve as pointers 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 region imaged on the fluoresent screen. Figures 25 and 26 i l l u s t r a t e t h e i r use. The f i r s t diagram i s a tracing of a region of a 6 day skin a l l o g r a f t (score: 2) on a W/WV host, and depicts the graft bed as i t can appear at low magnification. Notice that the collagen bundles (dotted areas) are at t h e i r highest density i n the upper region of the diagram while the f a t c e l l s (hatched area) are present i n the lower edge of the diagram. Thus, the c e l l s , debris and blood vessels of the c e n t r a l area l i e between the dermis and a layer of fat c e l l s . This area i s then the graf t bed. The importance of th i s method of o r i e n t a t i o n i s most evident when examining areas which are d i f f i c u l t to d i s t i n g u i s h as graft bed by t h e i r i n t e r n a l components. This i s i l l u s t r a t e d i n figure 26 which i s a tracing of an area i n an 8 day skin a l l o g r a f t (score: 3) on a w/+ host. Here at lea s t 50% of the material i s labeled debris (figure 30) and then, even 64 Figure 25: Tracing of a region of a 6 day skin a l l o g r a f t (score: 2) This diagram depicts the g r a f t bed as i t appears at low magnifi-cation. The g r a f t bed consists of a heterogeneous population of c e l l s , areas of e x t r a c e l l u l a r material, including collagen (dotted areas) and debris, and blood vessels (dark areas). Notice i t l i e s between a region having a high density of collagen bundles (dermis) and fa t c e l l s (hatched area). B- activated lymphocyte L- small lymphocyte P- neutrophil E- eosinophil F- f i b r o b l a s t M- macrophage H- immunoblast 65 Figure 26: Tracing of a region of an 8 day skin a l l o g r a f t (score: 3) This diagram depicts the graft bed as i t appears at low magnifi-cation, Notice that, i n contrast to figure 25, the major component of the graft bed i s the e x t r a c e l l u l a r material, p r i m a r i l y the debris. I d e n t i f i c a t i o n of most of the c e l l s i s impossible due to marked c e l l u l a r necrosis. 6 5 c 66 though boundaries of s i n g l e c e l l s are outlined, only about 20% can be i d e n t i f i e d (refer to figures 26, 29 and 30A). I t i s sometimes impossible to know i f blood vessels are present or not. In other g r a f t s with a score of 3 t h i s state of the graft bed i s heightened to the point where the r e -gion may be considered a mass of debris where c e l l s are d i f f i c u l t to d i s -t i nguish at a l l (as i n figure 30B). Under these circumstances the guide-l i n e s (presence or absence of collagen bundles, fat c e l l s or muscle c e l l s ) become very important i n assuring oneself that one i s , i n f a c t , i n the graft bed. (2.) General c h a r a c t e r i s t i c s of the graft bed As depicted i n figures 25 and 26, the graft bed consists mainly of a heterogeneous population of c e l l s , areas of e x t r a c e l l u l a r material, i n -cluding collagen and debris, and blood vessels. The amount and condition of these components varies somewhat within a g r a f t but d i f f e r s markedly between 6 day a l l o g r a f t s (score: 2) and 8 and 10 day a l l o g r a f t s (score: 3). The o v e r a l l appearance of the g r a f t beds i s the same i n g r a f t s with the same score whether on mutant or non-mutant hosts. The following i s a des-c r i p t i o n of the general c h a r a c t e r i s t i c s of the graft beds of 6 day (score: 2) and 8 and 10 day (score: 3) a l l o g r a f t s . (a.) Graft bed of 6 day a l l o g r a f t s (score: 2) The majority of the c e l l s comprising the graft bed of a 6 day a l l o -g r a f t do not show any signs of necrosis (figure 27 and 28). The proximity of the c e l l s to one another varies considerably from one area of the g r a f t bed to the other. Figures 25, 27 and 28 are examples of the two main kinds of d i s t r i b u t i o n noted. In some regions, as i n figure 25 and 27, the c e l l s occur s i n g l y or i n groups of 2 to 4, separated by distances of .1 u to 4 u. 67 Closer spacing of the c e l l s often occurs around the blood v e s s e l s . In other regions of the graft bed the proximity of a c e l l to i t s neighbors i s less than .1 .u (figure 28), creating masses of c e l l s w i t h i n the graft bed. Figure 28 shows a section of such a c e l l mass. Apposing c e l l membranes often follow the same contour and, i n some cases, appear blurred, due probably to oblique sectioning. With the c e l l s so close there i s l i t t l e room for e x t r a c e l l u l a r material although sometimes a l i t t l e collagen and very small areas of debris are present. Within the e x t r a c e l l u l a r spaces of the graft bed there are blood vessels, collagen, debris and a few red blood c e l l s (figure 25). The blood vessels traverse the gra f t bed. Their endothelial c e l l s do not show any signs of necrosis or degeneration. A cross section of the lumen usually shows i t to contain a few red blood c e l l s and, sometimes, one or two white blood c e l l s (figure 25). Occasionally the white blood c e l l s l i e between the endothelial c e l l s and the basement membrane of the blood v e s s e l w a l l , suggesting they are moving into or out of that v e s s e l . The material i n the e x t r a c e l l u l a r spaces labeled debris consists of eith e r vacuoles of varying s i z e and electron density, almost c e r t a i n l y de-ri v e d from broken-down c e l l s , membrane-bound granules (some c h a r a c t e r i s t i c of eosinophils or ne u t r o p h i l s ) , other c e l l remnants, or a l l of these. The amount of debris present varies from one area of the graft bed to another. However, scattered among the c e l l s of the g r a f t bed, as shown i n fig u r e 25, i t s presence i s never very extensive. (b.) Graft bed of 8 day a l l o g r a f t s (score: 3) The c e l l s of the 8 day graft bed are d i s t r i b u t e d i n much the same 68 Figure 27: Low power electron micrograph of the graft bed of a 6 day skin a l l o g r a f t (score: 2) Note the distance 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 to one another. Only a few of the c e l l s show any signs of necrosis. 69 Figure 28: Low power electron micrograph of the graft bed of a 6 day skin a l l o g r a f t (score: 2) This i s a section through a mass of c e l l s found w i t h i n the g r a f t bed. Notice that the c e l l s are so close t h e i r apposing c e l l membranes often follow the same contour. 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 material. 70 was as those found i n the 6 day graft beds. They may occur s i n g l y , or i n groups of 2 to 4, .1 u to 4 u apart, as i l l u s t r a t e d i n figures 26 and 29. Neighboring c e l l s may also be very close to each other r e s u l t i n g i n the formation of a small c e l l mass i n the graft bed (a portion of such a c e l l mass i s shown i n fig u r e 30A). A s t r i k i n g contrast which i s i l l u s t r a t e d i n the figures between the 6 day and 8 day graf t beds i s the condition of the c e l l s . The majori-ty of the c e l l s i n the graf t bed of an 8 day a l l o g r a f t (score: 3) show marked signs of necrosis i n c l u d i n g pyknosis, swollen cytoplasmic organelles, i n some cases, extrusion of eit h e r nuclear chromatin or cytoplasm, or both, and absence of e i t h e r the c e l l membrane or the nuclear membrane, or both. I d e n t i f i c a t i o n of these c e l l s i s impossible (figure 26). Further, these signs of necrosis are seen i n only a small number of the c e l l s of the 6 day a l l o g r a f t (figures 27 and 28). Again, i n contrast to the graft bed of 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 material, p r i m a r i l y the debris. I t consists of remnants of c e l l s , vacuoles of var-ious sizes and el e c t r o n density and granules (figures 29 and 30). Often so many c e l l s of the graft bed are breaking down that i t becomes f e a s i b l e to consider the whole graft bed as debris (figure 30B). The endothelial c e l l s of the blood v e s s e l walls also show signs of breakdown and, i n many areas, no longer completely enclose the lumen. The lumen i s us u a l l y o b l i t e r a t e d by masses of red blood 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 of the graft bed (a.) F i x a t i o n procedures and the preservation of the u l t r a s t r u c t u r e of the graft bed c e l l s 71 Figure 29: Low power electron microphage of the graft bed of an 8 day skin a l l o g r a f t (score: 3) Note the distance 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 to one another. The majority of the c e l l s i n the graf t bed show marked signs of necrosis including pyknosis, swollen cytoplasmic organelles, extrusion of e i t h e r nuclear chromatin or cytoplasm, or both, and an absence of either the c e l l membrane or the nuclear membrane, or both. I d e n t i f i c a t i o n of these c e l l s i s impossible. 72 Figure 30A: Low power electron micrograph of the graft bed of an 8 day skin a l l o g r a f t (score: 3) Similar to figure 28, t h i s i s a section through a mass of c e l l s found within the graft bed. However, notice that the majority of the c e l l s show marked signs of necrosis. Figure 30B: Debris Sometimes the c e l l u l a r necrosis i s so extensive i n an 8 day skin a l l o g r a f t (score: 3) that the whole graft bed may f e a s i b l y be considered debris. Note the remnants of c e l l s , vacuoles of various s i z e s and gran-ules . 73 Due to the extent of c e l l breakdown occurring i n some of the graft beds, i t i s important to consider the e f f e c t s of the f i x a t i o n procedures used. The method of f i x a t i o n followed was e i t h e r : (1) 6% glutaraldehyde i n phosphate b u f f e r , pH 7.4, followed by p o s t - f i x a t i o n i n 1% osmium tetrox-ide; or (2) Karnovsky's f i x a t i v e (paraformaldehyde plus glutaraldehyde i n phosphate b u f f e r , pH 7.4) followed by p o s t - f i x a t i o n i n 1% osmium tetroxide. With the former method of f i x a t i o n there i s a high incidence of c e l l breakdown i n the g r a f t bed of the, 6 d a y . a l l o g r a f t . 