A RADEOAUTOGRAPHEC STUDY 0F LYMPHOCYTE PROTEIN PRCDUCTEON EN SKiN ALLOGRQFT RBECTEOM Thesis for the Degree of Ph. D. WCHiGAN STATE UNIVERSETY DALE COfiWiN E’EERBOLTE 1973 M' , Mr- - . ‘ ‘ban 3 til {3 UHiVCI'SF y This is to certify that the thesis entitled A RADIOAUTOGRAPHIC STUDY OF LYMPHOCYTE PROTEIN PRODUCTION IN SKIN ALLOGRAFT REJECTION presented by Dale Corwin Peerbolte has been accepted towards fulfillment of the requirements for _P_b_L_Q4_degree in m \ Major professor Date 8/10/73 0-7639 ABSTRACT A RADIOAUTOGRAPHIC STUDY OF LYMPHOCYTE PROTEIN PRODUCTION IN SKIN ALLOGRAFT REJECTION BY Dale Corwin Peerbolte In an attempt to demonstrate that lymphocytes produce proteins within allograft beds, and also to make observations on the fate of those proteins, analyses were performed on 64 skin allografts and autografts placed on ten C3H and A/J strain mice. Tritiated thymidine given 24 hours prior to the grafting labeled the nuclei of blood lymphocytes and made possible their subsequent identification in the graft beds. Four days after grafting, when lymphocytes had entered allo- graft beds and begun to hypertrophy, tritiated leucine (H3L) was given to the animal to label the protein being produced. Allografts, autografts, and the surrounding tissues were analyzed at various time intervals ranging from one hour to six days following the H3L injection. The thymidine label, which localized mainly over nuclei, made it possible to identify hypertrophied lympho- cytes in allograft beds, even though reutilization of the label was shown to have occurred. By comparing the leucine label in allografts to that seen in the fibroblasts of Dale Corwin Peerbolte autografts, it was observed that a labeled material (presum- ably protein) was produced in the cytoplasm of hypertr0phied lymphocytes in allograft beds and subsequently was slowly released from the cells. In the graft tissue above the beds, allografts were continually accumulating protein (with a H3L label), while in autografts a decrease in the radioactivity indicated a normal protein turnover rate. It was concluded that the lymphocyte release of radioactive material (presumably protein), demonstrated in allograft beds, could account for the majority of the radio- active accumulation in allografted tissue, and therefore was a major factor in the akin allograft rejection reaction. A RADIOAUTOGRAPHIC STUDY OF LYMPHOCYTE PROTEIN PRODUCTION IN SKIN ALLOGRAFT REJECTION BY Dale Corwin Peerbolte A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1973 Q? ACKNOWLEDGMENTS The author wishes to express appreciation to the many individuals who assisted in the completion of this in- vestigation. Mention will not be made of each one, since this would likely require an additional volume, but these need not feel that their contributions were inferior. Special thanks are given to Dr. Bruce E. Walker, Chairman, and Drs. N. B. McCullough, C. K. Whitehair, C. W. Smith, and M. H. Ratzlaff, Committee members, for their interest, comments, and construCtive criticism throughout the project. I am indebted to Mr. Darwin Schmuck for the photo- graphic work, and to Miss Paula Hunter for typing the final manuscript. My wife and family deserve special recognition for their patience and encouragement. May God, our guide, con- tinue to enrich our lives together. ii TABLE OF CONTENTS INTRODUCTION 0 O O O O I O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . . MATERIALS AND METHODS I. II. III. IV. V. VI. RESULTS II. III. Animal S O O O O O O O O O O O O O 0 Skin grafting . ... . . . . . . . . Radioisotope injection . . . . . . .' Graft biopsy . . . . . . . . . . . . Histologic techniques . . . . . . . A. Skin grafts . . . . . . . . . . B. Blood smears . . . . . . . . . . C. Radioautography . . . . . . . . Statistical methods . . . . . . . . Graft survival . . . . . . . . . . . Histology . . . . . . . . . . . . . A. Allografts . . . . . . . . . . . B. Autografts . . . . . . . . . . . Radioautography . . . . . . . . . . A. Leucine labeling . . . . . . . . B. Thymidine labeling . . . . . . . C. Labeling within the graft itself D. Labeling within the graft bed . iii Page 14 16 18 18 18 19 19 21 21 22 28 29 38 40 43 55 DISCUSSION I.‘ Comments on techniques . . . . . . II. Histology of grafts and graft beds A. B. Allografts . . . . . . . . . . Autografts . . . . . . . . . . III. Radioautographic labeling . . . . IV. Radioautography of graft tissue V. Radioautography of graft bed cells SUMMARY . . . REFERENCES CITED 0 O O O O O O O O I 0 O O O O APPENDICES Appendix A. Appendix B. Appendix C. Appendix D. Comprehensive data for graft labeling O O .1 O O O I O O 0 Nuclear labeling in animals receiving H3T . . . . . . . . Cytoplasmic labeling in graft beds Distribution of isotope labels iv Page 68 70 70 70 71 73 76 82 83 88 93 103 108 Table 1. 2. 3. 10. 11. 12. 13. 14. 15. LIST OF TABLES Summary of radioisotopes injected . . . . . . . Graft biopsy schedule . . . . . . . . . . . . . Distribution of tritiated leucine label between cyt0plasm and nuclei . . . . . . . . . . . . . Distribution of tritiated thymidine label between cytoplasm and nuclei in animal number 7 Radioactivity within grafted tissue . . . . . . Mean cytoplasmic density for animals given both tritiated thymidine and leucine . . . . . . . . Mean cytoplasmic densities for animals given only tritiated leUCine O I I O O O O O O O O 0 Grain counts of allograft tissue from animal 9 (leuCine) I O O O O O O I O I O O O O O O I O 0 Grain counts of autograft tissue from animal 9 (IGUCine) o o o o o o o o o o o o o o o o o o 0 Grain counts of allograft tissue from animal 10 (leuCine) I O I I I I O O O O O O O I O O O O 0 Grain counts of autograft tissue from animal 10 (leuCine) I O O 0 O O O O O O O O O O O O O I 0 Grain counts of allografts from animal 4 (H3T and H3L) 0 O O O O I O O O I. O O O O I 0 0 Grain counts of autografts from animal 4 (H3T and H3L) O O I O O O O I I O O O I O O O 0 Grain counts of allografts from animal 3 (H3T and H3L) . . . . . . . . . . . . . . . . . Grain counts.of autografts from animal 3 (HBT and H3L) O O O O O O O O O O O O O O O O O Page 15 16 39 4O 44 61 62 89 89 9O 90 91 91 92 92 Table Page 16. Endothelial nuclear counts for animal 7 (thymidine) O C O O O O O O O C O O I C I O O O 94 17. Basophilic cell nuclear counts of allografts from animal 7 (thymidine) . . . . . . . . . . . 95 18. Basophilic cell nuclear counts of autografts from animal 7 (thymidine) . . . . . . . . . . . 96 19. Endothelial nuclear counts for animal 4 (thymidine and leucine) . . . . . . . . . . . . 97 20. Basophilic cell nuclear counts of allografts from animal 4 (H3T and H3L) . . . . . . . . . . 98 21. Basophilic cell nuclear counts of autografts from animal 4 (H3T and H 3L) . . . . . . . . . . 99 22. Endothelial nuclear counts for animal 3 (thymidine and leucine) . . . . . . . . . . . . 100 23. Basophilic cell nuclear gounts of allografts from animal 3 (H3 T and H . . . . . . . . . 101 24. Basophilic cell nuclear counts of autografts from animal 3 (H3 T and H3L) . . . . . . . . . . 102 25. Cytoplasmic labeling in allografts of animal 4 104 26. Cytoplasmic labeling in autografts of animal 4 105 27. CytOplasmic labeling in allografts of animal 3 106 28. Cytoplasmic labeling in autografts of animal 3 107 29. Leucine label distribution “. . . . . . . . . . 109 30. Nuclear and cytoplasmic grain densities in animal 9 (leucine) . . . . . . . . . . . . . . 110 31. Thymidine label distribution (animal 7) . . . . lll vi Figure 1. 3. 4. 5. 6. 7. 12. 13. 14. 15. 16. 17. 18. LIST OF FIGURES C3H mouse which had been given three allografts and three autografts . . . . . . . . . . . . . . Low power photomicrograph of a graft immediately after placement . . . . . . . . . . . . . . . . Allograft three days after placement . .'. . . . Allograft ten days after placement . . . . . . . Autograft three days after placement . . . . . . Autograft ten days after placement . . . . . . . Thin epithelium of a three day allograft . . . . Normal epithelium from the surrounding area skin Basophilic cell layer in a six day allOgraft bed Basophilic layer in a six day autograft bed . . High power photomicrograph of basophilic cells in the graft bed of a six day autograft . . . . Basophilic cells in a six day allograft bed . . Panniculus carnosus beneath the graft area . . . Panniculus carnosus beneath uninjured skin . . . Oil immersion photomicrograph of large baprhilic cells beneath a six day autograft . . . . . . . Basophilic cells beneath a six day allograft . . Basophilic allograft bed cells from animal 10 (leucine group) one day after injection of H3L . Basophilic cells from five day autograft on animal 10 (leucine group) . . . . . . . . . . . vii Page 13 25 27 27 31 31 33 33 33 33 35 35 35 35 37 37 42 42 Figure 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 3:3. 344. Basophilic cell layer of a four day allograft bed from animal 7 (thymidine group) . . . . . . Basophilic cell layer of a 10 day allograft bed from animal 7 (thymidine group) . . . . . . Allograft tissue from animal 7 (H3T) six days after grafting . . . . . . . . . . . . . . Allograft tissue from animal 4 (H3T and H3L) four days after grafting and one hour after the H3L was inj eCted C O I O O O O O O O O O O Allograft tissue from animal 4 ten days after grafting O O O O O .0 O O O I O I O O O O O O 0 after grafting and one hour following H Autograft tissue from animal 4, four days L injection 0 O I I O O I I O I O O I I I O Autograft tissue from animal 4, ten days after grafting . . . . . . . . . . . . . . . . Normal skin from animal 4, eight days after grafting I O O O O C O O O O O O O O O O O O 0 Normal skin from animal 4, ten days after grafting O I O O O O I O O O O O O I O O O O 0 Blood smears from animal 7 (H3T) four days after grafting . C . . . . . . . . . . . . . . Blood smears from animal 7.(H3T) four days after grafting . . . . . . . . . . . . . . . . Allograft bed cells from animal 3 (H3T and H3L) five days after grafting . . . . . . . . . . . Allograft bed cells from animal 3, four days after grafting . .'. . . . . . . . . . . . . . Allograft bed cells from animal 3, six days after grafting O O O I O O O O O O O O O I O O Autograft bed cell from animal 3, four days after grafting . . . . . . . . . . . . . . . . Autograft bed cell from animal 3, six days after grafting . . . . . . . . . . . . . . . . viii Page 42 42 48 50 50 52 52 54 54 57 57 60 65 65 67 67 INTRODUCTION Immunology is one of the more important areas of medical practice today, for in its realm lie a myriad of diseases which have plagued man for centuries. Immunology basically involves the capability of the body to recognize and destroy a foreign substance which may cause it harm. This broad field has been divided into two general areas, namely, (1) humoral immunity, which involves such phenomena as antibody production and antigen-antibody interaction; and (2) cellular immunity, which involves sensitized cells, cell-to-cell interaction, and cell-bound antibody. Cellular immunity seems to function by guarding the body against foreign tissue grafts and certain forms of malignancy. These two have led to a dichotomy in cellular immunity research. In tissue grafts, the reaction is unde- sirable and needs to be inhibited if the graft is to survive, ‘whereas in malignancy, the reaction is desirable, and needs to be enhanced so that malignancy can be eliminated. Never- theless, in~both areas it is necessary to understand the basic mechanisms operative in the rejection reaction; so that;, provided with sufficient knowledge and ability to con- trol the processes involved, treatment of such ailments as cancer and terminal heart or kidney diseases could be greatly faci .1 itated . 1 One method for studying the graft rejection reaction which has been widely used is the skin allograft system. It serves as an adequate model for the process because the fea- tures of lymphoid cell infiltration, blast transformation, and foreign cell destruction are.similar to those occurring in other foreign tissue transplants, and can be easily ob- served, manipulated, and quantified. The present experiment seeks to clarify one aspect of the allograft rejection reaction, namely the role of hy- pertrophied lymphocytes of the graft bed in the reaction against allografted skin in mice. More specifically, the question is how these hypertrOphied lymphocytes and their products participate in the death of the foreign cells in skin allografts ig_gizg, and whether this parallels what they seem to do in allogeneic systems ig_!itgg. Objectives for the present study were: (1) to identify hypertrophied lymphocytes in the graft beds. (2) to identify proteins being produced by cells within the graft bed. (3) to trace these proteins and analyze their possible role in graft rejection. LITERATURE REVIEW The amount of research literature which has accumu- lated over the past years regarding the graft rejection re- ' action makes it inappropriate to attempt to review all of the related information available. Rather, the present re- view will select appropriate orginal research articles and pertinent recent reviews which are representative of many others and which are directly applicable to this study. In skin allograft rejection a series of events takes place which begins shortly after the grafted tissue is. placed, and which culminates in death of the foreign cells and their elimination from the body. Known events in the reaction include vascularization of the tissue; infiltration by lymphocytes, monocytes, and polymorphonuclear cells (Feldman, 1969); hypertrophy of the invading lymphocytes with accumulation of basophilic cytoplasm (Jakobisiak, 1971 and Walker and Goldman, 1963); followed by the progressive destruction of the grafted cells and the vascular tree (Feldman, 1969, Lykke and Cummings, 1970, and Liem and Jerusalem, 1970) . It is the origin and role of the infiltrating lym— phocytes which has been the focus of much attention. The Immediate origin of the ‘cells is the blood stream of the host. 3 Using tritiated thymidine to radioactively label short-lived cells, Walker and Goldman (1963) found that the radioactive cells in the graft bed were similar in labeling character- istics to the large lymphocytes of the peripheral circula- tion. Griffiths (1970) also labeled short-lived lymphocytes with tritiated thymidine and found the cells invading allo- grafts in large numbers. These cells are thought to be of bone marrow origin (Osmond, 1969), which, under the influ- ence of the thymus are rendered capable of recognizing antigen (Basten gE_§l, 1971 and Yunis 33 El; 1971). These thymus-dependant lymphocytes (T—cells) are distributed throughout the lymphatic system and peripheral circulation (Miller et _a_1_]_._. 