SHORT ARTICLES

SEQUENTIAL RAT-TO-MOUSE SKIN XENOGRAFTS

MICHAEL RAY, ANTHONY NICASTRI AND ELI A. FIUEDMAN The Departments of Medicine andPathology of the State University of New York,

Downstate Medical Center, Brooklyn, New York

MAMMALIAN xenograft rejection varies in intensity and histology depending on the com- bination of donor and recipient species. Mice respond to rat skin grafts with early and inconstant cellular infiltration and vascularisation prior to rapid destruction of the graft epidermis (Ben-Hur, Solowey and Rapaport, 1969). The relative contribution to xenograft rejection of preformed immune globulins and " new " antibodies resulting from graft sensi- tisation is unclear. In the present study, repetitive rat-to-mouse skin xenografting did not shorten the survival of subsequent xenografts. When mice were challenged at the same time with first-set rat skin xenografts and mouse skin allografts, a significant delay in allo- graft rejection was induced.

MATERIALS AND METHODS

Male adult A/J (Jackson Memorial Laboratories) mice were used as recipients of male adult Sprague-Dawley rat (Marland Farms) skin xenografts and male adult CBA/J mouse (Jackson Memorial Laboratories) skin allografts. For both rat and mouse donors circular grafts of back skin measuring approximately 1.3 cm were removed under ether anaesthesia and placed orthotopically on defects of the same size in recipients. Each graft was sewn to the graft bed and surrounding host skin with interrupted 6.0 silk sutures. Grafts were inspected daily for macroscopic signs of technical failure or rejection. A graft was considered rejected when 50 per cent. of its surface area was escharified with gross opacification and discoloration. Each graft was scored daily by two observers. Biopsies of three grafts in each group were obtained daily beginning on the sixth post-operative day for allografts and on the third post-operative day for xenografts. A search for preformed mouse anti-rat antibodies was performed on fresh I-pm frozen sections of Sprague-Dawley rat back skin. The sections were coated successively with freshly prepared A/J mouse serum and fluorescein- labelled anti-mouse gamma-globulin antiserum and examined with a Leitz fluorescence microscope. A/J mouse recipients were grafted as follows.

Group I . Control allografts. First-set skin allografts from CBA/J donors were observed in 19 animals and followed in 10 of 19 mice by second-set grafts five days after rejection of the initial graft.

First- and second-set rat skin grafts were studied in 20 mice, and ten of these received third-set xenografts five days after rejection of the second xenografts .

Group IZI. First-set allografts and simultaneous first-set xenografts were studied in 17 reapients.

Group ZV. Second-set allografts and first-set xeitografs. Following rejection of first-set grafts, 9 of the mice in Group I were grafted with simultaneous second-set allografts and first-set xenografts.

RESULTS

Group II. Sequential xenografts.

Control allografts. As shown in the table first-set allografts were rejected in 9.9f1.2 days and second-set grafts were rejected in 7-3kO.2 days. No first-set graft was rejected

Received 13 Jan. 1971 ; accepted 6 June 1972. J. PATH.-VOL. 109 (1973) 259

260

before the eighth day, whilst no second-set graft was viable beyond 8 days. Histological manifestation of allograft rejection consisted of lymphoid cellular infiltration of graft dermis and epidermis prior to the development of necrosis.

Sequential xenografts. There was no difference in gross appearance of first-, second-, or third-set xenografts, which lasted 7.3, 7.4 and 6-7 days respectively (table). Biopsies from each of the three sets of xenografts were remarkably similar, and all showed complete

TABLE Survival of skin rat-to-mouse xenografts and mouse allografts

MICHAEL RAY, ANTHONY NICASTRI AND ELI A . FRIEDMAN

9.91-1.2

7.3k0.2

Group

I 8-12

7- 8

I1

I11

w

Grafts

First-set

Second-set

allografts

allografts

First-set

Second-set

Third-set

xenografts

xenografts

xenografts

First-set

first-set

allografts

xenografts

and

Second-set allografts

and first-set

xenografts

Number of animals

19

10

20

20

10

17

17

9

9

Graft survival time (days)

for individual grafts

8 ,8 ,9 ,9 ,9 ,9 ,9 , 10,10,10, 10,10,10,10,11,11,11,12, 12 7,7,7,7, 7, 7, 7, 8, 8, 8