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 , including marked d i s t e n t i o n of the endoplasmic reticulum, g o l g i and nuclear membrane, broken cisternae within the mitochondria, chromatin clumping along the nu-c l e a r membrane, and separation of the cytoplasm from the nuclear membrane. Often i t was found that with two c e l l s side by side, the u l t r a s t r u c t u r e of one would show the above c h a r a c t e r i s t i c s while the other would show a l l the properties i n d i c a t i n g proper preservation. In the 8 day g r a f t s the incidence of c e l l breakdown was very high. With such a high and e r r a t i c incidence of c e l l breakdown i n the gra f t beds, the second method, Karnovsky's f i x a t i v e , was t r i e d . The amount of c e l l breakdown i n the 6 day a l l o g r a f t s decreased s i g n i f i c a n t l y (figures 25, 27 and 28), except for the neutrophil population. For some reason the neutrophils always show signs of breakdown. This i s evident i n figures 27 and 28 (notice the gap present between the cytoplasm and nucleus, empty vacuoles and broken mitochondrial c i s t e r n a e ) . Karnovsky's f i x a t i v e did not lower the extremely high incidence of c e l l breakdown i n the graft beds of 8 and 10 day a l l o g r a f t s (score: 3) (refer to figures 29 and 30). This 74 breakdown i s probably not due to improper f i x a t i o n . Due to t h i s condition of the c e l l s i n 8 day a l l o g r a f t s (score: 3) the problem of sampling occurs again. A search for c e l l s w ithin the g r a f t bed with s u f f i c i e n t preservation of 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 hosts on grafts given a score of 3 i s not possible. This also means that the comparisons of c e l l types between beds i n which the graf t s had a score of 2 and 3 have been made on the "general appear-ance" l e v e l only. (b.) Description of the c e l l types found i n the graft bed of 6 day a l l o g r a f t s (score: 2) on mutant and non-mutant hosts There i s a wide v a r i e t y of c e l l types present i n the graft bed, i n -cluding f i b r o b l a s t s , small lymphocytes, immunoblasts, activated lymphocytes, macrophages, eosinophils and neutrophils. The d i s t r i b u t i o n of any one c e l l type does not appear to follow an obvious pattern (figure 25). In other words, one c e l l type does not co n s i s t e n t l y appear associated with another s p e c i f i c c e l l type or a c e r t a i n kind of e x t r a c e l l u l a r m a t e r i a l . The only exception to t h i s i s the f i b r o b l a s t , which i s usually associated with c o l l a -gen bundles. There does not appear to be any difference between the mutant and non-mutant hosts i n either the kinds of c e l l s which enter the graft bed of a 6 day a l l o g r a f t (score: 2) nor i n t h e i r u l t r a s t r u c t u r e . The following i s a d e s c r i p t i o n of each c e l l type found i n the graft bed. Examples of the c e l l s are taken from graft beds of a l l o g r a f t s with a score of 2 on e i t h e r mutant or non-mutant hosts. 75 1. Small lymphocyte Small lymphocytes are present i n the graft beds of 6 day (score: 2) and 8 day (score: 3) a l l o g r a f t s . In electron micrographs these c e l l s range i n s i z e from 5 to 7 u i n diameter (figure 31A). 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 of the " r e s t i n g " or " i n a c t i v e " state by which they are commonly described. The nucleus has a considerable amount of heterochroma-t i n and a small nucleolus and i s surrounded by a narrow rim of cytoplasm (figure 31A). The contents of the cytoplasm vary. Centrio l e s are usually present and there are a few, small mitochondria. The Golgi apparatus, i f present, i s poorly developed and rough endoplasmic reticulum i s sparse. Ribosomes are usually free i n the cytoplasm and, sometimes, there are one or two small electron dense vacuoles present. Some of the small lymphocytes show minor changes i n t h e i r u l t r a s t r u c -ture. Notice i n figure 31B the increase i n the number of RER strands present i n the cytoplasm and the number of ribosomes and the proportion of these which are ribosomal aggregates. These changes may (as shown i n figure 31B) be accompanied by an increase i n the amount of euchromatin present i n the nucleus and by a more prominent nucleolus. Often times these c e l l s are larger suggesting they are medium or large lymphocytes. 2. Immunoblas ts The immunoblast (figure 32) i s one of the l e a s t frequently seen c e l l types i n the graft bed. I t i s found i n the g r a f t bed of 6 (score: 2) and 8 (score: 3) day a l l o g r a f t s on both mutant and non-mutant hosts. It appears to be very s e n s i t i v e to the f i x a t i o n procedure since, more often than not, the mitochondrial cisternae appear broken down within the matrix. These 76'. Figure 31A: Small lymphocyte found i n the gra f t bed of a .6 day skin a l l o g r a f t (score: 2). The bar represents 1 u. Figure 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 found i n the graft bed of a skin a l l o g r a f t re-moved 6 days following transplantation. The bar represents 1 u. 7C*. Figure 32: Immunoblast The most prominent c h a r a c t e r i s t i c of t h i s c e l l i s the abundance of polyribosomes within the cytoplasm. This c e l l i s present at the gra s i t e of 6 day skin a l l o g r a f t s (score: 2). The bar represents 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 electron micrographs. The nucleus consists p r i m a r i l y of f i n e l y scattered chromatin with a t h i n electron-dense band of clumped chromatin along the nuclear membrane. I t also has a prominent nucleolus. This nucleus i s large and round, often with an indentation at one end, and i s surrounded by a somewhat narrow rim of cytoplasm. Only a few cytoplasmic organelles are usually present, mito-chondria and a strand or two of RER. The most prominent c h a r a c t e r i s t i c of the immunoblast i s the abundance of polyribosomes within the cytoplasm ( r e f e r to figure 32). 3. Activated lymphocyte Another c e l l type present i n the graft bed i s the activated lympho-cyte described by H a l l et^ al_. (1967). These c e l l s vary markedly i n t h e i r s i z e but are usually larger than the small lymphocytes and appear with greater frequency than any other type of lymphocyte i d e n t i f i e d . H a l l et^ a l . (1967) judge the various u l t r a s t r u c t u r a l states as having a "le a s t d i f f e r -entiated" or a "more d i f f e r e n t i a t e d " appearance. Such states were also found i n the population of c e l l s comprising the graft bed. Figure 33A i s an example of a "le 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 c e l l as described by H a l l (1967). The nucleus i s large and has an i r r e g u l a r shape. There i s a moderate amount of chromatin clumping along the nuclear membrane and a nucleolus i s usually present. The cytoplasm i s much more developed than anything described thus f a r . Ribosomes, abundant i n the cytoplasm, usually occur s i n g l y , although a few polyribosomes are sometimes present. A few scattered strands of RER are present. Located i n the 79 Figure 33A: Activated lymphocyte found i n the graft bed of a 6 day sk i n a l l o g r a f t (score: 2) The 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 t h i s c e l l type. The bar represents 1 y. Figure 33B: Activated lymphocyte found i n the g r a f t bed of a 6 day skin a l l o g r a f t (score: 2). 80 c e n t r a l region of the c e l l i s the Golgi apparatus. I t i s usually w e l l developed, co n s i s t i n g of 2 or 3 lamellar stacks and associated v e s i c l e s , vacuoles and some small e l e c t r o n dense vacuoles (as i n fi g u r e 33B). Figures 34A and 34B are examples of activated lymphocytes with a more " d i f f e r e n t i a t e d appearance". There i s an abundance of ribosomes which mostly occur i n rosettes and, sometimes, i n short chains. RER also becomes more abundant. In some c e l l s i t i s generally d i s t r i b u t e d through-out the cytoplasm (as i n figure 34A) while i n others the strands are a r -ranged i n short stacks i n one area of the c e l l (as i n figu r e 34B). The number of mitochondria may also be up. As i n the " l e s s d i f f e r e n t i a t e d " activated lymphocytes, the gol g i zone i s well developed with a number of vacuoles present, some containing electron dense granules (figure 34A). Microtubules and m i c r o f i b r i l s are often present also. 4. Macrophage Another c e l l type present i n the graft bed of a l l o g r a f t s with scores of 2 and 3 on both mutant and non-mutant hosts i s the macrophage, which make up a su b s t a n t i a l portion of the c e l l population. The c e l l appears i n various u l t r a s t r u c t u r a l states which have been separated i n t o "non-activa-ted" and an "activated" form. Figure 35 i s an example of the "non-activated" form. The diameter of these c e l l s i s 10 to 14 u . The nucleus i s horseshoe-shaped and consists of dispersed chromatin with a t h i n electron dense band of clumped chromatin along the nuclear membrane. The volume r a t i o of cytoplasm to nucleus i s high. The cytoplasm contains scattered strands of RER, a g o l g i zone, several mitochondria and electron dense vacuoles of varying s i z e . There 81 Figure 34A: Activated lymphocyte found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2) This i s an example of an activated lymphocyte showing a more " d i f f e r e n t i a t e d appearance". Note the abundance of ribosomes i n the cytoplasm. The bar represents l ' u . Figure 34B: Activated lymphocyte found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2) This i s an example of an activated lymphocyte showing a more " d i f f e r e n t i a t e d appearance". Note the short stacks of rough endoplasmic reticulum. The bar represents 1 p . Bio, 82 Figure 35: Monocyte, or "non-activated" form of macrophage, found i n the graf t bed of a 6 day skin a l l o g r a f t (score: 2) The bar represents 1 u. 83 are a large number of small v e s i c l e s also. The c e l l membrane has a very uneven contour due to the number of pseudopods present. Examples of the more "activated" forms are shown i n figures 36A, 36B and 36C. These c e l l s also have diameters of 12 to 15 u i n electron micrographs. The nucleus shows an i r r e g u l a r i t y of i t s surface and con-tains more clumped chromatin than the "non-activated" form. I t also has a well developed golgi zone, several mitochondria, scattered strands of RER and large numbers of small v e s i c l e s i n i t s cytoplasm. In contrast to the cytoplasm of the "non-activated" form are the increased density, s i z e and number of ele c t r o n dense vacuoles and the reduced number of surface processes. Figures 36A and 36B are examples of two u l t r a s t r u c t u r a l states of this activated form. Figure 36C shows a macrophage engulfing another c e l l . Such ass o c i a t i o n with c e l l s or debris i s not uncommon. 