1971a) and, upon contact with a foreign graft, migrate to the draining lymph nodes (Tilney and Gowans, 1971 and Zatz and Lance, 1971). Here (or in the spleen) the antigen stimulated cells are thought to interact with effec- tor cells (Giroud gt_gl. 1970, Miller gt al..l97lb, and Lonai and Feldman, 1971a) which are then released by way of the thoracic duct (Chanana gt_gl. 1969) where they rejoin the circulation. The nature of the antigen-sensitive and effector cell ixiteraction has generated much interest and speculation. Bunch of the research invOlves in vitro models of rejection SYstems to study the various cell populations involved. T- oells, B-cells (non-thymus dependant), and macrophages inter- act; in the presence of antigen and produce a cell type which is capable of destroying target cells which have the sensi— tizing antigen. Whether T-cells render B-cells cytotoxic (Grant 23 a1. 1972, Billingham, 1969, and Miller 33 al. 1971b), or whether the T-cells themselves are the effectors (Miller 22 31. 1971c' Golstein gE_§1. 1972, Cerottini 22 31. 1970, and Wagner 33 31. 1972) or whether macrophages are the effectors (Salvin gt El- 1971, Kramer and Granger, 1972, and Evans and Alexander, 1971) remains to be determined. How- ever, it has been found that all three are required in an interaction to produce the optimal response ig,vitgg (Feldmann,l972, Gisler and Dukor, 1972, and Lonai and Feldman, 1971b)- Once these activated effector cells have re-entered the circulation, they infiltrate the graft area (Goren gt_§l. 1972) and destroy the foreign cells. The mechanism of this destruction involves cell-mediated cytotoxicity which is even less well understood than the interaction at the lymph nodes. Controversy exists as to whether the action is immunospecific or nonspecific (Moller and Lapp, 1969 and .Hayry and Defendi, 1970) and whether or not it requires dzirect cell contact (Benacerraf and Green, 1969). The most Eilausible explanation at this time is that there appear to 1x3 at least two processes by which effectors can destroy floreign tissue. One process involves direct immunospecific aetion in which cell-to-cell contact is mandatory, and the ot‘her process involves a nonimmunospecific cytopathic effect, whidch also acts as an inflammatory agent (Billingham, 1969). In_!itgg_cell mediated cytotoxicity can be detected by Cr51 release from susceptible target cells (Peter and Feldman, 1972). This cytotoxic activity was found to be parallel to the time sequence noted for rat skin allograft cytotoxicity in 2322 in that it begins at five days and peaks at seven to eight days after grafting. This time sequence is also indicated in the deterioration of the vas- cular endothelium of the graft (Liem and Jerusalem, 1970), which lends support to the nonspecificity of the reaction, since the vascular endothelium is derived from host tissue. The cytotoxic activity is characterized by being independant of complement, independant of anti-immunoglob- ulin sera, and susceptible to sera prepared against thymo- cytes (Cerottini §t_al. 1971 and Cerottini 22 21° 1970). Such a substance, therefore, probably acts in a different manner than most antibodies. Several attempts have been made to isolate and study this reactive "lymphotoxin". Kolb and Granger (1970) found a substance in mice which was heat and pH stable, acting like a protein with a molecular weight of 90-150,000. It (Lid.not require complement, and its heat stability indicated tiiat it was not an immunoglobulin. Furthermore, these in- ‘Westigators stated that there were differing physical and Chemical properties for such a substance in different species Qf animal. Kramer and Granger (1972) found that mouse macro- Phages could be induced i_n_ vitro to produce a cytotoxic substance which cross-reacted with antibody specific for mouse lymphotoxin. An extensive investigation into the nature of mouse lymphotoxin by Williams and Granger (1969) indicated it to be a negatively charged protein of 90-100,000 molecular weight which acts progressively over 36-48 hours, is temperature and concentration dependant, does not bind strongly to target cells, and is nonimmunospecific in its action. The mechanism of action appears to be lysis of the cell membrane. If indeed such a substance is produced in the graft bed, it is obvious that this would contribute to the rejection process, and its nonspecificity would account for the destruction of host tissue (vascular endothelium) as well. In addition to cell-mediated cytotoxicity, there is evidence that systemic humoral antibody is produced against a primary allograft ig_zi!g. Canty and Wunderlich (1971) analyzed mouse serum daily following skin or tumor grafting and noted a rise in titer from O to 1/160 in seven days. This "serum cytotoxicity" was detected by release of Cr51 from target cells incubated with the sera. Additional evi- dence for a humoral allo-antibody is given by experiments in ‘Mhich spleen cells are cultured in the presence of allo- antigen, producing plague-forming cells (Fuji 2.12 31. 1971) Vfllich can inhibit allogeneic tumors in the presence of rabbit Complement (Cerottini gt _a_1. 1971) .' Hamilton‘gt; _a_1_.(1971) believe that the humoral mechanism is insufficient to reject an allograft by itself. Nevertheless, mouse skin allografts on alymphatic pedicels, although they survive longer, even- tually shrivel and die just as surely as do allografts in contact with sensitized lymphocytes (Tilney and Gowans, 1971). It appears then, that the basophilic cytoplasm of the hypertrophied lymphocytes of the graft bed (confirmed by electron microscopy to contain numerous free ribosomes [Walker et_§l, 1964]) may be producing a substance beneath the graft which acts as does lymphotoxin in_zi£52, and that this is a major factor in allograft rejection. Demonstration of protein production by the lymphocytes of the graft bed would support this hypothesis. That radioactive labeling techniques using tritiated leucine can demonstrate protein production has been estab- lished by numerous investigators as exemplified by Reid and Heald (1970). These investigators, performing fractionation studies, noted that tritiated leucine was incorporated only into the protein fraction of the tissue and not into the car- bohydrates or fats. Melchers (1970) noted that H3L was not incorporated into the carbohydrate fractions of immunoglob- Illin molecules, but did appear in myeloma proteins of plasma Cell tumors. Radioautography with tritiated leucine has been lused to demonstrate protein production in lymph node cells in reaponse to antigen (Slonecker, 1969) . No research has been reported which demonstrates pro- tein production by hypertrophied lymphocytes in an '_i_n‘ vivo‘ graft rejection system. MATERIALS AND METHODS I. Animals Animals used in this experiment were inbred A/J (H-Za) and C3H (H-Zk) adult female mice*, which were between 12 and 24 weeks of age and weighed 20-28 gm at the time of skin grafting. These mice were maintained in individual cages following grafting and were housed in air conditioned quarters and given food and water ad libitum. Ether was used for anesthesia‘in all Operative procedures. II. Skin grafting Allografts consisted of skin transplanted between A/J and C3H mice. Autograft controls consisted of skin grafts transplanted from one location to another on the same mouse. Hair was clipped from the dorsal thorax of anesthe- tized animals and areas larger than the anticipated graft were shaved and cleansed with 70% alcohol. Surgical steps were carried out alternately on donor and host in order to ruinimize the time between removal of the graft from the donor and placement on the host. Cleaned instruments were soaked in.7o% alcohol before operating and care was taken not to 'hJuch the graft'or graft bed except with cleaned instruments *Jackson Memorial Laboratories, Bar Harbor, Maine. 9 10 during the grafting procedure. The skin in a 1 cm2 area was held gently with a forceps and a cut made on three borders of the graft with a small scissors to the depth of the pan- niculus adiposus. One corner of the resulting flap was lifted slowly with the forceps while the panniculus adiposus was split beneath the graft with a sharp scalpel. Thus the panniculus carnosus with its relatively rich vascularity was left intact in the graft bed. When the flap was completely separated, the fourth border was cut and the graft removed and immediately placed on the host whose graft bed had been prepared in the same way. The graft and surrounding skin were then covered with a layer of Flexible Collodion*, which was allowed to dry before the animal was returned to its cage. This method of split thickness grafting was the same as that recommended by Billingham (1961), except for the Collodion dressing. A total of 10 mice was used in this eXperiment and each was given either six or eight skin grafts (three allo- grafts and three autografts, or four allografts and four autografts) as illustrated in Figure l. Allografts were E?laced on the animal's right side and autografts were placed <1n its left. This was done in order to conserve isotope and animals, and also to provide an internal control so that an allograft and autograft biopsided at a given time would be frxnn the same animal. It also provided a control for the \ *J.Ir. Baker Chemical Company, Phillipsberg, New Jersey. 11 possibility that cells of different animals might react in a slightly different time sequence. With several grafts on a single animal, the timing between biopsies would yield a more acurate estimate of cell and protein turnovers than if a single animal were sacrificed at each given time. Figure l. 12 C3H mouse which had been given three allografts and three autografts. Two autografts (on the' left) have been removed for histologic analysis, as has one of the allografts. Two allografts remain in place on the upper right, and one auto- graft on the upper left. rafts the [YSiSI 5 autr 13 14 III. Radioisotope injections Four animals serving as the main experimental group were injected with both tritiated thymidine* and tritiated leucine**. The thymidine was given subcutaneously in three equal doses at four hour intervals. Skin grafting was per- formed 24 hours following the last injection so that hema- topoietic tissues would be labeled and no unbound label would be present at the time of grafting (Walker and Goldman, 1963). Two of these mice were given a total of 300‘pc each and the other two were given a total of 600,nc each of H3T. Tritiated leucine injections were given on the fourth day following grafting, in order to label the protein produced by the hypertrophied lymphocytes of the graft bed, which first appear on day three or four (walker and Goldman, 1963 and Jakobisiak, 1971), and whose products first appear on day five (Peter and Feldman, 1972). A single dose of 200 no of H3L was given subcutaneously to each of the two mice which had received 300,uc H3T, and a dose of 500,pc H3L was given to each of those receiving 600,pc of H3T. A thymidine control group consisting of three animals was given H3T as above in three subcutaneous injections at four hour intervals 24 hours before grafting.- Two of these mice were given a total of 300‘pc of H3T each, and one was ‘ *Thymidine-methyl-H3, specific activity 2.0 curies/mM, NeW‘England Nuclear, Boston, Massachusetts. *VL~leucine-4, 5-H3(N), specific activity 5 curies/mM, ”3” England Nuclear, Boston, Massachusetts. 15 given a total of 600 pc of H3T. mice of this group. No leucine was given to the A leucine control group consisting of three animals was not given thymidine before grafting, but did receive tritiated leucine on the fourth day after grafting, as had the experimental group. of H3L and two were given 500 no of H3L each. marizes the radioisotope doses in all animals. Table 1. Summary of radioisotOpes injected. One of these mice was given 200 pc Table 1 sum- Animal # Strain H3T H3L I l C3H 300 pc 200 pc Experimental 2 A/J 300 no 200 no group 3 C3H 600 no 500 no 4 A/J 600 pc 500 no 5 A/J 300 no Thymidine 6 C3H 300 no control group _ 7 A/J 600 no 8 C3H 200 pc Leucine 9 A/J SOO‘pc control group 10 C3H 500,no 16 IV. Graft biopsy In the experimental group and the leucine control group, at least one allograft and autograft pair were re- moved at each of the following times: one hour and two hours after H3L injection on day four after grafting, and days five, six, eight, and ten after grafting. In the thy- midine control group at least one allograft and autograft pair were removed at each of the following times:‘ days three, four, five, six, eight, and ten after grafting. Table 2 contains a complete biopsy schedule for all animals. Table 2. Graft biOpsy schedule. An X indicates one allo- graft and one autograft were removed at this time. Animal # Strain 3 4 5 6 8 10 Days after grafting l C3H X* X X 2 A/J X X X 3 C3H X** X X 4 A/J X* X X 5 A/J X X X X 6 C3H X 7 A/J X X X X X 8 C3H X* X X X 9 A/J x* x x 10 C3H X** X X ‘ *One hour after H3L injection. *TTwo hours after H3L injection. 