9,10,10,10,10,10,10,11, 11,12,12,12,14,14,15,16, 16

7, 7, 7, 7, 7, 7, 8, 8, 8

6, 7, 7, 7, 7, 7, 7, 8, 8

7.31-0.9

7 . 9 1 0 9

6.7*0.7

11.912.1

7.61-0.6

6- 9

6- 9

6- 8

9-1 6

6- 8

7-3+06

7.1 1 0 . 6

7- 8

6- 8

infarct-like necrosis by the third post-grafting day. Lymphoid cell intiltration, though prominent in the granulation-tissue bed, did not invade the graft itself. Sinusoidal vessels from the graft bed showed some growth into the graft. The slight vascularity and the presence of well preserved erythrocytes in capillaries well within the graft were well correlated with the gross appearance of " viability " beyond the third day. Nuclear pyknosis in the epidermis and its appendages was followed by progressive autolysis, desquamation of epi- dermis, apparent condensation of dermal collagen, and sometimes superinfection accom- panied by exudation of polymorphonuclear leucocytes.

Allografts and xenografts. A small but significant delay (Pc0.02) in the rejection of a first-set allograft to ll.9f2.1 days was produced by the simultaneous grafting of a first-set xenograft (table). None of the control first-set allografts survived more than 12 days, whilst 5 of 17 allografts placed alongside first-set xenografts were rejected from the 13th to the 16th day. Xenograft rejection in this group was not retarded. No delay in rejection of second- set allografts was produced by simultaneous grafting with primary xenografts.

Imrnunofluorescence tests. Neither nuclear nor cytoplasmic coating of rat skin tissue

SEQUENTIAL RAT-TO-MOUSE SKIN XENOGRAFTS 261

with normal A/J mouse serum was found, nor was specific fluorescence of blood vessels, basement-membrane or other intercellular substances detected.

D~scussroiv The timing and character of allograft rejection observed in the primary and secondary

CBA/J to A/J grafts used in this study are consistent with those in other allograft systems including Medawar’s (1944) original observations in the rabbit. The appearance of xeno- grafts during rejection in the present study confirms the description reported by Ben-Hur et al. (1969). Though gross changes were well correlated with microscopic events in allografts undergoing rejection, the poor and inconstant vascularisation of xenografts made it difficult to assess the value of gross signs of early rejection such as cyanosis, oedema, and failure to blanch on pressure, all of which are dependent on loss of previously established blood flow.

Inability to detect by gross examination any acceleration of rejection of second- and third- set rat-to-mouse xenografts probably reflected the short period of viability of first-set grafts ; by the third post-grafting day, the primary skin xenograft was necrotic with only poor blood supply, which was gradually lost over the next 3 4 days. The detection of any acceleration of rejection due to heightened immunity may require more sensitive techniques than gross and histological examination of grafts.

That rejection of xenografts in some systems is due, at least in part, to immunological mechanisms, may be inferred from the protracted graft survival facilitated by immuno- suppressive agents such as antilymphocyte serum in mice (Monaco et al., 1966) and azathio- prine and prednisone for patients given chimpanzee and baboon kidney grafts (Pearce et al., 1964; Starzl et al., 1964). The rapidity of rejection in the rat-to-mouse system suggests a possible contribution of preformed antibodies to graft destruction, but no such antibodies were found by the indirect immunofluorescence method employed in this study.