5. Eosinophil The eosinophil i s another c e l l type found i n the g r a f t bed. In some regions i t makes up a s u b s t a n t i a l portion of the c e l l population while i n others, as i n figure 25, there are only one or two c e l l s . The c e l l i s 9 to 13 u i n diameter i n electron micrographs (figures 37A and 37B). The nucleus of an eosinophil i s lobed, usually 2 or 3 lobes are present i n a section, and the chromatin i s densely packed along the nuclear membrane. Within the cytoplasm are a few strands of RER, a few empty v e s i c l e s of varying s i z e and several mitochondria. Also present are the granules which characterize the eosinophil. These are membrane-bound, electron-dense v e s i c l e s varying from .5 to 1 u i n diameter. Most contain a s t i l l denser el e c t r o n band within t h e i r c e n t r a l area which appears i n sections as a 84 Figure 36A: "Activated" macrophage found i n the gra f t bed of a 6 day skin a l l o g r a f t (score: 2). The bar represents l u , Figure 36B: "Activated" macrophage found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2) Notice the increased number of electron dense vacuoles compared to figure 36A. The bar represents l u . Figure 36C: "Activated" macrophage i n the process of phagocytosis within the graft bed of a 6 day skin a l l o g r a f t (score: 2). The bar represents l u . 85 Figure 37A: Eosinophil found i n the graft bed of a skin a l l o g r a f t removed 6 days following transplantation (score: 2) Note that the electron dense granules contain a l e s s dense el e c t r o n band wit h i n them. The bar represents l y Figure 37B: Eosinophil found i n the graft bed of a 6 day skin a l l o g r a f t (score: 2) Note that the electron dense granules contain a s t i l l denser e l e c -tron band within t h e i r c e n t r a l area which appears i n sections as a rec-tangle. The bar represents l y Figure 37C: Neutrophil found i n the graft bed of a 6 day sk i n a l l o -graft (score: 2) Figure 37D: Degenerating neutrophil found i n the graft bed of a skin a l l o g r a f t removed 6 days following transplantation (score: 2). The bar represents l y 86 square or rectangle (figure 37B). In some, a less dense band occupies the same p o s i t i o n (figure 37A) i n the granule. 6. Neutrophil Another c e l l type found i n the graft bed of a l l o g r a f t s on the mutant and non-mutant hosts i s the neutrophil. This c e l l i s always present, and sometimes, i t makes up a substantial portion of the c e l l population (figure 25). The nucleus i s multi-lobed and consists almost e n t i r e l y of clumped chromatin (figures 37C and 37D). Within the cytoplasm are a few mitochondria and some cl e a r vacuoles approximately .01 y i n diameter. A golg i i s usually located c e n t r a l l y i n the c e l l , as seen i n figu r e 37C. Also i n the cytoplasm are a number of electron dense granules approximately .1 to .3 y i n diameter which are a c h a r a c t e r i s t i c of the neutrophil. Often the neutrophils appear as i n figu r e 37D. This c e l l shows signs of degenera-t i o n . The nuclear membrane i s swollen and within the cytoplasm there are a large number of empty v e s i c l e s as w e l l as electron dense vacuoles of varying s i z e . The background matrix of the cytoplasm appears to have clumped with-i n the l i m i t s of the c e l l membrane. 7. Fibroblast F i b r o b l a s t s (figure 38B and 38C) are also part of the c e l l population of the graft bed. In ele c t r o n micrographs these c e l l s are 11 to 13 y i n diameter and are often associated with collagen bundles. They have a large oval nucleus which contains f i n e l y dispersed chromatin and one or two pro-minent n u c l e o l i . The prominent feature of the cytoplasm i s the abundance of RER as shown i n the i n s e r t (figure 38A). There are also a number of 87 Figure 38A: Fibroblast found i n the graft bed of a s k i n a l l o g r a f t removed 6 days following transplantation (score: 2) The bar represents l y . Figure 38B: Stacks of rough endoplasmic reticulum of a f i b r o b l a s t i n the g r a f t bed of a 6 day skin a l l o g r a f t . The bar repre-sents l y . Figure 38C: F i b r o b l a s t found i n the graft bed of a s k i n a l l o g r a f t removed 6 days following transplantation (score: 2) The bar represents l y . 88 mitochondria present, and a well developed golgi i s often centrally l o -cated. Sometimes there are a few electron dense granules i n the cytoplasm. The shape of the fibroblasts is very irregular, depending on their asso-ciation with other cells or collagen bundles. The essential features of the ultrastructure of each c e l l type found i n the graft bed ofaallografts on both mutant and non-mutant host are summarized in table IX. Table IX E s s e n t i a l features of the u M t r a s t r u c t u r e of 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 mutant and non-mutant hosts C e l l type approximate s i z e N u c l e i N u c l e o l i Ribosomes RER Go l g i V e s i c l e s & Vacuoles M i t o - Other chondria Small Lym-phocyte a. " r e s t i n g " s t a t e b. w i t h "minor change 5 to 7y i n diameter round and i n -dented; l a r g e amount of he-terochromatin small f r e e very z: 'G~ poo r l y few e l e c t r o n sparse deve- dense vacu-loped oles present few, c e n t r i -s m a l l o l e us-u a l l y present 5 to 8y i n diameter i n c r e a s e i n the amount of euchromatin more pro-minent increase i n increase # & % aggre-in # of gates strands present same as above same as above Immunoblas t 9 to l l y i n diameter round & f i n e l y s c a t t e r e d chro-. prominent m a t i n ; t h i n r electron-dense band at nuclear membrane abundance of p o l y -ribosomes 1 or 2 strands few e l e c t r o n - few dense v e s i -c l e s A c t i v a t e d Lymphocyte a. l e s s d i f -f e r e n t i a t e d more d i f f e r -e n t i a t e d 8 to lOu i n diameter l a r g e & i r r e -gular shape; moderate a-mount of clumping  present abundant- few s c a t - w e l l few e l e c t r o n occur tered d e v e l - dense vacu-s i n g l y strands oped o l e s ; l o t of v e s i c l e s present 8 to l l y i n diameter same as above present abundant- more abun- w e l l l o t of both; i n -most occur dant d e v e l - some e l e c - creased i n r o s e t t e s oped tron dense i n num-granules bers (continued) 00 Table IX (continued) C e l l type Approximate s i z e Nuclei N u c l e o l i Ribosomes RER Golgi Vesicles & Mito- Other Vacuoles chondria Macrophages a. non-activa- 10 to 14u ted Horse-shoe-shaped; dis-persed chro-matin present few scattered strands present electron dense va-cuoles of varying s i z e ; large & small v e s i c l e s several c e l l mem brane un even con tour b. activated 12 to 15u i r r e g u l a r shape more clumped chro-matin present few scattered strands well devel-oped increased several reduced #, density and s i z e of e l e c -tron dense vacuoles # at sur-face pro-cesses Neutrophil 10 to 12u lobed; chro-matin dense-l y packed absent few scattered short strands cen-t r a l l y located clear va- few cuoles; # of • dense gran-uoles Eosinophil 10 to 13u lobed; chro-matin denser l y packed absent few few strands present few e l e c -tron v e s i c l e s containing electron dense bands several Fibroblast 11 to 14u l a r g e ; o v a l ; f i n e l y d i s -persed chro-matin 1 or 2 prominent few abundant well devel-oped few e l e c -tron dense granules several 91 DISCUSSION The s k i n a l l o g r a f t response: general remarks With regard to the skin a l l o g r a f t response, only very few points have become clear as to what i s happening at the graft s i t e . Prendergast's (1964) studies, along with the work of others ( H a l l , 1967; Lance and Cooper, 1972; Moore and H a l l , 1972; 1973; Najarian and Feldman, 1962), c l e a r l y demonstrate that only a small percentage of the c e l l s i n f i l t r a t i n g the gr a f t s i t e are s p e c i f i c a l l y s e n s i t i z e d to the sk i n antigens. These c e l l s have ar i s e n i n the regional lymph node i n response to the presence of the g r a f t . Prendergast estimates, on average, that 5.6% of the i n f i l -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 . The r e s t of the graft bed c e l l population consists p r i m a r i l y of non-specific e f f e c t o r c e l l s , i ncluding those of monocyte-macrophage lineage 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) and non-s e n s i t i z e d lymphocytes. Accumulation of both the s p e c i f i c and the "uncom-mitted" e f f e c t o r c e l l s appears to be an e s s e n t i a l component of the skin a l l o -graft response (Billingham and S i l