17 At each time interval, the animal was anesthetized with ether and the graft area cleaned gently with 70% alcohol. The same procedure for instrument cleaning and graft handling were used for biopsy as had been used in grafting (above). Care was taken not to disturb adjacent grafts or remove their Collodion coating. The skin in the area adjacent to a graft was lifted with a forceps and incised with a small scissors. A cut was made through the skin to the depth of the skeletal musculature. The incision was made in the non-grafted host skin completely surrounding the graft so that the biopsy in- cluded normal skin on all sides of the graft. The section 'was then lifted with the forceps and attachments to the skeletal musculature were severed with a#scalpel in an attempt to include all available tissue with the biopsy. The tissue was then placed in 10% buffered formalin for fixation. Topical antibiotic powder* was applied to the resulting wound and covered‘with gauze which was then held in place by a layer of Flexible Collodion. Blood smears were taken at the time of each biOpsy in order to correlate the radioauto- graphic phenomena of the peripheral circulation with that of the graft bed biopsied.’ samples were taken by stabbing the Cleansed tail vein with the point of a #ll scalpel blade. *Neosporin brand of Polymyxin B, Bacitracin, and Neomycin, 'Buliroughs Wellcome and Company, Incorporated, Tuckahoe, New York. fi 18 Histologic techniques - A. Skin grafts After fixation in 10% buffered formalin for 24 hours, the tissues were dehydrated in ethanol, infiltra— ted, and embedded in a glycol methacrylate mixture*. This method was chosen because of'the necessity of making two micra sections so that the resolution under light microscopy would be sufficient to allow accurate dis- tinction between cytoplasm and nuclei of the cells in- volved, since thymidine would label nuclei and leucine presumably would label cytoplasm, where most protein pro- duction would occur. Once polymerization was complete, the tissues were sectioned at two micra with a Sorvall JB—4 microtome**. Sections were floated in double dis- tilled water on cleaned slides and dried on a hot plate at 50°C. Slides were then processed for radioautography (see C below). Following developing, slides were stained in hematoxylin and eosin and cover slipped with either Histoclad*** or Permount****. B. Blood Smears Blood smears were made on cleaned slides and allowed to air dry. Slides were then dipped in a 0.05% *JB-4 Plastic Embedding Kit, Ivan Sorvall, Incorporated, Newton, Connecticut. *WIvan Sorvall, Incorporated, Newton, Connecticut. ***Clay-Adams Incorporated, New York, New York. ****Fisher Scientific Company, Fair Lawn, New Jersey. 19 solution of Parlodion* in absolute ethanol in order to insure adherence to the slide. Thus prepared, the slides were coated for radioautography (see C below). After de- veloping, the slides were stained by flooding for 20 min- utes with Giemsa stain diluted with phosphate buffer 1/20. Slides were then rinsed with distilled water, al- lowed to dry, dipped in xylene, and cover slipped with Histoclad. C. Radioautography Prepared slides were coated in a dark room with Kodak type NTBZ nuclear track emulsion**. The procedure for coating, drying, storage, and deve10ping of slides was that detailed by Walker (1959) except that the cel- loidin coating was omitted. Slides were exposed for varying intervals from one to eight weeks, depending on radioisotope dosage. VI. Statistical methods All grain counts were performed on slides which had been exposed for eight weeks in the case of mice given 300 no of H3T and/or 200‘uc of H3L, and four week exposures were used for thoSe given 600,uc H3T and/or 500.nc of H3L. Counts Were performed on cellsfound in randomly selected 2500;;2 areas of the graft bed. ‘ *Mallinckrodt Chemical Works, St. Louis, Missouri. *113astman Kodak Company, Rochester, New York. 20 The thymidine label was evaluated by counting silver grains located over cell nuclei and measuring the nuclear area, thereby determining the nuclear grain density. Nuclear area was determined by estimating the nuclear area to the nearest S‘pz in comparison to an ocular grid on the micro— scope which was composed of lOO‘pz squares. Reutilization of H3T label by proliferating fibroblasts was accounted for by ‘ comparing the nuclear grain density with that seen in endo- thelial cell nuclei (the reutilization index). Graft bed cells with a nuclear density greater than the reutilization index plus twice its standard deviation were considered to be hypertrophied lymphocytes. The leucine label was evaluated by measuring cytoplas- mic area and counting silver grains (cytoplasmic grain den- sity) for cytoplasm directly associated with a radioactive nucleus. Cytoplasmic area was determined by comparison to an ocular grid and estimated to the nearest S‘pz. Comparisons of cytoplasmic counts and densities for a given animal over a given period of time were analyzed according to mean and stan- dard deviation, using P{0.05 for statistical significance. Formulae used to calculate these data are given in the appendices. RESULTS I. Graft survival All animals used in this experiment remained healthy throughout the manipulations and suffered no obvious adverse systemic effects from the radiation used nor from the multi- ple grafting and biopsying. The clear Collodion coating over the grafts allowed observations of the grafts and any changes occurring without disturbing the grafted tissue. This coating was also effective in holding the grafts in place until biopsy. Even allografts, which would likely have been sloughed sooner, were maintained up to ten days after grafting. All grafts were observed to "take" initially; in- deed at three to four days after grafting, the only differ- ences seen between allografts and autografts were the varia- tions in pigmentation between C3H and A/J skin. As time intervals increased between four and ten days after grafting, allografts were observed to become more darkly colored and dried, as well as shrunken in size, whereas autografts main- tained the healthy appearance noted at three to four days after grafting. II. Histology Histologic observations were aided greatly by the JB-4 plastic embedding technique. Sections cut at two micra permitted a great degree of resolution, especially at high 21 22 power and under oil immersion. Whereas in seven micra para- ffin sections the plane of focus must be raised and lowered in order to view all cells of the section, the two micra sections showed all the details of all the cells present, without readjusting the focus. It was still necessary, how- ever, to raise the plane of focus in order to view the silver grains in the layer of emulsion. Nevertheless, the greater resolution was also beneficial in this situation, because it could easily be determined whether a given silver grain was originating from the nucleus or cytoplasm of the cell beneath. In contrast, using paraffin sections, one would have to con- sider the possibility that a layer of cells deep to the sur- face layer was producing the radioactivity observed. Microscopic observations of the grafts and graft beds revealed several notable features. Allografts and autografts are described separately and appropriate comparisons drawn. A. Allografts At three to four days after grafting, the grafted tissue was still easily discernable from the host tissue (compare Figures 2 and 3). The graft itself now had only a thin layer of epithelium, many of the cells of which appeared to be necrotic, but some still appeared essen- tially normal. The epithelia of allografts and normal skin are illustrated in Figures 7 and 8. Several neutro- phils were seen to have'invaded the graft and were espec- ially numerus in areas adjacent to host skin at the edges 23 of the graft. Typically, a densely packed area of neu- trophils was situated within the junction of host and grafted skin (see Figure 3). ‘The layers of dermis and panniculus adiposus were evident within the grafted skin, but as the base of the graft was approached, an increasing ,. proportion of cells was encountered with basophilic cyto- plasm and medium to large basophilic nuclei. Cells of this type also comprised a dense layer of three to four cells in depth between the graft panniculus adiposus and the host panniculus carnosus. Individual cells had a variety of nuclear shapes and sizes, but many nuclei were noted averaging about 30).:2 in area and containing numer- ous small nucleoli. These cells appeared to be flattened, with their broad surface parallel to the surface of the graft (Figures 12 and 16). Several larger basophilic cells were also noted here. The panniculus carnosus con- tained numerous muscle fibers which were smaller in diameter than those of adjacent uninjured skin, and which occasionally had centrally placed nuclei (Figures 13 and 14). Below the panniculus carnosus was a layer of widely spaced basophilic cells, this layer being somewhat thicker in the area immediately beneath the graft than beneath uninjured skin. By eight to ten days, the graft, although re- maining in place, was completely necrotic with no evidence of living cells present (see Figure 4). The dense layer Figure 2. 24 Low power photomicrograph of a graft immediately after placement. The intact skin is present on the right, and the grafted skin on the left. Note that the panniculus carnosus of the host re- mains intact in the graft bed. I .3mm I 25 Figure 3. Figure 4. 26 Allograft three days after placement. The graft is on the right and has a thin, necrotic epithe- lium. The dark area at the edge of the normal epithelium is a clump of neutrophils, typically seen filling the junction between graft and host skin. I .3mm_¥] Allograft ten days after placement. The graft (above) is necrotic and contains many neutrophils (black area). The host epithelium has grown in from the left (arrows) and separates the graft from the host. I .3mm #1 27 28 of basophilic cells was still present between graft and host tissue, and, in addition, host epithelium was seen growing into this area from the adjacent host skin (Figure 4). The pattern of neutrOphil infiltration did not seem consistant in all animals, since some allografts had increasing concentrations with increasing time, and in others the concentration of neutrophils did not change appreciably from the three or four day level. B. Autografts The grafted tissue of autografts was also dis- cernable from the host tissue, but not for the same rea- sons as allografts. Here the grafted epithelium remained healthy looking without evidence of necrosis (Figure 5). The graft-host junctions were filled with neutrophils as in allografts, but there were fewer neutrophils invading the graft itself. The layer of basophilic cells was pre- sent between the graft panniculus adiposus and host panniculus carnosus (Figure 10), but did not appear as densely packed as that beneath allografts. Consequently the basophilia seemed less prominent in this area for autografts than it did for allografts. The individual cells in this area appeared generally larger than those in allografts, with nuclei averaging about 50lp2 in area, with some as large as 100,112 (Figure 11). These nuclei contained one or two large nucleoli, compared to the more numerous and smaller nucleoli noted in cells beneath 29 allografts (see Figures 15 and 16). The host panniculus carnosus was similar to that noted beneath allografts in that it had numerous small fibers, some of which con- tained centrally placed nuclei. The loose fascial layer beneath the panniculus carnosus looked the same as that observed for allografts. Autografts biopsied at later time intervals did not have the changes noted for allo- grafts. Here the grafts continued to look healthy, and the distinctions between graft and host tissue gradually diminished. At day ten after grafting, the only differ- ence seen was the elevation of the grafted tissue above the level of the normal skin (Figure 6). III. Radioautography As detailed above, some of the animals in each ex- perimental group were given at least twice as much radioac- tive isotope as the others, and consequently could be ob- served after a shorter exposure time. In addition to this exposure and dosage difference, tissues from those mice given the lower dosages (300‘pc tritiated thymidine and/or 200,uc tritiated leucine) were processed before the JB-4 embedding technique was perfected. As a result, many of the sections were too small or too poorly oriented to provide the volume of data needed to yield statistical significance. Neverthe- less, these preliminary data suggested all the main points described below, and the subsequent statistical analyses, 30 Figure 5. Autograft three days after placement. The graft (on the left) has a thinner epithelium than normal skin, but most of its