The limited vascularity of skin xenografts has precluded the demonstration of host sensitisation when the skin is used as the “ indicator ” graft. Thus, Perper and Najarian (1966) noted that in a sheep-goat system, sensitisation with a skin xenograft shortened the viability of a “ second-set ” kidney xenograft to 3 days from a control first-set rejection time of 14 days. The reverse experiment, in which a kidney was the sensitising xenograft and skin the “ second-set ” graft, showed no shortening (6 days), as compared with control first-set grafts (7 days). Even though no vascular communication is formed with a skin xenograft, as in the rat-to-rabbit system studied by Krakower and Greenspon (1967), host sensitisation may occur as evidenced by the development of rabbit anti-rat antibodies.

Another type of immunological effect of a skin xenograft may be shown when it acts as a competing antigen to a skin allograft, as in the present study in which primary allograft rejection was delayed by simultaneous xenografting from 9.9 i 1.2 days to 11 -9 f2-1 days. Adler (1957) found the guinea-pig’s antibody response to rabbit globulin reduced by haemo- cyanin administered as a competing antigen. Terino, Miller and Glee (1964) produced toler- ance to mouse allografts by injecting five different bacterial strains at the time of grafting. The failure of a competing xenograft to delay rejeclion of a second-set allograft is consistent with the relative resistance of established allograft immunity to suppression as compared with initial sensitisation.

SUMMARY Male adult A/J strain mice were given full-thickness first-, second-, and third-set rat

skin grafts. No difference between the survival of primary skin xenografts (7.3f0.9 days) and either second-set (7.9 1 0 . 9 days) or third-set (6.7 f0.7 days) xenografts was found. Biopsies of xenografts showed poor vascularisation and minimal cellular infiltration when compared with mouse skin allografts of the same age. It is suggested that the limited vascular supply of skin xenografts prevents the expression in this organ of “ accelerated ” rejection.

Initial rat-to-mouse xenografts, when placed at the same time as first-set mouse allo- grafts, retarded allograft rejection to ll .91t2.1 days, as compared with 9.9f1-2 days for

262

control grafts. The delay in first-set allograft rejection, induced by the synchronous grafting of skin xenografts, was interpreted as an effect of competing antigens.

This work was supported in part by a grant from the Kidney Foundation of New York.

MICHAEL RAY, ANTHONY NICASTRI AND ELI A. FRIEDMAN

REFERENCES ADLER, F. L. 1957. Antibody formation after injecting heterologous immune globulin 11.

Competition of antigens. J. Immun., 78, 201. BEN-HUR, N., SOLOWY, A. C., AND RAPAPORT, F. T. 1969. The xenograft rejection

phenomenon. I. Response of the mouse to rabbit, guinea pig and rat skin xenografts. Israel J. Med. Sci., 5, 1.

KRAKOWER, C. A,, AND GREENSPON, S. A. 1967. Skin reactive heteroantibodies produced by orthotopic skin xenografts. Transplantation, 5, 207.

MEDAWAR, P. B. 1944. A second study of the behavior and fate of skin homografts in rabbits. J. Anat., 78, 176.

MONACO, A. P., WOOD, M. L., GRAY, J. G., AND RUSSELL, P. S. 1966. Studies on hetero- logous antilymphocyte serum in mice. 11. Effect on the immune response. J. Immun., 96, 229.

PEARCE, C. W., DEWITT, C. W., SMITH, P. E., HEWITT, R. L., FLINNER, R. L., AND CREECH, O., JR 1964. Renal heterotransplantation in man. Ann. Sravg., 160, 384.

PERPER, R. J., AND NAJARIAN, J. S . 1966. Experimental renal heterotransplantation 11. Closely related species. Transplantation, 4, 700.

STARZL, T. E., MARCHIONO, T. L., PETERS, C. N., KIRKPATRICK, C. N., PORTER, K. A., RIFKIND, D., OGDEN, D. A., HITCHCOCK, c. A., AND WADDELL, w. R. 1964. Renal heterotransplantation from baboon to man : experience with six cases. Transplantation, 2, 752.

TERINO, E. O., MILLER, J., AND GLEE, W. W. L. 1964. Tolerance induction and skin graft prolongation by competing antigens. Surgery, St Louis, 56, 256.