v e r s , 1971; Giroud et a l . , 1970; Lambert and Frank, 1970). Neither the cause of t h i s massive accumulation of c e l l s at the graft 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 of ac t i o n which r e s u l t i n the destruction of the grafted tissue are known. The only h i n t as to what may be happening a r i s e s from i n v i t r o s t u-dies. Of primary i n t e r e s t here are the tissue culture studies of lympho-cyte a c t i v i t y which demonstrate three important properties of the 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 only to the stimulating 9 2 antigen, thus demonstrating a capability for specific recognition. Second-ly, upon proper stimulation the lymphocyte i s capable of cytotoxicity, damaging the target c e l l by some unknown mechanism. Further, upon recog-nition of specific antigen the lymphocyte, in vitro at least, is capable of releasing any one or more of a number of effector molecules, referred to as lymphokines (Dumonde et a l . , 1969). Included in this group of affector molecules are substances that inhibit macrophage migration (David and David, 1972; Lamelin, 1971; Pick and Turk, 1972; and Pick, 1974), that are chemo-taxic for neutrophils (Ward et a l . , 1970), eosinophils (David and David, 1972) or mononuclear cells (David and David, 1972), that stimulate unsensi-tized lymphocytes to transform to blast cells and divide (Falk et a l . , 1970; Dumonde et a l . , 1969), that stimulate macrophages, increasing phagocytotic a b i l i t y (Nathan ejt a l . , 1973) and "arming" them for direct cytotoxicity (Evans and Alexander, 1972; Evans and Grant, 1972; Lohmann-Matthes et a l . , 1973) , that stimulate the proliferation of "granulocytic and macrophage precursor populations" (Parker and Metcalf, 197&). These properties of the lymphocyte suggest that the lymphocyte popu-lation, especially the specifically sensitized portion, may play an important role i n the accumulation and activation of the cells at the graft site. Upon interaction with and recognition of the specific antigen within the graft site the few sensitized lymphocytes present produce and release effec-tor molecules capable of either attracting or activating, or both, macro-phages, eosinophils, neutrophils and non-sensitized lymphocytes. Such ac-t i v i t y would, in fact, place these sensitized lymphocytes in an i n i t i a t i n g role i n the mediation of graft destruction. 93 The lymphocyte: A l i g h t microscope study Light microscope studies 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 popula-t i o n (Medawar, 1944; Simar and Betz, 1970; Billingham and S i l v e r s , 1971). The observations and data on c e l l counts presented i n t h i s thesis are con-s i s t e n t with t h i s f i n d i n g . Depending on the time elapsed since transplan-t a t i o n , the lymphocyte can account for up to 60% of the host c e l l popula-t i o n i n f i l t r a t i n g the gr a f t s i t e (table V I I I ) . Since l i g h t microscope studies of the graft bed have been l i m i t e d , i n the majority of cases, to paraffin-embedded ti s s u e sections i n which the surrounding ti s s u e compon-ents i n t e r f e r e with 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 sub-populations has not been possible. By e x t r a c t i n g the c e l l s from the graft bed, as i n t h i s study, i t becomes passible to i d e n t i f y d i f f e r e n t types of lymphocytes, based on the morpho-l o g i c a l descriptions from studies of .blood smears (Elves, 1966; Ling, 1968) and of blood and lymphoid c e l l s i n tissue c u l t u r e . The lymphocyte popula-t i o n i s o l a t e d here has been divided i n t o the following categories; small lymphocyte, large lymphocyte; transformed lymphocyte and b a s o p h i l i c c e l l . I t was hoped that this more precise i d e n t i f i c a t i o n of lymphocytes would provide some in s i g h t as to the kinds of lymphocyte a c t i v i t y occurring wi t h i n the graft s i t e . However, accumulative information indicates 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 of the lymphocyte i s not possible by s t r i c t l y morphological c r i t e r i a since a group of morphologically 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 quite diverse. Therefore, i t may be misleading to base conclusions about lymphocyte a c t i v i t y on s t r i c t l y morphological 94 c r i t e r i a . In t h i s study, small and large lymphocytes, s t r u c t u r a l l y the same as those of mouse blood and lymph (Schermer, 1967), appear to peak i n con-centration, r e l a t i v e to the other c e l l types, on the s i x t h day following transplantation (chart I ) . This i s consistent with the findings of Jaboisiak (1971) who, using mice, s i m i l a r l y i s o l a t e d the c e l l s i n f i l t r a -t i n g a skin graft and found that the number of lymphocytes i s highest 6 days following transplantation. The small lymphocyte, i d e n t i f i e d with the l i g h t microscope, has been at t r i b u t e d a rather diverse number of metabolic a c t i v i t i e s . Many authors consider the small lymphocyte to be a " r e s t i n g " or " i n a c t i v e " c e l l due to i t s undeveloped cytoplasm and large amount of heterochromatin (Zucker-Franklin, 1969). I t i s suggested that these c e l l s are i n an "un-stimulated" state. They can then be viewed as c e l l s with the p o t e n t i a l for i n i t i a l recognition of antigen or s p e c i f i c recognition of the s e n s i t i -zing antigen and with the a b i l i t y to d i f f e r e n t i a t e further following proper stimulation. Data from i n v i t r o tissue culture studies of lymphocytes suggests that the "morphologically" small lymphocyte as seen with the l i g h t microscope i s not s t r i c t l y " i n a c t i v e " as far as only 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 a c t i v i t y . In p a r t i c u l a r , Wilson (1965, 1967) states that small lymphocytes, removed from the regional lymph node or thoracic duct lymph of mice which have previously rejected a s k i n a l l o g r a f t , are capable of destroying monolayer cultures of target c e l l s containing the antigenic determinants of the s k i n donor. Other r e -search groups have also c i t e d the small or large lymphocyte as responsible 95 i n v i t r o f o r s p e c i f i c target c e l l l y s i s (Able and Rosenau, 1970) and, i sometimes, for release of e f f e c t o r molecules (Lawerence and Landy, 1969). Another category of lymphocytes noted i n this l i g h t microscope study i s the transformed lymphocyte. I t has not been described i n other l i g h t microscope observations as part of the c e l l population i n f i l t r a t i n g the gr a f t bed. This i s probably due to the technique of 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 of instances i n both i n v i t r o and i n vivo studies. They have been described as a r i s i n g from small or large lymphocytes upon stimulation with PHA i n tissue culture studies ( B i b e r f e l d , 1971; Elves, et^ al., 1964). H a l l and co-workers demon-stra t e t h e i r appearance i n the efferent lymph of a lymph node stimulated by either soluble or transplantation antigens, and suggest that the c e l l s have arisen from small lymphocytes stimulated within the lymph node. In v i t r o studies of lymphocyte c y t o t o x i c i t y describe c e l l s of s i m i l a r morpho-logy as a r i s i n g from small lymphocytes i n the presence of heterogeneic lym-phocytes (mixed lymphocyte culture) (Andersson and Hayry, 1973; Hayry et 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 small lymphocytes i n the presence of the s e n s i t i z i n g antigen (Heise and Weiser, 1969; Granger and Klob, 1968). In these instances the c e l l s have been termed b l a s t c e l l s . I t also appears they are capable of s p e c i f i c or non-specific target c e l l l y s i s and the re-lease of e f f e c t o r molecules (Granger and Klob, 1968; Andersson and Hayry, 1973; Shacks et a l . , 1973). Another lymphocyte i d e n t i f i e d i n this l i g h t microscope study i s r e -ferred to as a b a s o p h i l i c c e l l due to i t s very b a s o p h i l i c cytoplasm. No comparable c e l l type i s mentioned i n other l i g h t microscope i n v e s t i g a t i o n s 96 of the s k i n a l l o g r a f t response or tissue culture studies of lymphocyte a c t i v i t y . Again, this i s probably due to the technique of i n v e s t i g a t i o n . Jakobisiak (1971) describes a "basophilic c e l l " as part of the i n f i l t r a t i n g c e l l population at the skin graft s i t e , but h i s d e s c r i p t i o n i s not c l e a r and the proportion 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 are quite d i f f e r -ent from those of the b a s o p h i l i c c e l l described i n t h i s study. The very b a s o p h i l i c s t a i n i n g property of these c e l l s suggests they are taking an a c t i v e part i n the a c t i v i t y occurring within the graft s i t e . Some of the i n v i t r o t i s s u e culture studies of 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 small lymphocytes when newly formed are b l a s t c e l l s ( i . e . transformed lymphocytes). However, i n the older cultures these b l a s t c e l l s become smaller i n s i z e and simultaneously, more e f f e c t i v e as k i l l e r c e l l s (Andersson and Hayry, 1973; Hayry et a l . , 1972). Since the b a s o p h i l i c c e l l forms such a large portion of the c e l l population perhaps they are the equivalent of--'the. ""older" c e l l s of Andersson and Hayry (1973), a c t i v e l y synthesizing material as part of the cell-mediated immune response to s k i n a l l o g r a f t antigens. The lymphocyte: An electron microscope study The findings from the electron microscope study concerning the i n -f i l t r a t i n g c e l l s are discussed i n a separate section from the l i g h t micro-scope study, since combining the information would require an attempt to r e l a t e the c e l l types observed i n each study to one another. This I do not think would be v a l i d without a s p e c i a l study of t h i s point due to the grossly d i f f e r e n t means by which the tissues were treated and the c r i t e r i a by which they were c l a s s i f i e d and 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 population. 