cells are normal. I .3mm I Figure 6. Autograft ten days after placement. The epithe- lium of the graft (right) appears similar to normal epithelium (Figure 8). Numerous basophilic cells are present in the graft bed, but are less dense than those in allografts (compare Figures 9 and 10). I .3mm #1 31 32 Figure 7. Thin epithelium of a three day allograft. Most of the cells are already necrotic, as are the hair follicles (compare with Figure 8). I .lmm I Figure 8. Normal epithelium from the surrounding area skin. Here, all cells appear normal. Late autografts eventually regained this appearance (see Figure 6). I .lmm I Figure 9. Basophilic cell layer in a six day allograft bed. Cells appear smaller and more densely packed than in a comparable autograft bed (Figure 10). I 50 B I Figure 10. Basophilic layer in a six day autograft bed. Cells appear relatively large and are not as densely packed as in allografts. I 50 1.1 I 33 34 _ fi.om _. .coflumnsmfimcoo.ummaosc Hofiuoa on“ m>ma pom mNflm Hmeuos no can mhmnfim one .caxm omusnnfino nummcmn monocumo moazoflccmm _ .1 om a .nmmnmouonm on» no Eonuon may no muonwm Hmum>mm ow floaosc Hmnuomo muoz .Avd ousmwmv HmEHoc coon HmHHmEm mum mnmnfim .mmnm ummum on» cummcon momosumo msflcoflssmm _ I‘mm _ .Emmamouwo omummaoam may no coaumuomfluo HmHHmnmm on» muoz .waomaosc vowsfiecum 0: Spas .omn ammumoaam HHmEm mum mfiawo .wH musmflm moo xwm m ca mHHoo owaacmommm _§: mm . .mHHmo umos cw waomaoao ucocfiEonm on» muoz .ummumouom amp xwm o no own ummnm map cw maamo oaaficmommn .mH mmsmflm mo nmmumonoweouonm Hmzom swam .NH masons .HH museum 35 36 Figure 15. Oil immersion photomicrograph of large basophilic cells beneath a six day autograft. I 10 p I Figure 16. Basophilic cells beneath a six day allograft. Cells appear more flattened, with more dense nuclear material. Note parallel cell orientation which is also parallel to the graft (not visible). I 10.n4J 37 38 which were performed only on the data from the animals with the higher radioactive isotope dosages, were used to support the points as described. A. Leucine labeling (Figures 17 and 18) In the leucine control group (those injected with only tritiated leucine) a rather consistant pattern of labeling was noted in that most of the silver grains were overlying cytoplasmic areas, rather than nuclear areas. Table 3 lists the percentages of label overlying cyto- plasm and nuclei for each graft on two of the leucine animals.“ Areas were randomly selected from the living tissue of the graft bed or surrounding normal dermis. There was no consistant change in this distribution pat- tern with increasing time after injection, nor was there any difference between allografts and autografts. Counts are tabulated in detail in Appendix D. 39 Table 3. Distribution of tritiated leucine label between cytoplasm and nuclei. Each count is from a randomly selected area of graft bed or adjacent normal dermis. Parentheses in— dicate grain densities (grainsfipz). animal allo- auto— days after % label over % label over number graft graft grafting* cytOplasm nuclei 9 X 4** 80 (.25) 20 (.14) 9 x 8 83 (.19) 17 (.11) 9 x 10 80 (.14) 20 (.12) 9 X 4** 85 (.19) 15 (.18) 9 x 8 82 (.19) 18 (.15) 9 X 10 82 (.17) 18 (.16) 10 X 4*** 86 14 10 X 5 80 20 10 X 6 78 22 10 x 4*** 78 22 10 X 5 78 22 10 X 6 86 14 *Animals were injected with H3L on day 4 after grafting. **One hour after H3L injection. ***Two hours after H3L injection. In order to obtain a more accurate estimate of the relative proportions of leucine in cytOplasm and nu- clei, the areas of these cell subdivisions must be consi- dered, thus actually comparing cytoplasmic radioactive density with nuclear radioactive density rather than ab- solute grain counts. This was performed for animal 9 and the values appearing in parentheses in Table 3 indicate only a slightly higher concentration of label in cyto- plasm than in nuclei. 40 B. Thymidine labeling (Figures 19 and 20) In the animals which were given only tritiated thymidine, a direct contrast to the leucine labeling pat- tern was noted. Here, the label was concentrated almost exclusively over nuclei, and was light over cytoplasm. Table 4 lists the percentages of silver grains overlying cytoplasm and nuclei for each graft on animal 7. This distribution was also consistant over time and between allografts and autografts (data given in Appendix D). Table 4. Distribution of tritiated thymidine label between cytoplasm and nuclei in animal number 7. Random areas of the graft beds or adjacent normal skin. allograft autograft days after % label over % label over grafting* cytoplasm nuclei X 4 23 77 X 6 24 76 X 8 19 81 X 10 21 ' 79 X 4 21 79 X 6 24 76 X 8 ’ 18 82 X 10 *Injected with H3T one day before grafting. .l— 4 _ mom _ Hm>aflm may no Ham umOEHm mono .fimaooc mflaum>o mowmum -meu mmmnu mo anon me pun» muoz .mcwamnma >>mmc 303m pom wanwmw> aaflmmm mum Amy moumoosmEhH pmwnm Iouuuwmmn mouse .Amooum wchflE nanny h Hmawcm Eoum own ummumoaam woo ca 8 mo Momma Hamo oaaflsmommm _ Now _ .moumoonmaha HmHHmEm I m .muhoozmfixa omega nonuummmn I m .Amsonm mcHo«E>£uv h Hogans scum own ammumoaao amp 950m m mo Momma Hamo UHHHnQOmmm . now _ may Ho>o mononucmocoo Honda .Emmamouho can .mumonmoaam cw md .Amsonm wcwoomdv 0H HmEHcm co ummnmoucm .om wusmflm hop m>fim Eoum maamo owaflnm0mmm _ mom _ .EmmHmogao mcwmaum>o oHnHmH>.mw dogma on» no umoz .Amm mo oowuomncfl Hmpmm amp oco Ansonm mcwoomav oa Hmecm scum 2. .ma mnsmem maamo can ammumoeem oeeeamommm .mH mnsmem .eH mesmem 42 43 C. Labeling within the graft itself Preliminary data indicated that allografts tended to accumulate label with increasing time, even though the tissue was becoming increasingly more necro- tic; whereas autografted tissue maintained a relatively constant label. This did not seem to be as true for animals given only H3T as for those given H3L. Subse- quent data confirmed these findings. Ten unit areas (areas of the microscope grid under oil immersion mea- suring 2500,112 each) were selected at random from the grafted tissue located between the graft epidermis above and the layer of densely packed basophilic cells of the graft bed below. Silver grains in these unit areas were counted and the mean calculated for each graft. These data are given in tabular form in Table 5 and in narra- tive form thereafter. Comprehensive data and statistical determinations are tabulated in detail in Appendix A. It should be noted at the outset that data are not consis- tant for different animals, i.e. grafts from different mice differ in grain counts even though type of graft, time after grafting, and isotope dosage are identical. Therefore, accurate comparisons can be drawn only from data for different grafts on the same mouse. Therefore the illustrations given are photomicrographs of grafts from the same animal (Figures 22-27). 44 cowuomuquSm mom Hmumm who mommnucwnmm cw monomflmaegg manuaflm>m mommwp ucoflowmmsmcH«*« mcofiuoonsw Amm mow3oaaow mason 039«« oofluomncw Am: mcwzoaaom noon mco« h.m o.oa Ah.mv v.HH Am.mv o.~H oH 9mm h N.m m.oH Am.mv ¢.HH Aw.oav m.mH m Bmm h m.m h.ma Am.~v m.HH AH.¢V v.ma m 9mm h m.oH o.mH wo.m~ m.mH «444Ammmv o.mH v 9mm (\w m.m e.hm h.vm h.@@ m Amm com 8mm m m.> m.~v >.mm m.om m Amm pom 9mm m m.m m.om H.mm o.~¢ ««v Ammm ocm.mmm m m.m m.~m m.~m o.h¢ 0H Amm ocm 9mm v m.m m.mm «.mm «co m Amm flaw 8mm v m.m m.hm m.~m «.mm «v Amm com 5mm v m.m m.¢m ~.wm m.nm m Amm oa m.m m.mm . m.hm H.Hm m Amm ca m.m N.mm Imqam Im.vH 44¢ Amm ca. «.5 ~.mm m.hm N.me OH Amm m m.m ~.vv m.v¢ m.mm m Amm m v.m mJWM *** mhba Ike HMS m mufi>fluomowomu mufi>wuom0fiomu mufl>fiuom0womu mom Aameuoc. ummumousm ammumoaam mcwummnm OQODOmHoHomu Hones: some weapon coma some some umymm mmmo Hmeficm .msmmw» ummum mo comm m: comm mmmum>m cm ca mcflmum H0>me mo Monaco on» mycomonmmu coma comm .msmmnu cayenne ensues muw>euomoeomm .m OHQMB 45 In animal 9, a comparison of allografts and autografts indicates that allograft tissue gradually in- creased in radioactivity, especially in the initial four day interval, the same time that autografts and normal skin dermis were decreasing their radioactivity. In animal 10 (given only H3L) the same pattern was noted, even though biOpsy intervals were only 24 hours. The in- crease in autograft radioactivity seen between four and five days is the only instance where an autograft was ob- served to increase in grain density, and possible reasons will be discussed below. Animals 3 and 4 were given both tritiated thymi- dine and leucine; however, a comparison of animal 3 with animal 10 and animal 4 with animal 9 (because these were biopsied at parallel time intervals) reveals that the addition of H3T did not consistantly increase the grain counts in the grafts, nor in the normal dermis. Radio- activity in allografts was again noted to increase (Figures 22 and 23) and in autografts and normal skin to decrease with increasing time after grafting and injec- tion (Figures 24-27). In these two animals (3 and 4), the grain counts over autografts and normal dermis were essentually parallel in their declines, as can be seen in Table 5. Background fog remained relatively constant for all time intervals and between different animals. The 46 graft radioactivity in animals given H3L was elevated to such an extent that these consistantly low fog levels did not present an important factor in evaluating changes in the graft radioactivity. However, in the case of animal 7 where only H3T was given, the graft radioac- tivity was only slightly elevated above background fog levels, and when these were subtracted, no consistant pattern of decrease or increase over time was seen. These data indicate that thymidine contributed an insig- nificant amount of radioactivity to the graft tissue (compare Figure 21 with Figures 22-27). Statistical significance (p(L05) was obtained for each of the increases seen in allografts except for that between day 8 and 10 in animal 9. Each of the decreases seen in autograft radioactivity was also statistically Significant except for those between day 5 and 6 in animal 10, and day 5 and 6 in animal 3. These calcula- tions are detailed in Appendix A. Two generalized statements can be made to sum- marize the above data. In animals given H3L four days after grafting: (l) allograft radioactivity increased with time, while autograft and normal dermis radioactivi- ty decreased; and (2) allografts were initially less ra- dioactive than either autografts or normal dermis, but at late intervals allografts were more radioactive than either autografts or normal dermis (see Figures 21-27). 47 Figure 21."Allograft tissue from animal 7 (H3T), six days after grafting. Epithelial cells are still intact and have incorporated some H3T. There is ' very little radioactivity below the epithelium. I 20“. I 48 49 13le .%Dfi>HDUMOflomu SH mmmmuo use pmmnm m moan mHHmo oeuonomc mo mgflmeH on» moflmucoo Boo Edflaosgfimm gnu nummcmn wommflu one Lfidfiamsuflmm mzu mo mcflmamu pmcu Ham ma cmmumouonm may no mon mcu pm Axv cflumumx «0 mafia saga one .mcwummum Hmnmm mmmo cm» .v Hmsflcw Eonm mommflu ummnmoHH¢ alhsmwlla .mmmum maumm was“ um muH>HuomoHocH mauuwa hnm> mm: Dunno was .oflquomc 080003 on mcwccflmmn we Esflamnuwmm .omuowncw mm3 Amm can Hound noon moo pom mcflpmmum umumm memo “you .xemm can ammo .mm ousmflm v Hmfiwcm Scum mammfiu pmmnmoHHm .mm mussem 50 51 flinmmWI|_ .lvm musmemc Hm>oH woo H50m on» scum aapsmfiam oomoouooo moo muw>wuooowoom .moflumoum “ovum mmoo sou .v Hofiwco Scum oommwu uwoumouso .IJmmMIIJ .lmm musmemv ummumoeam oanouomfioo onu mom oouoc uosu coop mHEMoo poo Eswaonuflmo nuon ca Hopooum ma mufl>wuooowoon one .QOfluooncfi Amm on» ocw3oaaom H50: oco poo.mcwpmoum Houmo mmoo Hsom .mm ouomfim .v Hofifico Scum oommwu amoumous¢ .em musmem 52 53 filflflMllJ .ouou Ho>ocusu neon loam Hofinos osu ou oop oouoom Ixo on canoz mm .mefihoo poo Ecwaonuemo coon mo euw>fiuooowoou ca omoonooo o macaw onmmfiu mane .mcwumoum Hopmo meow coo .v Hofieso scum wam HoEMoz addmmflll_ .coeuosooum cfiououm Hoauoc Heonu 30£m mwfinoo amoum one no mHHoo one .Homoa canonox onu oucw oououomuoocw coon o>o£ maoeuouofi o>wuoooHomu ocu Ecwaonvflmo osu ca coo .o>eao ouo mHHoo Had .mceumoum Houmo memo .hN ounmwm pomeo .v HoEHco Eoum cexm Hofiuoz .mm museem 54 55 D. Labeling within the graft bed As noted previously, the graft bed was charac- terized by the presence of basophilic cells of two main types; those whose nuclei were large and contained one or two large nucleoli; and those which had somewhat smaller nuclei with several small nucleoli (see Figures 15 and 16). Both of these cell types were seen in allo- graft and autograft beds, but there seemed to be a higher proportion of the smaller nuclei in allografts and larger nuclei in autografts. Still, these histologic criteria were insufficient to clearly determine which of the cells were hypertrophied lymphocytes from the blood. Radioautography aided the situation greatly. When tritiated thymidine was injected before grafting, the cells of the graft bed had a rather characteristic pattern of labeling. Four types of cells were labeled as follows: (1) neutrophils had 5-6 silver grains over the nuclei of about 80% of the cells; (2) large round cells with kidney shaped nuclei and varying amounts of pale- staining cytoplasm generally had 9-10 silver grains over their nuclei; (3) endothelial cell nuclei were observed with 2-4 grains over the nucleus; and (4) the large cells with basophilic nuclei and cytoplasm had a wide range of nuclear label from none, to a few; up to 12 grains per nucleus. In order to separate the hypertrophied lympho- cytes from the fibroblasts in this last population, com- parisons were drawn to the labeling seen in smears of 56 Figures 28 and 29. Blood smears from animal 7 (H3T), four days after grafting. Note that the large mono- cyte (with kidney shaped nucleus) in Figure 28 and the large round cell in Figure 29 are labeled, while the small lymphocytes in Figure 28 are not labeled. The neutrophil in Figure 29 has silver grains over it which are not visible in the photograph because of its dense staining. I 20“ I 57 58 peripheral blood. Three days after H3T injections, the neutrophils of the peripheral blood also showed 5-6 grains over about 80% of the cells. Monocytes and large lymphocytes had 8-10 grains over about 50% of the cells seen (Figures 28 and 29). Small lymphocytes were seen in great numbers, but only a few had more than one or two silver grains over the nucleus. ‘In the graft bed, there- fore, in order to identify the basophilic cells which had originated from blood lymphocytes, the cells would have to demonstrate a relatively high amount of radioactivity, greater than that possible by the reutilization of the degenerating neutrophil label. Cells with this heavy la- bel would then correspond to the large lymphocytes of the peripheral blood in their labeling characteristics. It was assumed that reutilization of neutrophil label would be to about the same degree in fibroblasts as in endothe- lial cells, since both of these must proliferate after the grafting in order to repair the injuries. Conse- quently, endothelial cells were used as an index of re- utilization. The mean nuclear grain density (average number of grains per);2 of nuclear area) of endothelial nuclei, plus twice its standard deviation was used as the cut off point; cells with the nuclear grain density above this point were assumed to have come from the blood. This procedure was carried out for each graft bed for all animals receiving tritiated thymidine, and the comprehen- sive data are tabulated in Appendix B. Figure 30 illus- trates these types of cells. Figure 30. 59 Allograft bed cells from animal 3 (H3T and H3L), five days after grafting. Note that the two endothelial nuclei present (E) have silver grains over their nuclei, evidence of thymidine reuti- lization. Using these cells as a reference, it appears that fibroblasts (F) have reutilized thymidine to about the same degree, whereas hypertrOphied lymphocytes (H) maintain a higher concentration of nuclear label. The cytoplasmic labeling is light in all of the cells shown here. p201: l 60 61 Once the hypertrophied lymphocytes were thus identifiable by their nuclear (thymidine) label, the animals also receiving tritiated leucine were analyzed in order to determine what happened to the protein in the cytoplasm of these cells in the graft beds. Cyto- plasmic areas were measured for each cell (to the nearest 5 p2) and grain densities calculated. For each allograft, the mean cytoplasmic density (average number of grains per‘nz'of cytoplasmic area) was calculated from 20 hypertrophied lymphocytes of the graft bed. Autografts had very few cells meeting the nuclear cri— teria for hypertrophied lymphocytes, therefore mean cyto- plasmic densities for each autograft were calculated from 20 basophilic cells regardless of origin or nuclear labeling pattern. The resulting data are outlined and tabulated below (Table 6) and in detail in Appendix C. Table 6. Mean cytoplasmic density for animals given both tritiated thymidine and leucine. hypertrophied ‘autograft bed animal days after lymphocyte cytoplasmic number grafting cytoplasmic density density (allograft) 4 4* 0.25 0.14 4 8 0.15 0.17 4 10 0.14 0.16 3 4** 0.19 0.15 3 5 0.15 0.15 3 6 - 0.13 0.15 *One hour after H3L injection. **Two hours after H3L injections. Table 7 . 62 In animals given both tritiated thymidine and leucine (animals 3 and 4, Table 6), the hypertrophied lymphocytes of the allograft beds had a decreasing cyto- plasmic density with increasing time. Each of the de- creases in Table 6 was statistically significant (p(.05) except between day 8 and 10 in animal 4. Cytoplasmic densities for autografts on the same animals did not de- crease, but remained relatively constant for the same time intervals. Typical allograft bed cells from these animals are illustrated in Figures 31 and 32. Typical autograft bed cells are illustrated in Figures 33 and 34. These cytoplasmic densities reflect only the labeling due to H3L and not to H3T. In animals given only H3thymidine there was no cytoplasm present which was labeled sufficiently to provide grain counts. Table 7 lists cytoplasmic densities for animals given only H3leucine, and these values were similar to those seen where both isotopes were given (Table 6). Mean cytOplasmic densities for animals given only tritiated leucine. animal days after allograft mean autograft mean number grafting cytoplasmic density cytoplasmic density 9 4 052'? 0.19 9 8 0.19 0.19 9 10 0.14 0.17 10 4** 0.20 0.20 10 5 0.20 0.18 10 6 0.16 0.17 *One hour after H3L injection. **Two hours after H3L injection. 63 Again the densities decreased more in allografts than autografts, but here there was no nuclear label and therefore a pure population of hypertrophied lymphocytes for allograft beds was not obtained, as it had been for values in Table 6. The above data for cytoplasmic labeling can be summarized in two sentences. 1) In animals given H3L, or H3L and H3T, cytoplasmic radioactivity declined more in allografts than in autografts. 2) In animals given both H3L and H3T, hypertrophied lymphocytes decreased in cyto- plasmic density in allograft beds, whereas cytoplasmic radioactive density was relatively constant in autograft beds (see Figures 31-34). Background fog was not an important factor in these calculations because, averaging 10 grains per 2500‘u2 area, it could contribute only .004 grains/p.2 to a calculation of grain density. 64 Figure 31. Allograft bed cells from animal 3, four days after grafting. Two hypertrophied lymphocytes (H) are present with heavy nuclear labels and moderate labels over cyt0plasm. I 202 I Figure 32. Allograft bed cells from animal 3, six days after grafting. The hypertrophied lymphocytes (H) now have light cytoplasmic radioactivity. I 20u I Figure 33. Figure 34. 66 Autograft bed cell from animal 3, four days after grafting. The nucleus has light radioactivity (either H3T or H3L), and the cytoplasm is quite heavily labeled. I 20H I Autograft bed cell from animal 3, six days after grafting. Both nucleus and cytoplasm are well labeled, but the nuclear density is not great enough to meet the criteria for a hypertrophied lymphocyte. The cytoplasmic grain density has decreased only slightly from that typical for the earlier intervals (Figure 33). I 20E I 67 DISCUSSION I. Comments on techniques The excellent success of the grafting technique was more the result of sustained practice than of any particular manipulation. Throughout the experiment, slight changes in the procedure were introduced in order to combat specific problems or eliminate more difficult steps by replacing with simpler ones. The grafting technique outlined above was the result, and, because of several problems yet remaining, it requires a bit of explanation and precaution. The greatest diffiCulties were encountered in anesthesia. It required a great deal of concentration and effort to simultaneously ad- minister ether to two mice, and at the same time proceed with the cross grafting. Perhaps a solution here would be to use a short-acting barbiturate or similar substance.. However, experimentation with these possibilities raised the addi- tional problems of varying responses to the same dosage in otherwise identical animals, and a longer waiting period for both induction and recovery than those for ether. The same comments hold true for the biopsy and em- bedding procedures. These techniques were also developed and modified as the experiment progressed. The final product, as outlined, was the best solution to several problems. Taking 68 69 a portion of normal skin completely surrounding the graft allowed for more accurate orientation of the tissue within the embedding medium, and consequently, more uniform orien- tation betweeh sections of all the grafts. The fact that only two out of the last 32 tissues processed contained in- sufficient graft tissue to provide accurate grain counts bears up the validity and accuracy of the technique, pro- vided the steps are carried out with appropriate care and experience. The plastic embedding technique was one main reason that such a minute task as separating nuclear from cytoplas- mic label could be undertaken. Without the increased resolution, the results could not have been as precise or accurate. Still, the problem of the emulsion being in a dif— ferent plane of focus remains. It is possible that a more dilute emulsion could have been used so that the silver grains would appear nearer their source; however, the radi- ation characteristics of tritium (to be discussed below) would also have to be taken into account. At any rate, re- search involving microscopic analysis of tissue has been en- hanced by using the glycol methacrylate embedding material, and these techniques are recommended where precise resOlution is desired. 70 Histology of grafts and graft beds A. Allografts The appearance described for allografts and graft beds is in agreement with the findings of other investigators. The beginnings of graft destruction are noted grossly after four days with increasing coloration, drying, and shrinkage. Microscopic evidence of graft destruction can be found even earlier in the accumula- tion of clusters of basophilic cells around the graft ' vasculature at three to four days (Feldman, 1969), and beginning necrosis of graft epithelium. The cells seen within the graft bed were the same types as those described in detail by Walker and Goldman (1963) and many other investigators. In allo- grafts, these cells were a combination of fibroblasts, proliferating in the area in order to repair the tissue injury, and lymphocytes which had entered the tissue from the blood and subsequently hypertrophied. As mentioned above, histologic criteria were indeterminate in separating these two populations since there was a continuous range of variation in amounts of basophilia, size of nuclei, and numbers and sizes of nucleoli. B. Autografts In autografts, the origin of the basophilic cells was determined to be mainly fibroblasts prolifer- ating locally in response to injury (Walker and Goldman, III. its 71 1963). The radioautographic evidence from the present experiment supported this conclusion, as discussed below. The muscle fibers of the panniculus carnosus beneath both allografts and autografts were noted to con- tain several fibers which were smaller in diameter, more basophilic, and contained a greater proportion of cen- trally placed nuclei than fibers beneath uninjured skin. The combination of these criteria (central nuclei, baso- philia, and small diameter) suggested that theSe fiberS‘ were regenerating (Walker, 1962), probably in response to the injury received at the time‘of grafting. The infil- tration of neutrophils into both allografts and auto- grafts indicated that this was also a non-specific re- sponse to injury. The fact that allografts had slightly more neutrophils than autografts was possibly a result of the chemotaxis from the degenerating, necrotic cells of the graft, rather than a cause of the necrosis. Radioautographic labeling The radioisotope tritium (H3) was chosen because of ready applicability to biological systems} Wilson (1966) describes tritium as a beta emitter of very'low energy. This fact has at least two implications for biological use. First, its use requires little shielding, and, second, it has great advantages in high resolution radioautography. Furthermore, it is in the class of radioactive compounds with the lowest toxicity. 