97 Lymphocytes have been noted as a major part of the c e l l u l a r i n f i l -t rate i n a l l electron microscope studies of the skin a l l o g r a f t response. Some discrepancy does appear between research groups as to the kinds of lymphocytes which are present (table X). In t h i s thesis the types of lymphocytes that could be i d e n t i f i e d at the u l t r a s t r u c t u r a l l e v e l included the small 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 activated lymphocyte. The small lymphocyte i s commonly seen with the electron microscope as being one of the c e l l s i n f i l t r a t i n g the skin a l l o g r a f t s i t e (Simar and Betz, 1970; Weiner, Lattes and P e a r l , 1969; A l l e g r a et a l . , 1968). They are e a s i l y i d e n t i f i e d by t h e i r s i z e , high nucleus/cytoplasm r a t i o , lack of cytoplasmic organelle development and abundance of heterochromatin (Elves, 1966; Ling, 1968). They are considered by some authors to be " i n a c t i v e " such that, i n t h i s case, they could be considered as contributing l i t t l e to the s k i n a l l o g r a f t response u n t i l properly stimulated. Of i n t e r e s t here are the small lymphocytes "with minor changes". These were also described as present i n the graft bed by Simar and Betz (1970) i n v e s t i g a t i n g u l t r a -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. In v i t r o studies i n d i c a t e these c e l l s could be taking an active part i n the destruction of the grafted t i s s u e . B i b e r f e l d (1971, 1973) describes c e l l s of s i m i l a r morphology as attaching to Chang c e l l s and bringing about t h e i r l y s i s i n the presence of PHA or antibody i n t i s s u e c u l t u r e . Ax et a l . (1968) describe c e l l s of t h i s morphology as capable of c y t o t o x i c i t y by d i r e c t c e l l contact. This c e l l has also been shown to be capable of producing hemolysis i n plaque tests (Harris et a l . , 1966). B i b e r f e l d (1971) describes c e l l s of this morphology as appearing i n cultures of " t y p i c a l " small lymphocytes, 2 hours following Table X C e l l types i d e n t i f i e d within the skin a l l o g r a f t s i t e i n a number of electron microscope studies Weiner et a l . (1964) A l l e g r a e_t a l . (1968) Weiner et a l . (1969) Simar and Betz Collen (1974) (1970) (to day 11) (days 5 and 7) (before 7 or 8 days) (between 2 to-. (day 6 and 8) 11 days) ( i n rabbits) ( i n rhesus monkeys) ( i n mice) ( i n mice) ( i n mice) graft r e j e c t i o n c e l l reticulum c e l l s small lymphocytes small lymphocytes small lymphocytes plasma c e l l s ( a f t e r lymphocytes bl a s t c e l l s (as des- transformed lympho- activated lympho-r e j e c t i o n f a r ad- cribed by H a l l , cytes cytes vanced) 1967) Did not see: macrophages macrophages immunoblast h i s t i o c y t e s monocytes eosinophils monocytes polymorphonuclear leukocytes plasma c e l l s (small number) immature plasma c e l l s plasma c e l l s Did not see: hemocytoblasts macrophage neutrophil eosinophil Did not see: plasma c e l l s VO CO 99 t h e i r incubation with PHA. I t i s i n t e r e s t i n g to note that i n cultures of PHA-stimulated lymphocytes, lymphotoxin (Williams and Granger, 1969) and macrophage migration i n h i b i t o r y f a c t o r (Lawerence and Landy, 1969) could be i s o l a t e d from the culture only 3 to 6 hours following i n t r o d u c t i o n of PHA, even though, with the l i g h t microscope, there did not appear to be any noticeable changes i n the morphology of the small lymphocyte. Another c e l l type found at the graft 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 state that a " t y p i c a l immunoblast" i s not part of the c e l l u l a r i n f i l t r a t e , during t h e i r study of the s k i n a l l o g r a f t response i n mice. However, i n my i n v e s t i g a t i o n , c e l l s characterized by the presence of a large nucleus, a large nucleolus, r e l a -t i v e l y l i t t l e rough endoplasmic reticulum and many free ribosomes are part of the c e l l u l a r i n f i l t r a t e . The immunoblast (also referred to as the hemocytoblast or pyronino-p h i l i c c e l l ) i s defined with these c h a r a c t e r i s t i c s by several research groups. Andre-Schwartz (1964), using r a b b i t s , and Binet and Hathe (1962), using mice, f i n d and describe these c e l l s which a r i s e within the regional 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 o r i -gin and possible fate show that they a r i s e from small lymphocytes (Gowans, 1962) and w i l l subsequently divide to give r i s e to medium and small lympho-cytes (Gowans et a l . , 1962; Andre-Schwartz, 1964), which are possibly the e f f e c t o r c e l l s of the sk i n a l l o g r a f t response (Prendergast, 1964; Andre et^ a l . , 1962). A c e l l of this morphology i s also described as present w i t h i n the regional lymph node responding to the presence of soluble antigen (Leduc et a l . , 1968; Avrameas and Leduc, 1970). I t i s suggested that these c e l l s are the most " p r i m i t i v e " c e l l type of the plasmacyte or lymphoplasmacyte 100 l i n e . Further, i t i s demonstrated that these " e a r l i e s t " c e l l s contain antibody s p e c i f i c for the stimulating antigen ( B o u t e i l l e , 1971). If these c e l l s are the d i r e c t r e s u l t of antigeneic stimulation of small lymphocytes, the presence of these c e l l s at the graft s i t e suggests that there may be some primary recognition^ of g r a f t antigen occurring with-i n the graft s i t e during the e f f e c t o r phase of the a l l o g r a f t response. This would be equivalent to that antigenic stimulation of small lymphocytes which occurs within the regional lymph node during the s e n s i t i z a t i o n phase. This i s sheer speculation. I t i s impossible to a t t r i b u t e a p o s s i b l e r o l e to the immunoblast since i t appears that, although morphologically i d e n t i c a l , i t i s c h a r a c t e r i s t i c of both the B and T c e l l l i n e s which are f u n c t i o n a l l y d i s -t i n c t . Another type of lymphocyte found at the graft s i t e i s the activated lymphocyte which makes up a major portion of the mononuclear c e l l s i n f i l t r a -t i n g the g r a f t s i t e . This i s consistent with the findings of Weiner, Lattes and P e a r l (1964) studying r a b b i t r e j e c t i o n of s k i n a l l o g r a f t s . They describe c e l l s of this morphology as present and part of the c e l l population moving through the venule endothelium i n t o the s k i n graft bed. Simar and Betz (1971) also f i n d these c e l l s i n the graft bed during the skin a l l o g r a f t response i n mice. This c e l l has been re f e r r e d to as a b a s o p h i l i c c e l l ( Hall et^ al_., 1967), transformed lymphocyte (Simar and Betz, 1971), gra f t r e j e c t i o n c e l l (Weiner, Spiro and R u s s e l l , 1964) and b l a s t c e l l (Wiener, Lattes and P e a r l , 1969). In every instance this c e l l i s characterized by a large number of free ribosomes, a w e l l developed Golgi apparatus and a rudimentary endoplasmic 101 reticulum. Lymphocytes with 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 research groups. This d e s c r i p t i o n 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, 1973; Elves, 1966; Douglas_et a l . , 1967). Further, i t i s c e l l s of t h i s morphology which appear i n the efferent lymph of lymph nodes stimula-ted by "soluble proteins, soluble extracts of b a c t e r i a and helminths, k i l l e d b a c t e r i a , viruses, heterologous red c e l l s , homologous lymphocytes, heterologous tumour implants and skin g r a f t s " ( H a l l et a l . , 1967; H a l l , 1967) . These c e l l s are very active m i t o t i c a l l y and have been shown to be immunologically s p e c i f i c for the antigen which induces t h e i r appearance ( H a l l and Smith, 1971; H a l l et a l . , 1967; Cunningham, et a l . , 1966). Again, i t appears that t h i s c e l l population c l a s s i f i e d morphologi-c a l l y to be composed of activated lymphocytes i s a c t u a l l y a composite of f u n c t i o n a l l y quite d i s t i n c t c e l l types. A number of experiments have demon-strated that "many, perhaps most" of the activated lymphocytes i n the ef-ferent lymph of a stimulated lymph node are destined for the lamina propria of the small i n t e s t i n e (Hall and Smith, 1970; Moore and H a l l , 1972). I t has also been suggested that the c e l l s are responsible for the propagation and a m p l i f i c a t i o n of the immune response throughout the lymphoid tissues ( H a l l et a l . , 1967) and, further, that they may become plasma c e l l s when they reach t h e i r destination. These c e l l s have been demonstrated as capable of producing antibody (Cunningham et a l . , 1966; P e t r i s and Karlsbad, 1971), possibly lymphotoxin (Forester et a l . , 1969) and other e f f e c t o r molecules. Tissue culture studies show c e l l s of t h i s morphology to be capable of d i r e c t target c e l l c y t o x i c i t y (Weiss, 1968; Koren and Ax, 1973). 