72 The fact that tritiated thymidine localizes mainly in nuclei is not surprizing, in view of the fact that the base thymine is incorporated only into DNA and not into RNA. The 18-24 percent of silver grains seen over cytoplasm can be accounted for partially by the background fog and also by the dispersion of the radiation originating from the nucleus. Thus a given beta particle from the nucleus may have been emitted at such an angle as to terminate in the emulsion over adjacent cytoplasm rather than directly over the source of radioactivity. Nevertheless, the 80 percent of label re- maining over nuclei gave a sufficiently precise nuclear label so that labeled and unlabeled nuclei were easily discrimi- nated. On the other hand, the percentages of label over- nuclei and cytOplasm for tritiated leucine (Table 3), al- though having opposite percentages of distribution from those of thymidine, did not take into account the relative cyto- plasmic area versus nuclear area. Therefore, a comparison of cytoplasmic and nuclear densities for an animal given only H3L was also performed. From the“data in Table 3, the con- clusion can be drawn that tritiated leucine was distributed almost uniformly between cytoplasm and nuclei, with only a slightly greater concentration in the cytoplasm. Therefore the 80 percent of label seen over cytoplasm (Table 3) is accounted for by the‘fact that cytoplasmic area also is pro- portionately larger than nuclear area in a given segment of 73 the graft or_graft bed. The nuclear label also indicates that leucine is incorporated into nuclear as well as cyto- plasmic'proteins. The fact that amino acids are incorporated into body proteins must be balanced by the possibility that "pools" of amino acids may be present within cells without synthesis into proteins (White, Handler, and Smith, 1964). However, the present experiment deals with the comparison of two cell A 3..” -. ' types within the same animal and their relative, rather than absolute, protein turnovers. Thus the pooling effect, al- though not necessarily identical in different cell popula- tions, should be similar enough in both populations to eli- minate its significance in these results. From the above considerations, and the fact that leucine is incorporated on- ly into the protein fraction of tissue (Reid and Heald, 1970), it was concluded that the H3leucine distribution reflected the protein production and turnover in the cytoplasm and nu- clei of the cells to be discussed. IV. Radioautography of graft tissue The relative labeling of graft tissues proved inter- esting and intriguing. Table 5 indicated that allografts initially had a lower radioactivity than autografts, but con- tinued to increase in radioactivity so that at late time intervals they were more radioactive than the corresponding autografts. In order to eliminate the variation due to differing areas of the graft, the areas counted were chosen 74 at random between, but not including, the epithelium and the graft bed. The epithelium itself was not counted because its turnover rate is too high to detect any additional migra- tory or accumulating protein. Also, the epithelium was necrotic on most allografts and activily producing protein in autografts. Thus, elimination of these areas from the counting provided more uniform counts between the two types of grafts, and also a more accurate estimate of protein turn- over for similar areas of grafted tissue. Counts of the grafts for the animal given only H3T were also performed and indicated that there was only a small contribution of H3T to either allograft or autograft labeling. This suggested that the labels seen in the other animals were due to the presence of proteins. A wide variety of labeling was seen for the same time interval after grafting (4 days), especially in allografts. It was noted in animals receiving both H3T and H3L that these initial allografts were much more radioactive than those on animals receiving only H3L. But, as stated above, H3T does not contribute a great deal to the graft labeling, and therefore some other factor(s) must be Operating. Likewise, injections of H3L preceding the biOpsy by either 1 or 2 hours did not make a consistant difference in the labeling seen. One consideration is the fact that some of the allografts were not as necrotic as others at the same time intervals, thus could still be producing protein whereas others may have already ceased protein production. 75 A second possibility lies in the relative degrees of vascu- larity in the grafts. These grafts were biopsied and H3L was given on day 4 after grafting, which is the same time as revascularization of grafts takes place (Feldman, 1969). Therefore a given graft which was well vascularized may have been able to take up and incorporate circulating H3L, whereas a graft not completely vascularized at the same time would not receive a comparable supply of H3L. These considerations made it imperative to compare grafts only on the same animal, and even so, there is a possibility that grafts on the same_ animal are not vascularized to the same degree. Neverthe- less, the fact that all autografts (except one) were ob- served to decrease in radioactivity with time within a single animal, while all allografts increased in radioactivity is too consistant to attribute to chance. Furthermore, the allograft tissue of 2 out of 4 animals at the latest time intervals showed a level of radioactivity which exceeded even the highest radioactivity of autografts (at early intervals) for that animal. This last fact is explainable only by an accumulation of labeled protein within allografted tissue which did not occur in autografts. In addition, the accumu- lation must have originated from outside the graft, since no living cells remained within allografted tissue after 8 days to release any more protein. Comparison of autografts to normal skin showed a parallel decrease in both over time, which is explainable as an expression of the normal turnover 3 76 rate of protein produced within these tissues, and would be expected. The origin of the accumulating protein in allo- grafts will be discussed below. V. Radioautography of graft bed cells The basophilic cells of the graft beds were separable by the use of radioautography. Walker and Goldman (1963) ' ~ l7_ labeled at differing time intervals relative to grafting, but I I when H3T had been given 24 hours before grafting, they noted that only cells from the blood were labeled within the graft bed. When performing cell counts, however, they eliminated cells with less than 5 silver grains over the nucleus and also mentioned the probability that reutilization was occuring to account for the cells which were lightly labeled. Griffiths (1970) also mentioned heavy reutilization in allo- grafts when labeling had been performed to identify short- lived cells. The present study was consistant with these findings. A wide range of nuclear labeling was encountered in graft beds ranging from a few to 20 grains per nucleus (see Appendix B). If reutilization were indeed taking place (and it was obvious that it was by the number of endothelial cells seen with 3 to 4 silver grains over their nuclei) a simple cut off point using a certain minimum number of silver grains per nucleus might well lead to a fibroblast, which had reutilized a considerable amount of label in preparation for division, to be mistaken for a hypertrophied lymphocyte. Consequently it was decided to establish a different criterion 77 for determining whether a cell had originated from the blood. It was based on the assumption that endothelial cells and fibroblasts would be proliferating at a similar rate and therefore also reutilizing label at a similar rate. In order to include the nuclear size in the criteria, it was based on nuclear grain density (grains per,u2) rather than an absolute number of grains. The assumption of a similar reutilization rate in these two cell populations is justified because Walker and Goldman (1963) also labeled animals after grafts had been in place for several days and examined the tissue 2 hours after the injection of H3T. They found fibroblasts and endothelial cells to be the only cell types proliferating in the graft bed area, since both of these populations were heavily labeled. In the present experiment, therefore, the endothelial nuclear label was used as an”index of H3T reuti- lization. A nuclear label must have been above the 95 per- cent fiducial limits for endothelial nuclei in order to have definately originated from blood lymphocytes. In addition to increasing the amount of calculations involved, this technique also likely eliminated the cell which may have in- filtrated from the blood with a sizable grain density but hypertrophied to twice its size and therefore halved its density. Nevertheless, as noted in Appendix B, Table 18, more than half of the cells counted were still above the cut off point in allografts whereas in autografts the earliest graft had 30 percent and the latest 20 percent of the cells 78 counted being above the cut off point. These cells in auto- grafts were also very small, corresponding to small lympho- cytes rather than the large hypertrophied lymphocytes of the allografts. Once these criteria were established, only data for hypertrophied lymphocytes were tabulated for allografts, while in autografts all large radioactive cells were counted. This additional manipulation was justified, however, when comparing the cytoplasmic protein turnover for the various populations (hypertrOphied lymphocytes versus fibroblasts). Table 6, which contains data for a pure population of hyper- trophied lymphocytes in allograft beds, indicates more con- sistant and greater decreases in cytoplasmic densities than does Table 7 which contains the mixed population of all large basophilic cells. The analysis of cytoplasm to determine the protein turnover rates of these two different populations was carried out for animals receiving both H3T and H3L. This also involved a great deal of calculation, but the results were rewarding. Hypertrophied lymphocytes in the allografts initially had a higher cytoplasmic density than did cells in autograft beds. This indicates that they were incorporating H3L and therefore presumably producing protein at a faster rate than fibroblasts. However, at later intervals, the den- sity for hypertrOphied lymphocytes had dropped below the corresponding fibroblast cytoplasmic density. This can be accounted for only by infering that the protein produced had left the cells. W ‘ ”mi-4‘— h I. 79 It is easy to hypothesize that the presumed protein production by the hypertrophied lymphocytes is responsible for the accumulating protein of the allograft, and that this solves the mechanism of lymphocyte involvement in graft re- jection. However, some additional statistical manipulations indicated that this simple explanation is probably inade- quate. Examination of grafts from animal 3 indicated about 20 unit areas of graft tissue were present in each section of tissue. Assuming this to be true for all time intervals, the absolute increase in number of silver grains seen from early to late intervals can be calculated to be about 500 grains. Likewise it was noted that there were about 80 hypertrophied lymphocytes in each section of graft bed. Assuming that the nuclear labeling criteria accounted for only 50 percent of the lymphocytes actually present, and that the average amount of cytoplasm present per cell on a 2 micra section was 30‘p2, the absolute number of silver grains released by these cells in labeled protein between the early and late intervals would be 288 grains. Thus the graft appeared to be accumu- lating more protein than the lymphocytes were producing. There may have been an additional source of protein outside of the hypertrOphied lymphocytes which also contributed to the graft rejection process, since there was no such accumu- lation in autografts. One possible explanation lies in the fact that plasma cells were seen at late intervals in allo- graft beds (Russell and Monaco, 1965). Presumably these Wynn—2-? 80 cells could have incorporated and stored H3L while it was available, and later released it in a protein against the graft, but this seems unlikely. Another consideration is the amino acid pooling, mentioned earlier. If a pool of labeled amino acid were present in the cytoplasm of a cell at an early interval, it would have been washed out by the fixation process. However, at the later intervals, this pool could have contributed to additional labeled protein production. An additional source of protein may have come from the sera, which could include a specific substance against allografted tissue. This last possibility is supported by recent in_zizg findings of systemic humoral antibody against primary skin allografts in mice (Canty and wunderlich, 1971). However, it is unlikely that this protein could be radioac- tive, because tritiated leucine would be available for incor- poration into tissue proteins for only a few hours after injection on day 4, and this systemic antibody does not appear until day 5. Another explanation for the descrepancy between allograft protein accumulation and graft bed protein production may be the loss of some of the small molecules of the graft bed during fixation. The present experiment lends no clue to the identity of the substance produced by the hypertrophied lymphocytes of the graft bed other than that it is a protein. This is in accord with‘in_vitro studies in which investigators isolated 81 and analyzed such a protein (Kolb and Granger, 1970 and Williams and Granger, 1969). It might be possible, in some future experiment to collect late skin allografts whose hosts have been injected with H3leucine and with chroma- tography or appropriate separation columns, identify the substances which are labeled according'to their physical properties. Additional experimentation should also be per- formed to try to find the source of additional protein pro- duction, or loss from the graft bed, since it is likely that graft rejection, being the complex process it is, cannot be completely understood or controlled without such knowledge. SUMMARY The essential conclusions of this research are: (1) lymphocytes from the peripheral blood, having hyper- trophied within the graft bed of allografts, produce and release a substance which is probably a protein during the graft rejection process; (2) there is an accumulation of radioactive label (probably protein) in the dying allograft tissue during the graft rejection process, and (3) these phenomena (protein release by graft bed cells and protein accumulation in graft tissue) do not occur in autografts. These findings support the conclusion that lymphocytes are involved in the graft rejection reaction, and suggest that this involvement may be in the form of a protein produced within the graft bed which migrates into the graft and ad- heres to allogeneic cells. 82 REFERENCES CITED REFERENCES C ITED Basten, A., J. F. A. P. Miller, N. L. Warner, and J. Pye 1971 Specific inactivation of thymus-derived (T) and non- thymus-derived (B) lymphocytes by 125I-labeled anti- gen. Nature (London), 231: 104-106. L Benacerraf, B. and I. Green 1969 Cellular hypersensitivity. Annual Review of Medicine 1969, pp. 141-154. Billingham, R. E. 1961 Free skin grafting in mammals. Transplantation of Tissues and Cells. Ed. by . Billingham and Silvers. The Wistar Institute Press Philadelphia. pp. 22-23. Billingham, R. E. 1969 The role of the lymphocyte in trans- plantation immunity. Anat. Rec., 165: 121-124. . Canty, T. G. and J. R. Wunderlich 1971 Quantitative assess- ment of cellular and humoral responses to skin and tumor allografts. Transplantation, 11: 111-116. Cerottini, J. C., A. A. Nordin, and K. T. Brunner 1970 Specific in vitro cytotoxicity of thymus-derived lymphocytes sensitized to alloantigens. Nature (London), 228: 1308-1309. Cerottini, J. C., A. A. Nordin, and K. T. Brunner 1971 Cellular and humoral response to transplantation antigens. I. Development of alloantibody-forming cells and cytotoxic lymphocytes in the graft-versus- host reaction. J. Exp. Med., 134: 553-564. Chanana, A. D., E. P. Cronkite, D. D. Joel, L. M. Schiffer, and H. Schnappauf 1969 Studies on lymphocytes. XII. The role of immunologically committed lympho- cytes in rejecting skin allografts. Transplantation, 7: 459-467. Evans, R. and P. Alexander 1971 Rendering macrophages speci- fically cytotoxic by a factor released from immune lymphoid cells. Transplantation, 12: 227-229. Feldman, J. D. 1969 Graft rejection. Arch. Int. Med., 123: 713-718. 83 84 Feldmann,M. 1972 Cell interactions in the immune response in vitro. II. The requirement for macrophages in lymphoid cell colaboration. J. Exp. Med., 135: 1049-1058. Fuji, H., M. Zaleski, and F. Milgram 1971 Allogenic nucle- ated cells as immunogen and target for plaque- forming cells in mice. Transplant. Proc., 3: 852-855. Giroud, J. P., W. E. Spector, and D. A. Willoughby 1970 Bone marrow and lymph node cells in the rejection of I skin allografts in mice. Immunol., 19: 857-863. . *“ Gisler, R. H. and P. Dukor 1972 A three-cell mosaic culture: in vitro response by a combination of pure B- and T-cells with peritoneal macrophages. Cell. Immunol., 4: 341-350. Golstein, P., H. Wigzell, H. Blomgren, and E. A. J. Svedmyr 1972 Cells mediating specific in vitro cytotoxcity. II. PrObable autonomy of thymus-processed lympho- cytes (T cells) for killing of allogeneic target cells. J. Exp. Med., 135: 890-906. Goren, G., G. Berke, and I. Urca 1972 Localization of 51Cr labeled sensitized lymphocytes at the allograft region: a possible diagnostic test in organ trans- plantation. Surgery, 71: 513-521. Grant, C. K., G. A. Currie, and P. Alexander 1972 Thymocytes from mice immunized against an allograft render bone marrow cells specifically cytotoxic. J. Exp. Med., 135: 150-164. Griffiths, A. 1970 Skin transplantation and the types of lymphocytes involved in rejection. J. Anat., 107: 194. Hamilton, D. N. H., J. E. Castro, and J. M. Gaugas 1971 Cell-mediated and humoral mechanisms in allograft and xenograft skin rejection. Br. J. Surg., 58: 858-859. Hayry, P. and V. Defendi 1970 Mixed lymphocyte cultures pro- duce effector cells: model in vitro for allograft rejection. Science, 168: 133-135. Jakobisiak, M. 1971 Quantitative data concerning the devel- opment of the cellular infiltration of skin allograft in mice. Transplantation, 12: 364-367. 85 Kolb, W. P. and G. A. Granger 1970 Lymphocyte in vitro cytotoxicity: characterization of mouse lymphotoxin. Cell. Immunol., 1: 122-132. Kramer, J. J. and G. A. Granger 1972 In vitro induction and release of a cell toxin by immune C57B1/6 mouse peritoneal macrophages. Cell. Immunol., 3: 88-100. Lewis, A. E. 1966 ‘Biostatistics, Reinhold Publishing Cor-> poration, New York, p. 213. Liem, P. L. and C. Jerusalem 1970 .Blood supply of allogeneic skin transplants before and during graft rejection. ACto MOrph. No l 7: 356-3570 Lonai, P. and M. Feldman 1971a Cooperation of lymphoid cells . in an in vitro graft reaction system. II. The "bone marrow-derived" cell. Transplantation, 11: 446-456. Lonai, P. and M. Feldman 1971b Studies on the effect of macrophages in an in vitro graft reaction system. Immunol., 21: 861-867. Lykke, A. W. J. and R. Cummings 1970 _Increased vascular permeability in the primary allograft response in the skin of the rat. J. Pathol., 101: 319-327. Melchers, F. 1970 Biosynthesis of the carbohydrate portion of immunoglobulins. KinetiCs of synthesis and secretion of H3-1eucine, H3-galactose, and H3-mannose labeled myeloma protein by two plasma-cell tumours. Biochem. J., 119: 765-772. Miller, J. F. A. P., A. Basten, J. Sprent, and C. Cheers 1971a Interaction between lymphocytes in immune re- sponses. Cell. Immunol., 2: 469-495. Miller, J. F. A. P-, J. Sprent, A. Basten, N. L. Warner, J. C. S. Breitner, G. Rowland, J. Hamilton, H. Silver, and W. J. Martin 1971b Cell-to-cell interaction in the immune response. VII. Requirement for differ- entiation of thymus-derived cells. J. Exp. Med., 134: 1266-1284. Miller, J. F. A. P., K. T. Brunner, J. Sprent, P. J. Russell, and G. F. Mitchell 1971c Thymus-derived cells as killer cells in cell-mediated immunity. Transplan. P., 3: 915-917. 86 Moller, E. and W. Lapp 1969 Cytotoxic effects in vitro by lymphoid cells from specifically tolerant animals. Immunol., 16: 561-566. Osmond, D. G. 1969 The non-thymic origin of lymphocytes. Anat. Rec., 165: 109-112. Peter, H. H. and J. D. Feldman 1972 Cell-mediated cytotox- icity during rejection and enhancement of allogeneic skin grafts in rats. J. Exp. Med., 135: 1301-1315. Reid, R. J. and P. J. Heald 1970 Uptake of H3-leucine into proteins of.rat uterus during early pregnancy. Biochem. Biophys. Acta, 204: 278-279. 1- Russell, P. S. and A. P. Monaco 1965 The Biology of Tissue Transplantation. Little, Brown, and Company, Boston, P. 18. Salvin, S. B., S. Sell, and J. Nishio 1971 Activity in vitro of lymphocytes and macrophages in delayed hypersen- sitivity. J. Immunol., 107: 655-662. Slonecker, C. E. 1969 H3-1eucine incorporation in antigen- ically stimulated rat popliteal lymph node cells. Anat. Rec., 165: 363-378. Tilney, N. L. and J. L. Gowans 1971 The sensitization of rats by allografts transplanted to alymphatic ped- icels of skin. J. Exp. Med., 133: 951-962. Wagner, H., A. W. Harris, and M. Feldmann 1972 Cell-mediated immune response in vitro. II. Role of thymus and thymus-derived lymphocytes. Cell. Immunol. 4: 39-50. Walker, B. E. 1959 Radioautographic observations on regener- ation of transitional epithelium. Tex. Rep. Biol. Med., 17: 375-384. Walker, B. E. 1962 A radioautographic study of muscle re- generation in dystrophic mice. Am. J. Path., 41: 41—53. Walker, B. E. and A. S. Goldman 1963 Thymidine-H3 radio- autography of skin grafts in mice. Tex. Rep. Biol. Med., 21: 425-441. Walker, B. E., R. D. Yates, and D. Duncan 1964 Cell mor- phology in tissue underlying skin grafts. Anat. Rec., 149: 651-670. 87 White, A., P. Handler, and E. L. Smith 1964 Principles of Biochemistry, 3rd ed., McGraw-Hill Book Company, New York, p. 507. Williams, T. W. and G. A. Granger 1969 Lymphocyte in vitro cytotoxicity: mechanism of lymphotoxin-induced target cell destruction. J. Immunol., 102: 911-918. Wilson, B. J. 1966 The Radiochemical Manual, 2nd ed., The Radiochemical Centre, Amersham, p. 42. Yunis, E. J., O. Stutman, and R. A. Good 1971 Thymus, immunity, and autoimmunity. Ann. N. Y. Acad. Sci., 183: 205-220. Zatz, M. M. and E. M. Lance 1971 The distribution of 51Cr labeled lymphocytes into antigen stimulated mice. Lymphocyte trapping. J. Exp. Med., 134: 224-241. fllflx‘lnfl _‘n APPENDICES ’L [.51 Hi1; APPENDIX A Appendix A. Comprehensive data for graft labeling This appendix lists the actual grains counted for randomly selected areas (2500,p2) of the graft tissue. Areas were located within the graft tissue between the epidermis and graft bed. This would include the dermis and panniculus adiposus. Means were calculated for the ten areas from each graft. Standard deviations were calculated using the formula sd= “((X - X)2/(n - 1). Grafts of the same type for the same animal at different time intervals were compared for statis- tically significant changes by applying the t test using the formula t=(Xl - X2)/s' /(l/n1) + (l/nz) , where s'=./(n1 - l)V1 + (n2 - l)V2/(nl -‘l) + (n2 - l) or, because n1 = n2 = 10 in this case, s'= ./(V1 + V2)/2 and t=(X1 -‘X§)/s'(.45). The appropriate tables were then con- sulted (Lewis, 1966) for p values. 88 89 Table 8. Grain counts of allograft tissue from animal 9 (leucine). Days after grafting . ~ 4.1 8 10 17 38 54 15 44 46 19 46 45 grain counts 17 43 40 (grains/ZSOWuZ) ‘22 36 46 16 31 40 20 35 38 18 36 41 21 38 37 23 43 45 Mean . ~.18.6 38.9 43.2 Standard deviation 2.7 4.6 5.0 t 11.95 2.00 p {.001 (.10 Table 9. Grain counts of autograft tissue from animal 9 leucine). Days after grafting 4.1 8 10 47 39 40 38 insufficient 46 37 grain counts 44 31 (grains/ZSOQuz) tissue 41 4o 44 34 available 42 41 46 37 45 43 51 38 Mean J 44 o 6 ' ' ‘ 37 o 8 Standard deviation 3.2 3.5 t 4. 48 p ,,. (.001‘~ wxl—m 'l‘I 90 Table 10. Grain counts of allograft tissue from animal 10 (leucine). Days after grafting 4.2 5 6 18 38 58 11 36 52 . 12 22 53 grain counts 16 30 48 (grains/2500u2) 12 37 67 14 27 61 19 28 53 14 23 68 18 33 60 V1 11 37 ' 57' Mean 14.5 31.1 57.6 Standard deviation 3.1 6.0 6.5 t 7.77 9.44 ~ p ' (.001 (.001 Table 11. Grain counts of autograft tissue from animal 10 (leucine). Days after grafting 4.2 5 6 24 40 37 24 38 33 33 43 33 grain counts 32 38 38 (grains/ZSOQpZ) 33 39 43 38 35 39 35 36 35 32 33 35 34 39 31 33 35 38 Mean 31.8 37.6 36.2 Standard deviation 4.5 2.9 3.5 t 3.42 0.96 p ' (.005 (.50 91 Table 12. Grain counts of allografts from animal 4 (H3T and H3L). Days after grafting 4.1 8 10 37 47 44 53 41 insufficient 41 grain counts “ 43 43 (grains/2500p2) 33 tissue 48 50 49 39 available 53 48 51 42 47 41 44 Mean 38.8 47.6 Standard deviation '5.9 4.1 t I 3.86 p (.005 Table 13. Grain counts of autografts from animal 4 (H3T and H3L). Days after grafting . 4.1 8 10 61 36 30 51 40 35 60 41 31 grain counts 49 44 30 (grains/ZSOQpZ) 50 44 34 48 38 28 54 38 31 47 34 36 54 36 38 54 33 32 Mean ‘ 52.8 38.4 32.5 Standard deviation 4.8 3.8 3.1 t 7.39 ' 3.74 p (.001 (.005 Table 14. Grain counts of allografts from animal 3 (H3T and H3L). 92 Days after grafting 4.1 5 6 40 52 61 47 56 68 ,51 44 74 grain counts 39 51 63 (grains/ZSOQpZ) 37 48 63 43 47 67 45 49 65 41 52 71 40 56 70 37 50 65 Mean 42.0 50.5 66.7 Standard deviation 4.5 3.8 4.1 t 4.54 9.14 p (.001 (.001 Table 15. Grain counts of autografts from animal 3 (H3T and H3L). Days after grafting 4.1 5 6 55 35 35 58 38 36 65 42 38 grain counts .62 37 40 (grains/2500p?) 53 37 29 54 32 30 50 31 38 51 37 35 52 33 35 51 34 31 Mean 55.1 35.7 34.7 Standard deviation 5.0 3.3 3.7 t 10.14 0.64 p 5.001 ').50 4 . . $14374.» é .. APPENDIX B Appendix B. Nuclear labeling in animals receiving H3T In animals receiving only H3T, or both H3L and H3T, nuclear labeling characteristics were used to identify hyper- trophied lymphocytes of the graft beds. ‘Endothelial cells were used as an index of reutilization of the H3T label, and it was assumed that fibroblasts would reutilize to about the same degree. Therefore, taking the mean nuclear density of endothelial cells and adding twice its standard deviation produces a cut off point; and cells with nuclear densities higher than that point are assumed to be hypertrophied lym- phocytes originating from the blood. Data for endothelial cell nuclei, cut off points, hypertrophied lymphocyte nuclei (allografts), and basophilic nuclei (autografts) are pre- sented in this appendix. All nuclear areas are rounded to the nearest Sluz. Symbols used are as follows: # = grain count lg? = area in square micra #Auz = grain density 4.1 = one hour following H3L injection on day 4 4.2 = two hours following H3L injection on day 4 93 94‘ Hm. 1mm. (ms. 1%. peace No use vO. mO. mo. «O. GOHpoH>oo pnoocowm .V 2.. . OH. H. S. FHnooor sows NH. mN m I I OH. ON N mH. ON M NH. om O OH. Om O OH. ON m ON. mN O NH. , mN m ON. ON O NH. OO O mN. Om e Poul I IN. I .m I Imef I ONI Is... I mHh I low. I m I INTI I .mmI ImI nnsooo mH. ON m ON. ON O. NH. om O OH. Ov h HoHHosuooco OH. ON N ON. .ON m OH. OO e OH. ON N uwonmoucm ON. ON o ON. .ON v ON. ON O ON. mH s OH. ON m NH. mN m ON. ON v ON. ON v mH. om O (OH. om MI ON. mH m mH. ON m NQ\* NQ * 1— N.H&\* NH‘ . * % MPH/Q»... . NH. , * _ . NHK\# . I .NHK. I fl mm, 3. mm. em. uoHom too new JMb. «O. NO. «O. cOHuoH>oo ouoocoum NH. OH. H. mm. EH33 zoos eH. Om m hN. OH O ON. ON b OH. OH H «N. mN O meal I Ime I m IIIIIIIIIIIIIIIIIII Paul I OHI INI OH. OH H OH. OH H ON. ON m mpcsoo OH. OH H ON. OH ,N OH. mN O ON. ON v HoHHonuooco 50. mH H ON. mH m ON. mH m ON. ON O DOMHOOHHN mH.I I ImH I .m I IOM.I I MHI In! I bNh I lmH I M I IOH.I I MN! lvl ON. mH m OH. ON N ON. OH N , ON. mN w mO. ON H. OH. mN O ON. mH m ON. ON O OH. ON N mH. ON m mH. ON m mN. Om 5 low. Jme rm Om. OH N - OH. 3 v «N. N O N1; NIH * )N:\* Na 1* — NQV* NR * — New." N: H OH ... O _I O _ o mcHumon‘Houmo axon (I) .AoQHoHehsav s Hocho How muccoo HooHosc HoHHoADOOcm .OH oHnoe OO OO OO OO usHom mmo poo o>ono m NN. ON. NN. om. AOH oHnoe Eouwv usHoo Ono poo OO. ON O NO. ON O OO. Om NH Om. ON O ON. ON N NO. Om HH 5 OO. ON HH Om. Om OH ON. ON N Om. ON O 9 OO. Om NH NN. OH O ON. ON N Om. Om OH ANansoo msHmnososH .OION. I I MNI IOI I NN.”. I IO..O. I .N..HI INm..I I OOI IOI I em... I IOm I .HIHI Ho .898 5.; NO. ON O ON. Om N NN. OO O OO. ON O HoHoss OO. ON O ON. Om N ON. OH OH Om. OO NH HHoo oHHHseonnm ON. OO HH ON. ON O ON. Om N NN. OO HH ON. OO OH NN. OO OH ON. Om N ON. OO NH MN... I I .3... .OH I .OH.