102 The f i n a l c e l l type to be considered i n the lymphocyte section i s the plasma c e l l . " T y p i c a l " plasma c e l l s as described by Avrameas and Leduc (1970) with the "cartwheel" nucleus and abundant endoplasmic r e t i c u -lum were not observed i n the g r a f t bed of s k i n a l l o g r a f t s 6 to 8 days f o l -lowing transplantation p r i o r to epidermal necrosis. This i s s i m i l a r to the findings of Wiener, Lattes, and Pearl (1969) and Wiener, Spiro and Russell (1964) who found that plasma c e l l s did appear i n the c e l l i n f i l t r a t e but only a f t e r the r e j e c t i o n response was complete. In contrast, Simar and Betz (1971) describe both " t y p i c a l " plasma c e l l s and "young plasma c e l l s " as an important part of the g r a f t bed c e l l population although they do not s t i p u l a t e at what stage following transplantation of the s k i n they are pre-sent . When viewed with both the electron microscope and the l i g h t micro-scope there appear to be a large number of kinds of lymphocytes wit h i n the graft s i t e . Further, each type appears, i n i n v i t r o studies anyway, to be capable of s p e c i f i c recognition of the s e n s i t i z i n g antigen and either l y s i s of target c e l l s , synthesis and release of lymphokines (or antibody), or both. Thus, although the presence of these c e l l s does not reveal p o s i t i v e l y how destruction of the graft occurs, since t h e i r possible a c t i v i t i e s are too diverse, i t does not rule out the p o s s i b i l i t y of an important r o l e played by the lymphocyte i n the mediation of graft destruction through s p e c i f i c recognition, release of e f f e c t o r molecules and d i r e c t target c e l l l y s i s . Attempts to elucidate the mechanisms of these lymphocyte a c t i v i t i e s are themselves f i e l d s of intense research and controversy. 103 The class of lymphocytes which mediate g r a f t destruction are 0 bearing T (of thymus o r i g i n ) c e l l s . The means by which T c e l l s s p e c i f i -c a l l y recognize antigenic determinant s i t e s i s not c l e a r . In i n v i t r o studies, intimate contact between the e f f e c t o r T c e l l and the target c e l l appears to be necessary before l y s i s of the target c e l l w i l l occur (Ax et a l . , 1968; Hayry et a l . , 1972; Wilson, 1967; Cohen and Feldman, 1971). I t i s suggested that intimate contact occurs v i a a s p e c i f i c antigenic recog-n i t i o n unit on the lymphoid c e l l surface. I d e n t i f i c a t i o n of th i s receptor has proven to be very d i f f i c u l t , although i t i s f e l t that the receptor must be an immunoglobulin (Crone et a l . , 1972; Feldman, et a l . , 1972). It i s suggested that early 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 antigen or non-specific inducer may " t r i g g e r " the lymphocyte to lyse the target c e l l or release e f f e c t o r molecules. I t i s suspected that cyclic-AMP acts as a modulator of lymphocyte a c t i v i t y (Hadden et a l . , 1970; Henney, 1973; Strom et a l . , 1973). From the various i n v i t r o systems used to in v e s t i g a t e lymphocyte c y t o t o x i c i t y , several 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 target c e l l l y s i s following an i n i t i a l s p e c i f i c recognition of the s e n s i t i z i n g antigen; non-specific c y t o l y s i s me-diated by a c e l l toxin (Granger and Klob, 1968; Granger and Williams, 1971; Heise and Weiser, 1969); s p e c i f i c c y t o l y s i s possibly r e q u i r i n g continued c e l l contact (Andersgon and Hayry, 1973; Forman and Moller, 1973; Koren and Ax, 1973) : and, non-specific l y s i s i n v o l v i n g antibody bound to the target c e l l s (Perlmann and Holm, 1969; Perlmann and Perlmann, 1970; Perlmann et a l . , 1974) . An important point to note i s the suggestion that l y s i s may be 104 non-specific, as suggested by the involvement of c e l l toxins, or s p e c i f i c , as suggested by the demonstration of only s p e c i f i c l y s i s of the s e n s i t i z i n g membrane-bound antigen i n both i n v i t r o and i n vivo studies. This i s a matter of considerable controversy ( E i f e and August, 1973; K l e i n and K l e i n , 1972; Singh et a l . , 1973; Svedmyr and Hodes, 1970). With regard to the s k i n a l l o g r a f t response there has been one im-portant i n vivo study which strongly suggests that the l y t i c action of lymphocytes wit h i n the s k i n graft i s highly s p e c i f i c (Mintz and S i l v e r s , 1970). Allophenic mice are used as skin donors. Thus, a g r a f t w i l l con-t a i n two homozygous sub-populations of c e l l s with d i f f e r e n t a l l e l e s at the H-2 locus. When placed on a member of one parental isogeneic s t r a i n , i t was found that the c e l l s carrying the same H-2 antigens as the host survived while c e l l s with foreign H-2 antigens were destroyed. There was s e l e c t i v e s u r v i v a l of melanoblasts and h a i r f o l l i c l e c e l l s within an area of lympho-cyte c y t o l y s i s against foreign H-2 antigens. In v i t r o t i s s u e culture studies indicate that s p e c i f i c recognition of antigen by lymphocytes may also r e s u l t i n the release of soluble sub-stances which have varying b i o l o g i c a l e f f e c t s (discussed previously) and may 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 involve large numbers of non-specific c e l l s i n the homograft response. The e f f e c t o r mole-cules are synthesized subsequent to lymphocyte stimulation (David and David, 1972; Pick and Turk, 1972). I t i s not known how many d i f f e r e n t molecules can be produced by the same immune lymphocyte. The macrophage: A l i g h t microscope study Another major component of the mononuclear c e l l population found at 105 the gra f t s i t e and described i n t h i s study are the macrophages. A number of l i g h t microscope studies have c i t e d the macrophage as being a major part of the host c e l l population i n f i l t r a t i n g the g r a f t s i t e . Radiotracer studies in d i c a t e that, as i n delayed h y p e r s e n s i t i v i t y reactions (Volkman and Gowans, 1965), these c e l l s are non-sensitized e f f e c t o r c e l l s of bone-marrow o r i g i n (Kongshavin and Lapp, 1973; Giroud et a l . , 1970). In t h i s study the c e l l s c l a s s i f i e d as macrophages, although showing t r a i t s charac-t e r i s t i c of macrophages (including low nuclear/cytoplasmic r a t i o and a non-ba s o p h i l i c cytoplasm) did vary somewhat i n t h e i r morphology. This i s con-s i s t e n t with the findings of Jakobisiak (1971) who designated t h i s category of c e l l s macrophage-like. The v a r i a t i o n s i n morphology wit h i n the macro-phage population 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 there i s an abundant supply of s t i m u l i , including t i s s u e debris and activated lymphocytes, which as demonstrated by i n v i t r o studies, can t r i g g e r a l t e r a t i o n s i n macrophage morphology, as well as ac-t i v i t y (Nathan et_ a l . , 1973; Nelson, 196 ; Williams and Mayhew, 1973). I t has been c l e a r l y demonstrated that the proportion of histochemi-c a l l y active macrophages i n the c e l l u l a r i n f i l t r a t e of mouse skin a l l o g r a f t s increases markedly during the homograft response, as demonstrated by quan-t i t a t i o n of acid phosphatase and lysosmal protease a c t i v i t y of the i n f i l -t rate c e l l s (Poulter e_t a l . , 1971) . Light microscope studies show that macrophage a c t i v a t i o n i n the presence of proper s t i m u l i i s expressed by i n -creased c e l l s i z e , increased non-specific phagocytic a c t i v i t y and increased p r o l i f e r a t i o n (Nathan et a l . , 1973). These c h a r a c t e r i s t i c s are expressed by macrophages removed from animals during delayed h y p e r s e n s i t i v i t y reactions, by macrophages involved i n i n v i t r o c e l l mediated immunity and by macrophages 106 activated 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 (MAF) (Evans and Alexander, 1972; Nathan e_t a l . , 1971, 1973; Godal et a l . , 1971). Thus, depending on the functional state of the c e l l , entrance i n t o the g r a f t s i t e could be followed 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 into macrophage; m i t o t i c d i v i s i o n of macrophage or the acquiring of 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 factors explain the f i n d i n g of a morphologically diverse macrophage population. The data gathered on c e l l counts i n t h i s study suggest that the macrophages may be at t h e i r highest concentration, with respect to the other c e l l types, 8 days following transplantation. This i s consistent with the findings of other research groups. G i l l e t t e and Lance (1971) f i n d , using C57B1 mice, that the highest accumulation of ^"'"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 following transplantation. S i m i l a r l y , Poulter et a l . (1971), again using HALB/c mice, f i n d that 10 days following transplantation the highest proportion of the c e l l s at the graft s i t e i s formed by histochemically active macrophages. In contrast, Jakobisiak (1971), i s o l a t i n g the graft bed c e l l s from skin grafts on A/PI mice, noted t h e i r peak i n number occurs on the s i x t h day following transplantation. The s l i g h t differences i n r e s u l t s may l i e i n the techniques and c r i t e r i a used to locate and i d e n t i f y the macrophages at the g r a f t s i t e . The macrophage: An electron microscope study Some features which are c h a r a c t e r i s t i c of macrophages at the u l t r a -s t r u c t u r a l l e v e l are low nucleus/cytoplasm r a t i o and the presence of a number of phagocytic vacuoles and lysosomes. 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 to be a c t i v e l y phagocytosing, or are otherwise stimulated. Such an environment i s supplied 107 by the s k i n g r a f t s i t e . The macrophages were found to be at varying stages of a c t i v a t i o n , as outlined by Nathan et a l . (1971). The more activated macrophages contained a larger number of mitochondria and lysosomes i n t h e i r cytoplasm (Williams and Mayhew, 1973). Some monocytes were also noted, suggesting that the functional state of the macrophage can vary within the graft s i t e , r e s u l t i n g i n a morphologically diverse macrophage population. The r o l e of the macrophage i n the s k i n a l l o g r a f t response as a non-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 c l e a r , although i t i s an e s s e n t i a l one since absence of the non-sensitized population of c e l l s at the g r a f t s i t e w i l l r e s u l t i n g r a f t s u r v i v a l (Lambert and Frank, 1970). The macrophages were often observed i n the act of engulfing c e l l s or c e l l u l a r debris. Phagocy-t o s i s i s well accepted as an a c t i v i t y of the macrophage. I t has also been suggested by i n v i t r o studies, that the macrophages, when appropriately "armed", are capable of c e l l to c e l l contact l y s i s . This "arming" probably occurs i n association with the immune lymphoid system (Hersey, 1973; Granger and Weiser, 1964 and 1966; Evans and Alexander, 1972; Evans and Grant, 1972). Since activated lymphocytes are present within the g r a f t s i t e macrophage a c t i v a t i o n could occur and, therefore, possibly macrophage target c e l l l y s i s . The arming i s immunologically s p e c i f i c . I t i s not c l e a r l y understood how the macrophage s p e c i f i c a l l y lyses the target c e l l . The polymorphonuclear leukocytes: A l i g h t and e l e c t r o n microscope study Polymorphonuclear leukocytes, both neutrophils and eosinophils, are present at the graft s i t e . Following transplantation of allogeneic skin, there are two instances when the primary c e l l type i n f i l t r a t i n g the grafted tissue i s the neutrophil; during the period of primary wound healing and \ 108 during the f i n a l stages of graft r e j e c t i o n . This was demonstrated i n t h i s study and has been shown i n both l i g h t and electron microscopy by a number of research groups (Wiener, Lattes and P e a r l , 1969; Medawar, 1944; Simar and Betz, 1971). The neutrophil i s the c h a r a c t e r i s t i c c e l l type i n early inflammatory exudates of nonspecific inflammation. "This could explain early accumulation, and p o s s i b l y also the l a t e r one, as due to tissue damage and trauma r e s u l t i n g from the process of transplantation or ti s s u e destruc-t i o n . The cause of i t s accumulation at inflammation s i t e s i s not c l e a r (Harris, 1960). The main function of the neutrophil i s phagocytosis (Cochrane, 1968) and t h i s i s probably i t s main occupation within the graft s i t e and t i s s u e s . The periods during a l l o g r a f t r e j e c t i o n of i t s highest accumulation are also the periods of the most severe tissue damage and, thus, accumulation of damaged c e l l s and c e l l u l a r debris. The neutrophil engulfs and, when p o s s i -b l e , digests the i n j e s t e d p a r t i c l e s . Destruction of the i n j e s t e d material i s mediated by i n t r a c e l l u l a r enzymes contained within the c h a r a c t e r i s t i c granules of the neutrophil. The enzymes contained within these lysosomes include acid phosphatase, alkaline-phosphatase, collagenase, ribonuclease, deoxyribonuclease, l i p a s e , ^-glucuronidase and p r o t e o l y t i c enzymes. Also included are basic proteins (one e f f e c t s degranulation of mast c e l l s while the other 3 increase vascular permeability) and a n t i b a c t e r i a l substances, lipozyme and phagocytin (Cochrane, 1968). Phagocytosis of the tissue debris and immune complexes, i f present, r e s u l t s i n degrees of degranulation of the neutrophil. I t has been demon-strated with the electron microscope that there i s fusion of the lysosome granule with the phagocytic vacuole which r e s u l t s i n the emptying of the 109 enzyme contents into the vacuole containing the engulfed p a r t i c l e or immune complex. Phagocytosis of p a r t i c l e s and t h e i r subsequent degranulation may lead to conditions which favor i n s t a b i l i t y of the lysosomal membranes. Such i n s t a b i l i t y may occur to the extent that the enzyme constituents are re-leased in,greater amounts into the surrounding t i s s u e s . The release of these materials i n t o surrounding g r a f t t i s s u e s could be instrumental i n the f i n a l stages of graft destruction. I t has been c l e a r l y demonstrated i n various forms of immune ren a l disorders that a t t r a c -t i o n of neutrophils by the complement system followed by engulfment of immune complexes can r e s u l t i n the release of the lysosome constituents into the surrounding tissue structures (Cochrane, 1968). I t seems as though, while carrying out t h e i r job of ri d d i n g the tissues of b a c t e r i a or immune complex-es, the very constituents of these c e l l s destroy the surrounding tissue s t r u c -tures. The p o s s i b i l i t y e x i s t s that the conditions at the graf t s i t e may be such that i n s t a b i l i t y of the lysosomal membrane r e s u l t s and degranulation of the neutrophils occurs, releasing enzyme contents into g r a f t t i s s u e s . This could f e a s i b l y aid i n the f i n a l destruction of the grafted t i s s u e . Another c e l l type noted i n t h i s study at present i n the c e l l u l a r i n -f i l t r a t e during skin graft destruction i s the eosinophil. Neither the kine-t i c s nor the means by which i t accumulates at the s i t e , are c l e a r . An accumulation of eosinophils i s usually associated with anaphylaxis or p a r a s i t i c i n f e s t a t i o n , although eosinophils have also been noted around tumours, i n healing wounds, i n burnt tissues and i n the presence of immune complexes (Archer, 1963). The function of the eosinophil and i t s r o l e i n immunological reactions remains unknown (Gleich et a l . , 1973; Mahmoud e_t a l . , 1973). The eosinophils phagocytose material, although t h e i r e f f i c i e n c y 110 i s l e s s than that of the neutrophil (Cotran and L i t t , 1969). These c e l l s contain c h a r a c t e r i s t i c granules which may be designated lysosomes. Similar to neutrophils and macrophages, engulfment of p a r t i c l e s by eosinophils i s followed by membrane fusion of enzyme-containing vacuoles with the phagocy-t i c vacuole and, subsequently, enzyme degradation of the engulfed material (Cotran and L i t t , 1969). Information gathered thus f a r i n d i c a t e s the con-tents of the eosinophil lysosome i s s i m i l a r to that of the neutrophil except that the eosinophils lack a n t i b a c t e r i a l enzymes, lysozyme and phagocytin, and have much higher concentrations of peroxidase (Gleich ej: a l . , 1973). Much of the problem i n the e l u c i d a t i o n of eosinophil a c t i v i t i e s l i e s i n tech n i c a l problems (Gleich, et a l . , 1973 ; Mahmoud et a l . , 1973). Thus, a l l that can be said about the eosinophil and the skin a l l o g r a f t response i s that the eosinophil i s present i n the c e l l population i n f i l t r a t i n g the graf t s i t e . I l l The W mutant Information gathered i n t h i s study about the skin a l l o g r a f t r e -sponse of the W mutant i s of two kinds. F i r s t , i t was found that, 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 graft bed on the mutant do not d i f f e r from those i n f i l t r a t i n g the graft bed on the non-mutant host. Second, the data obtained from the c e l l counts indi c a t e there i s a s i g n i f i c a n t difference between the mutant and non-mutant hosts i n the frequencies of these c e l l types at the g r a f t s i t e . The following discus-sion i s concerned with the possible cause of 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 suggests, i f anything, about (1) the mechanism of the ski n a l l o -graft response and (2) the e f f e c t of the W gene as re l a t e d to a l l the pheno-typ i c c h a r a c t e r i s t i c s . The emphasis of the discussion i s placed p r i m a r i l y on the lymphocyte population. There are two reasons for t h i s . F i r s t , i t i s reasonably c l e a r from i n vivo r a d i o t r a c e r experiments (Prendergast, 1964; Gowans, 1962), lymphocyte depletion experiments ( M i l l e r et a l . , 1971) and experiments i n v o l v i n g t r a n s f e r of adoptive immunity (Billingham et a l . , 1954; Mitchenson, 1954), and from i n v i t r o tissue c u l -ture studies, that the lymphocyte population i s a component i n the skin a l l o -graft response, and, further, as suggested previously, that i t s r o l e may be a c e n t r a l one. A host's response to a ski n a l l o g r a f t may be divided into three phases: 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. Available information suggests that each phase i s dependent on lymphocyte a c t i v i t y . S e n s i t i z a t i o n involves recognition of the graft antigens and es-tablishment of s e n s i t i v i t y to them. The immuno-competent c e l l , r e t a i n i n g the a b i l i t y to recognize and respond to the presence of antigen i s the 112 lymphocyte. This phase Is followed by a c e n t r a l phase during which ampli-f i c a t i o n of the f i r s t component occurs along with production of s p e c i f i c a l l y s e n s i t i z e d e f f e c t o r c e l l s . Again, the c e l l undergoing d i v i s i o n i n response to the presence of antigen and becoming s p e c i f i c a l l y s e n s i t i z e d to that a n t i -gen i s the lymphocyte. The 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 of the a l l o g r a f t response during which host c e l l s accumulate within the gra f t and t h e i r a c t i v i t y r e s u l t s i n graft destruction. Based on lymphocyte tiss u e culture studies, i t i s suggested that the i n i t i a t i o n of t h i s phase i s dependent on the s p e c i f i c a l l y s e n s i t i z e d lymphocytes. These c e l l s enter-ing the graft s i t e recognize the skin antigens there to which they have been s p e c i f i c a l l y s e n s i t i z e d . This recognition can t r i g g e r target c e l