“ I IOM I w. I INH.I I Om... IOI I .mN... I IOm I .N. I ON. OO OH OH. OO N NH. OO O NN. OO OH NH. OO O OH. OO O OH. ON O ON. OO O OH. ON O OH. OO O OH. OH O ON. OO NH OH. OO O OH. OO O OH. OO O OH. OO O OH. OO O OO. OO O - OH. OH O . OH. OO O NQ\* N1 O 7— Na\# II“: * _ Nn<* N2. * _ N:\* Na * OH _. m _ O _ O msHumon Hopmo mama .HocHoHfihcuv N HoEHso Scum mDmMHOOHHm mo musooo HooHosc HHoo OHHHcmomom .NH oHnoe ON ON ON OO ucHoe Oeo poo o>ono w HN. ON. ON. . . ON. HOH OHnoe sosue usHoo OOo poo OO. OO NH NO. ON O NO. ON O OO. OO OH ON. ON O NO. ON OH MN I ONI IOI I MO... I IOm I O I IONV I OOI IOIH. I OO.“ I IOm I .mHI ON. OO OH OO. OO NH OO. OO NH OO. OO NH ON. OO O OO. OO O OO. OO NH NO. OO OH ON. OO O NN. OO O OO. OO O ON. OO OH ON. OO O ON. OO OH NN. OO HH ON. OO OH ANHHOsoo OanoososH ON I OOI IOI I .OH. I IOM I .H.HI IN.N..I I .O.OI IOI I NNI.. I IOM I .N.HI Oo sooso :3 ON. ON O OH. OO N oN.. OO OA NN.. OO O HOHoos ON. OO NH OH. OO N ON. OO N NN. OO O HHoo oHHHsoomom OH. OO N NH. OO O ON. OO O ON. OO NH OH. OO N NH. OO O ON. OO OH ON. OO N Eh I MOI IOI I NHh I IO» I .OHI IOm.I I OO.. IOI I ON... I IOm I O. I NH. OO O OH. OO O NH. . .OO O ON. OO O OH. OO O OH. ON O NH. OO O ON. OO O OH. OO O OH. OO O OH. OO O OH. OO O OH. OO O OH. OO NH OH. OO O OH. OO N OH. OO O NH. OO O OH. OO O OH. OO O NQ\* NQ # fl NQ\* NQ * NQ\* N1 * NHH\* N: * OH _ O O . O mcHumon Houmo memo .AocHOHshcuv N HoEHso ECHO mumoumouso mo mucsoo HooHocc HHoo OHHHcQOmom .OH oHnoe 97 OH. ON. .MN. ucHom,mmo #50 OO. OO. OO. coHumH>oo oumocoum NH. OH. . IOH. OUHmcoo coo: NH. ON O OH. ON N OH. ON O OH. on O OH. ON O OH. ON O OH. ON N NH. ON m ON. OO O OH. ON m ON. ON O ON. OO N OHIIINIIOIIIOHIIINIIOIIIOOIIINIIOI megs OH. ON N OH. ON O OH. ON O HoHHonuooco OH. OO O OH. OH N OH. ON N umosOousO OH. OO O OH. ON N OH. ON O OH. on O OH. om O OH. OH N OH. OO O ON. ON O ON. ON O NQVO N1 O ‘ NQVO N: * NQVO Nq O ON. ON. NN. ucHom,mmo #50 OO. OO. OO. GOHpoH>oo onoocoum OH. OH. HN. MuHmcoo coo: OH. ON O NH. ON N ON. ON O ON. ON O ON. ON O ON. ON O ON. ON O OH. ON O ONO _ON O ON. ON O OH. on m ON. Om N OOIIINIIm:IIOHIIIRIIOIIIOHIIINIIN Odds ON. OH O ON. ON O ON. ON O HOHHonuooco OH. ON N ON. . OH O OH. OO O OOOnOoHHO OH. ON O OH. ON N ON. OH N ON. ON O NO. Om N ON. ON O OH. ON O OH. ON N ON. OH N NQVO N1 * N§\* N1 O N:\* N1 O OH O H.O msHumoum Houmo mama .AosHosoH one osHOHENan O HoaHso How mpcsoo HooHosc HOHHonuoosm .OH oHnoe 98 OOH OOH OOH ueHoe Omo poo o>ono O ON. ON. NN. AOH oHnoe Eoumv ueHom «mo poo OO. ON HH OO. om NH OO. OO NN OO. ON O OO. OO OH OO. OO OH OO. OO OH OO. OO OH OO. OO NH Om. ON O mm. on OH OO. OO ON MONIIIOMIIOIIIMONIIIOMIIbHIIIbOhIIIOMIIMI Om. OO OH NO. ON O . OO. OO NH Om. ON O HO. Om HH NO. ON OH Om. ON O OO. OO NH Om. ON O OO. OO OH Om. OO NH Om. OO NH ANuHmooo mchooHocH hmhlllohllelllbmhlllohllblllhmhlIIOHIIMI m0 “350ch NO. ON O OO. OO NH mm. Om OH HoHows NO. ON O OO. OO NH NO. OO OH oumomnmENH Om. ON O ON. ON N OO. OO NH . .mNhIIIOMIINIIINNhIIIOhIlbIIIbOhIIIOMIIMI ON. ON N NN. OO O ON. ON N ON. ON N ON. OO OH ON. ON N ON. OO OH ON. OO OH ON. OO HH ON. OO OH ON. ON O ON. OO OH ON. on O . llmN. Ow OH ON. OO HH NQVO N1 O _ NQ\O N1 O fl NQVO N1 O , OH PI O fit H.O mcHumoum Houmo mama .AHOm woo emmv O HoEHso ECHO mumoumoHHo mo mucsoo HmoHosc HHoo oHHHemommm .ON OHoOe OH . OH OH eaHom OOo poo o>ono O OH. ON. ON. HOH OHnoe sosme uOHom mmo poo ON. roO OH NO. ON O OO. ON O ON. OO O ON. ON O OO. ON O OH. OO N ON. OO N ON. OO HH. OH. OO N ON. OO OH ON. OO OH OH...IIIOOIIOHIIIONNIIIOOIIMIIIONNIIIOOIINI OH. OO O ON. OO O.. ON. ON O OH. OO N OH. OO N ON. OO OH OH. OO O OH. OO O ON. OO O OH. OO OH OH. OO N ON. OO O Hmanooo OonoonoeH ”HI II I.. Inn-MN II I b I II .I. bHII I. II IOI“ I II IWI II I I le II II Inc-NI I .I. N I HO Hmcho adv OH. OO O NH. OO O ON. OO OH HOHomo OH. OO OH NH. OO O OH. OO O . OH. OO N OH. OO O OH. OO N HHoo oHHHsmomOm OH. OO O OH. OO O OH. OO O OHNIIIONIIOIIIMHNIIIONIIOIIIIOthllohIIOI OH. OO O OH. OO O NH. OO O NH. OO O OH. OO O OH. OO O NH. OO N OH. OO O OH. OO O OH. OO O OH. OO O OH. OO O OH. OO O OH. OO (IO (I NH. OO O NaxO N: O NaxO N: O _ NaxO N1 O OH O _ H.O mcHumoum Hopmo mama .AHmm can emmv O HoEHco ECHO mumouwouco mo muccoo HooHosc HHO.o oHHHsmomom .HN oHnoe 100 mm. . mm. mm. ucfiomflmMo ufiw mo. . .lmm. 1. mo. QOOQMO>m© wumvcmwm , OH. OH. ON. OuOmamO cam: ON. OH N ON. ON O ON. OH O ON. OH O ON. ON O ON. ON O ON. ON O NH. OO O ON. OH O ON. ON O ON. ON O ON. ON O bNhIIIOMIIMIIIbNhIIIOMIIMIIIthIIIOMIIwI 3550 OH. ON O OH. ON O OH. ON O HOOHmOuocam OH. ON O NH. ON O ON. ON O OOOuOoOOO ON. ON O OH. ON N ON. ON O OH. ON O OH. ON O ON. OO O ON. ON O OH. ON O ON. ON O Na; N: O _ Na; N: O 4 Na; N: O NN.. NN. HN. ucOom Ono pmm IIMO. NO. NO. coOumO>mO OuOOcmuO OH. . ma. Na. huwmcmc cum: OH. ON O OH. ON O OH. ON -O OH. OH N OH. ON O NH. OO O OH. ON N OH. ON O OH. ON O OH. ON O ON. ON O OH. ON O MHhIIIOMIIwIIIMHhIIIOmIINIIImHhIIIOMIIO... 358 ON. ON O NH. OO O NH. OO O HOOHmsuoOcm ON. ON O ON. ON O OH. OO O uOOuOoHHO OH. ON O OH. ON O ON. OO O OH. ON O ON. ON O ON. ON O OH. ON O , ON. OO O , ON. ON O HO O... O P Ni ”O O _ Om; O: O m _ m _ d.v chummnm umumm mama .mmcflosma can uqOUOshspv m Hmfiwqm Mom mucaoo HmmHoaa Hmwam£powcm .NN manna OOH OOH OOH ucOom «mo ago m>oam w NN. NN. . HN. ANN mHnma aoumv ucHom mmo #90 OO. OO NH NO. OO HH OO. OO OH OO. ON O OO. OO OH OO. OO NH OO. OO OH OO. OO OH OO. OO NH OO. OO OH NO. ON O OO. OO OH mOhIIIOMIIMIIIbOhIIIOMIIOIIImOhIIIOMII.O.HI NO. ON O ON. ON N OO. OO OH OO. OO O ON. ON N OO. OO OH OO. OO O ON. ON N OO. OO oH NN. OO O NN. OO O NO. ON O bah!llohllblllhm...llloMllmlllbmhllloMllblauHmcmv mchmeocH NN. OO O NN. OO O OO. OO O O0 umcuo OOO NN. OO O ON. OO OH NN. OO O OmHosn mpmoosmsOH ON. OO OH ON. OO OH NN. OO O OmOOmouuummOO ON. OO OH ON. ON O ON. OO OH ”NbIIIOMIIMIIIMNhIIIOhIIbIIIthIIIOMIIwI ON. OO O ON. OO O ON. OO O ON. OO O ON. OO O ON. OO N ON. OO N ON. OO O ON. OO N ON. OO N ON. OO N ON. OO O ON. OO N ON. OO O . ON. OO O NSO N: O A Na; N: O fl NEO N: O O ._ m _ ~.O oaHummum umumm mama .HHmm can Emmy m Hmchm Eoum mummumoHHm mo muanou HmmHonc HHwo OOHOnm0mmm .mN anme 102 OH OH ON ucOom «no uso m>onm m ON. ON. - ON. ONN OHOOO souOO ucOom Omo use OO. OO OH OO. OO OH OO. OO NH NN. OO O OO. OO NH ON. OO HH ON. OO OH OO. OO O NN. OO O ON. OO O NN. OO O NN. OO O mNhIIIONO...IOIIImNhIIIOMIIOIIIMNhIIIOMIIOI ON. OO N ON. OO O ON. OO O ON. OO O ON. OO O ON. OO O ON. OO N ON. OO O ON. OO OH ON. OO O ON. OO OH ON. OO O bN...IIIOmIIOIIIbNhIIIOMIImHIIIbNhIIIONIIMIOOOOOOOO 9:339: ON. OO O ON. OO O ON. ON O O0 umOuo cHO ON. ON O ON. OO O OH. OO OH OmHosa ON. OO OH ON. OO O OH. OO O HHmo oOHOOmoOOm ON. OO O OH. OO N OH. OO N thIIIOMIIOIIImHhIIIOMIImlIIOthIIOmIIMI NH. OO O OH. OO N NH. OO OH NH. OO O OH. OO O OH. OO O NH. OO O NH. OO OH OH. OO O OH. OO O OH. OO O OH. OO N OH. OO O OH. OO N OH. OO O NaxO N: O A NEO N1 O _ NOQO N1 O m — m _ ~.O mcHummum Hmumm mhmn .AHmm can Emmy m HmEHcm.Eoum mummnmousm mo mucsoo HmmHos: HHmo OHHHSQOmmm .ON mHQMB APPENDIX C Appendix C. Cytoplasmic labeling in graft beds In animals receiving both H3T and H3L, hypertrophied lymphocytes of allografts were observed to have an initially higher cytoplasmic density than the basophilic cells in the autograft beds. At late intervals, allograft cytoplasmic density declined to below that for autografts. This appendix presents the data from which these conclusions were drawn. All cytoplasmic areas were rounded to the nearest 5‘p2. Sym- bols used are as follows: #=grain count ‘p?=area in Square micra #An2=grain density 4.l=one hour following H3L injection on day 4 4.2=two hours following H3L injection on day 4 These cytoplasmic data are for the same cells whose nuclear data are given in Appendix B, although not in the same order. 103 OO.v HOO.v O NHO. NO.N msHm> O OO. OO.. OO. coOumO>mO unmoamum .OH. OH. ON. . ._.OuOOcmO cums OH. OO O NH. OO O NN. OH O OH. OOH OH ON. OH N NN. oO O NH. OO O OH. OO O ON. ON O OH. ON O OH. OO O NN. OO OH .mH....IIIO.N.II.mIII.O.H...I.....O.N.II.N.IIIMNhIIIOMIIwI OO. OO O OH. OO O NN. OO HH OH. OO N OH. ON O ON. ON O HH. ON O ON. OO O ON. OO O oH. ON N NH. OO O HO. OO HH mHhIIIOMIIMIII.O.HhIIIOMIIMIIIONhIIIOHIIwI . 3560 ON. ON O OH. ON N ON. ON O OOEOOHmouOo OH. ON N NH. ON O ON. ON O mpOooOmeOH NH. OO O OH. ON N OO. OO NH OmOsmouuummOm OH. OO O OH. ON N ON. OO OH thIIIOmIlm.IIImHhIIIOHIImIIImNhIIIOMIImHI OH. ON O ON. OH N ‘ OO. OH O OH. ON O OH. ON O NN. OO O OH. OH H ON. OH O ON. OO O OH. ON O OH. OH N OH. ON O OH. OH N . ON. ON O ON. OO OH NEO N: O _ Na; N: O Na; N: O OH _ m H.O mcwummum Hmumm mmmo .O HMEHcm mo mummnmoHHm cH chHmnmw UOEmMHmoumU .mm mHnma OO.v HO. , O OO.O OO.N msHO> O OO. OO. OO. aoOuOO>mO OHOOcOuO OH. NH. OH. OOOOOOO :Omz OH. OO O ON. ON O OH. ON- ,- O OH. OO O OH. ON. O ON. OO O OH. OO N ON. OH O NH. OO O ON. OO O OH. OH N OH. ON O mHhIIIOMIIOIIImHhIIIOHIIMIIIMHhIIIONIIMI OH. OO O ON. OH N OH. OO O ON. OH O ON. ON O OH. OO O OH. OO O OH. OO O OH. OO O OH. OO O OH. OO N OH. OH H .mH...IIIO.N...ImullehIIIOMIIMIII.H.H.wIIIO.O.HI....H.HI OH. ON O ON. ON O OH. ON O Oucsoo OH. OH N ON. OH O NH. OO O OOEOOHmouOo ON. OH N ON. OH N NH. ON O HHmo OOHHOmoOOm ON. OO O ON. OH N OH. OO O mHhIIION .O.IllmH...IIIOMIIMIIIbNhIIIOHIIMI NH. OO O OH. ON O ON. OO O ON. OH O ON. OH N ON. OH N OH. OO O NH. OO O OH. OO O OH. OO O ON. OO O OH. OO O OH. OO O OH. OO N OH. ON O Na; Na O L Na; NA O NSO N: O OH F m H.O mcfiummum Hmumm mama .O HmEHcm mo mummumouom cH mcHHmQMH OHEmmHmoumo .ON mHnme OO.v . HOO.v m 106 ON.N ON.O msHm> O OO. OO. OO. coOuOO>mO oumnzmum MH. mH. . . mH. huHmcmp cmmz OH. ON N OH. . OO O ON. ON O NH. ON O ON. OH O OH. ON O OH. ON N NH. ON O ON. OH N OH. ON O NH. OO O ON. ON O bNhIIIOMIIwIIIbNhIIIOmIIMIIIbOhIIIOMIIwI OH. ON O oH. ON N ON. OH O OH. . OO O OH. OO O OH. OO N OH. OO O NH. OO O OH. ON O NH. ON O OH. OO O OH. OO N MHVIIIOMIIMIII.N..H...IIIOMIIMIIIpNhIIIOHIImI 358 NH. ON O OH. OH N NH. OO O oOeOOHmouOo OH. OO O OH. ON O OH. ON O muOooOOEOH NH. OO O NH. ON O ON. ON O OmOOmouuummOm NH. ON O OH. ON O ON. ON O .mHhIIIOMIIMIIIMHhIIIOMII.ONIIIMHhIIIOMIINI ON. OO O OH. ON O oN. OO O NH. OO N OH. ON O ON. OH O OH. ON N ON. OO O OH. ON N OH. OO O NH. OO O ON. ON O NH. OO O OH. OO O ON. ON O Na: N: O H NSO N: O O Na; N: O m _ m _ N.O mcHummnm Hmumm mama .m HmEOnm mo mummnmoHHm cH mcHHman OHEmMHmouho .ON mHnma 107 Lfiaéwu .0 r. - 13E: . . OO.A OO.A O wocmumMMOO on mocmnmmmHO on onm> u mo. mo. OO. coOuMO>mO pumpcmum OH. . OH. OH. muHmcmO cam: OH.. ON O OH. ON O NH. OO O ON. ON O OH. OH H OH. OO O OH. OH N OH. OH N OH. ON O NH. om m ON. OH N NH. ON m mHhIIIOHIIMiIIMHhIIIOMIIMIIINuH...IIIOMIIMI OH. ON O NH. ON m ON. OH N OH. om O OH. OO O ON. ON O ON. ON O NH. OO O OH. on O OH. ON O OH. ON O OH. OO O OflunufiuIN:lumfluuufiunmnuumflauufiunmn mfiao OH. OO N ON. ON O ON. ON O UOEOMHmoumo NH. OO O H OH. OO O OH. OO O HHmo oHHOnmoOOO OH. om O OH. om O NH. om O OH. OH N OH. OO N NH. ON m OH:unfinnmnunmfluuufinuOuu:OflulufillO: OH. ON N ON. ON O OH. ON O OH. on O OH. ON O ON. OH N NH. ON O OH. OO O OH. ON O OH. OH N OH. ON O OH. ON O OH. ON O OH. ON m . oH. on m NQVO N1 O _ NnVO N1 O _ N:\O Ni O O O m _ _ N.O chummum Hmumm mama .m HmEOnm mo mummumousm cH mnOHmQMH oOEmMHmouho .ON mHQMB ~W V'f‘lumhw Hi Q . I ‘l' . APPENDIX D Appendix D. Distribution of isotope labels. In animals given only H3T, the majority of silver grains were located over nuclei. whereas in those animals given only H3L most of the grains localized over cytoplasm. Random.2500‘p2 areas of graft tissue, graft beds, or normal dermis were analyzed and the grains located over cytoplasm and over nuclei were tabulated separately and are listed in this appendix. Abbreviations used are as follows: nuc.=nucleus cyt.=cytoplasm #=number of silver grains ‘n2=area in square micra %=percent of label 108 109 OO OH ON NN ON NN ON NN - OO ON . OO OHO ON O OO NH _ OO NH NO OH OO NH NO O NO ON OO ON OO OH OO ON OO NH OO NHO NHH NH OO OH ON ON .OO NO OO HH OO OH .uh0 .05: .ymo .05: .um0 .05: .uh0 .05: . .u>0 .05: .uw0 .05: O O N.O O O N.O m:Hummum Hmuwm mmwp m:HpmmHO Hmumm mama OH HOEH:4 NO OH NO OH . OO OH OO ON - OO NH . OO ONO OO ON OO ON OO OH NN OH OO NH OO NH NN OH ON OH ON O ON ON OO OH OO O O OO OH OO OH OO OH OO HN HO NH OO OH .umo .05: .um0 .05: . .u>0 .05: .umo .05: .pao .05: .uh0 .05: OH O H.O OH O H.O m:HumOHO :mumm mhmw O:Hummnm Hmumm mmmo O HOEH:¢ mummnmou5< mummuOOHHd aoHuanuumHu HmOMH waHosmq .ON mHnms OH. . OH. OH. - _ NH. HH. OH. NOVO 110 OO O OO O OO O ON O . OO O OO O ON O OO O OO O OO O OO O OO O OO O OO O OO O ON O OO O OOH OH OO O OO O OO O OO O ON O OO N ON O ON O OO O ON O ON O OO N NR. O Ni O N1 O Na O NQ O N: O H0H05z .OH. OH. OH. OH. OH. mm. OOOO ON O ON O OO N OO O OH N OH O OO O ON O OO N ON O ON O OO OH OO O OO O OO N ON O ON O ON O OO O ON O OO O ON O ON O OO O ON O ON O OO O ON O ON O ON O OO N OH N OO O OO N ON O ON O OO O ON O OO O ON O OO N ON O ON O OO N ON NH ON O ON O OH O ON O ON O ON O ON O ON O OH O OO O ON O ON O OO O OH O ON O NR O NR O N1 O N1 O N: O N: O EOMHmoumU OH O H.O OH O H.O m:wummum Hmumm mama O:Oummum nouns mama mummumou5¢ . mummumoHH¢ .A0:H050HV O HOEH:m :O m0OUHm:0U :Hmum OHEmmHmoumu ©:m Hm0H05z .OO 0Hnme lll HN ON OH NO ON ON HN ON O O OO ON OO OH .NO OH OO O ON NH OO ON OO OH HO OH OO NH OO OH NO OH OO O O ON OH NO OH OO NN ON OH OO HH OO O OO NH ON mummumou5¢ HN ON OH HO ON ON ON NN O OH OO N OO OH NO ON NN NH OO HH OO OH ON ON NO O ON N OO NN OO NH OO O OH OO O ON HN OO OH OO OH NO O OO ON OO OH OO .pmu .05: .umo .05: .uw0 .05: .uM0 .05: muumuOoHHm OH O O:Hummnm Hmumm mama .ON Hmefigmv aoflusnfluumflu Hman quOOEOaa .Hm mHnma 11Ii I|‘ ,‘ ‘r "I "I n H R H "I “ Ill" 42853 031