l l y s i s or release of e f f e c t o r molecules, r e s u l t i n g i n the accumulation or a c t i v a t i o n of other c e l l types. I t i s the accumulation of these c e l l s w i t h i n the grafted tissue which brings about i t s destruction. Secondly, the lymphocyte population has already been shown to be effected by the presence of the W gene by other research groups. Wong (1967) showed that the W lymphocyte migrates more slowly than non-mutant lymphocytes on normal f i b r o b l a s t culture c e l l s . Work by Shearer and Cudcowitz (1967) demonstrated that the W mutant has a reduced number of precursor c e l l s to the antibody-forming c e l l , suggesting there i s a d e f i c i e n c y i n the lymphocyte B c e l l l i n e . These observations follow from the other phenotypic character-i s t i c s of the W gene where e i t h e r the migration or p r o l i f e r a t i o n , or both, 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 part of the lymphocyte population 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 destruction of grafted t i s s u e , then an a l t e r a t i o n i n the 113 properties of this c e l l population, say, due to the presence- of 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 of the skin a l l o -g r a f t response. In the present study, i t was found that the concentration of transformed lymphocytes was s i g n i f i c a n t l y higher (P < .005) i n the graft s i t e of the mutant than of the non-mutant host on a l l of the days i n v e s t i -gated. McNay (1974) has demonstrated that a s k i n a l l o g r a f t on a mutant host has a shorter s u r v i v a l time than i t would on a non-mutant. Thus, t h i s i n -creased tempo of the skin a l l o g r a f t response may be due to the increased number of transformed lymphocytes. As discussed previously, several f u n c t i o n a l a b i l i t i e s have been at-tributed 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 properties include a b i l i t y to divide, giving r i s e to more of the same c e l l type; a b i l i t y to revert i n s i z e ; a b i l i t y to k i l l by d i r e c t contact; a b i l i t y to k i l l by secretion of lymphotoxin; a b i l i t y to produce other lympho-kines; and a b i l i t y to produce antibody. Thus, although t h i s morphologically d i s t i n c t population of c e l l s i s obviously quite diverse 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 to s p e c i f i c a l l y recognize the s e n s i t i z -ing antigen and,in some manner, respond to i t s presence. The presence of a larger number of c e l l s within the graft s i t e of the mutant host, compared to the non-mutant host, responding to the a v a i l a b l e antigen could r e s u l t i n a shorter gr a f t s u r v i v a l time on the mutant host, as noted by McNay (1974). An a l t e r a t i o n i n the response of the lymphocyte population to the presence of a skin a l l o g r a f t , might, i f the idea of i t s r o l e as i n i t i a t o r i s true, also be expected to r e s u l t i n an al t e r e d tempo of e i t h e r the accu-mulation or a c t i v a t i o n , or both, of the other c e l l types at the graft s i t e . 114 Analysis of the c e l l counts obtained i n t h i s study indicated that there i s a difference between the mutant and non-mutant host i n f i l t r a t i n g popu-l a t i o n s i n the frequencies of other c e l l types within the g r a f t s i t e (table VII) . These findings concerning the cell-mediated immune response of the W mutant suggest that the lymphocyte may indeed play an important i n i t i a t i n g r o l e i n the skin a l l o g r a f t response, as i n v i t r o studies suggest. Further, i t suggests an in -; vivo means of i n v e s t i g a t i n g the phenomenon may be a v a i l -able with the use of the W mutant. Separate i n v e s t i g a t i o n s of each c e l l type i n f i l t r a t i n g the skin graft for a comparison between mutant and non-mutant would substantiate or disprove the above speculations and p o s s i b l y give some i n s i g h t i n t o the role and mechanism of a c t i o n of the various c e l l types present within the gr a f t bed whose a c t i v i t i e s r e s u l t i n the destruc-t i o n of the skin a l l o g r a f t . The next question i s : why the increased number of transformed lym-phocytes within the mutant's g r a f t s i t e ? Presumably, i t i s due to increased stimulation of the lymphocyte population. Two possible explanations are a v a i l a b l e . In the f i r s t , emphasis f a l l s on the f a c t that the W mutant shows a d e f i c i e n t population of B c e l l s , thereby i n d i c a t i n g a reduced capacity to form antibody. Although i t i s c l e a r that antibody production i s not an obligatory part i n the skin a l l o g r a f t response, i t has been demonstrated i n a number of cases that antibody i s produced. I t s e f f e c t s vary. However, often i t seems to have the e f f e c t of enhancement. If such antibodies are produced normally i n the WBB6 l i n e during a skin a l l o g r a f t response (as i n the non-mutant) then comparison with a mouse l i n e (such as the W mutant) lacking the a b i l i t y to produce such antibody could r e s u l t i n an accelerated 115 r e j e c t i o n time by the W host. Presumably t h i s would be due to the increased number of antigenic determinant s i t e s a v ailable to the lymphocyte population during the e n t i r e a l l o g r a f t response, thereby stimulating a higher proportion of the lymphocyte population. In the second explanation as to the cause of the increased number of stimulated 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 that i t i s a d i r e c t r e s u l t of a sing l e e f f e c t of the W gene. This involves a consideration of a l l the phenotypic c h a r a c t e r i s t i c s of the W gene, i n c l u d -ing the "black-eyed white coat" pigmentation, s t e r i l i t y , anemia and an en-hanced c e l l u l a r immune response. A common basis which these phenomena share i s that the affected c e l l s are a l l at a point where external stimulation from t h e i r surrounding environ-ment may play an important part i n determining t h e i r subsequent development or a c t i v i t y . This 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 are 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 -action of the c e l l with antigen (external stimulation) which i n i t i a t e s a response by the c e l l of e i t h e r p r o l i f e r a t i o n , migration, increased metabolic a c t i v i t y , or a combination of these. As mentioned previously, there are three phases of the skin a l l o g r a f t response during each of which lymphocyte a c t i v i t y i s an important component. The increased number of stimulated lym-phocytesamay.vb.eydue to the e f f e c t of the W gene at any stage of lymphocyte a c t i v i t y i n this response. However, keeping i n mind the other phenotypic c h a r a c t e r i s t i c s of the W mutant where there appears to be a lack of migration or p r o l i f e r a t i o n , the i n i t i a l recognition response may be considered. I t i s known that lymphocyte recognition i s a surface phenomenon, although with re-spect to the T c e l l population, i d e n t i f i c a t i o n of the recognition units has 116 not been poss i b l e . The slower migration, or lack of i t , of possibly the melanoblasts, the germ c e l l s , and e s p e c i a l l y the lymphocytes, suggests that there may be an a l t e r a t i o n i n surface properties. In the case of T c e l l recognition, t h i s a l t e r a t i o n may r e s u l t i n increased s e n s i t i v i t y of the T c e l l s to the presence of antigen, r e s u l t i n g i n more c e l l s which r e -spond to the antigen or the T c e l l s which do respond undergo more m i t o t i c d i v i s i o n s . In e i t h e r case the r e s u l t would be the a v a i l a b i l i t y of more activated lymphocytes within the blood along with, p o s s i b l y , an increased response by the unsensitized immunocompetent c e l l s at the g r a f t s i t e . Both could r e s u l t i n a faster skin a l l o g r a f t ; response. This idea, that the primary e f f e c t of the W gene involves an a l t e r a t i o n i n the response of c e l l s to external stimulation, also f i t s as an explanation of the other phenotypic c h a r a c t e r i s t i c s of the W gene. It i s quite f e a s i b l e that f o r the proper completion of t h e i r develop-ment, melanoblasts, germ c e l l s and hematopoietic c e l l s , at some stage, r e -quire s p e c i f i c external stimulation from an environmental source. With re-gard to the melanoblast and hematopoietic c e l l l i n e s i t i s c l e a r that the defect due to the presence of the W gene i s an i n t r i n s i c property of the c e l l s , since the environment i n which these c e l l s are found w i l l support the normal development of non-mutant c e l l l i n e s (Mayer, 1970; Mayer and Green, 1968; Russell and Bernstein, 1967). The data gathered thus f a r i n d i c a t e s that i t i s possible that the defect i n the melanoblast, germ c e l l and hematopoietic c e l l l i e s i n i t s i n -a b i l i t y to migrate or p r o l i f e r a t e properly. The cause of the increased s k i n a l l o g r a f t response appears to l i e i n an increased response on the part of the lymphocyte to the presence of the s k i n antigens. This could involve pro-117 l i f e r a t i o n or migration. These apparently contradictory c h a r a c t e r i s t i c s occurring i n the presence of the W gene are explicable i f one considers the primary e f f e c t of the W gene to be an a l t e r a t i o n i n the surface pro-p e r t i e s of the 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-t e r n a l stimulation. 118 BIBLIOGRAPHY Able, M.E., Lee, J.C. and Rosenau, W. 1970. Lymphocyte-target c e l l i n t e r a c t i o n i n v i t r o . Amer. J . Pathol. 60: 421-428. 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