Celli, S

Demetris, AJ

Fung, JJ

Kelly, RH

Pan, F

Rao, AS

Starzl, TE

Sun, H

Tsugita, M

Valdivia, LA

D-Scholarship@Pitt

HEART AND LUNG XENOGRAFTS Mechanisms of Protection From Humoral Rejection by a Xenografted Liver S. Celli, L.A. Valdivia, R.H. Kelly, H. Sun, M. Tsugita, F. Pan, A.S. Rao, A.J. Demetris, J.J. Fung, and T.E. Starzl WE PREVIOUSLY reported the immunological pro-tection from humoral rejection afforded to a ham-ster heart that was heterotopically placed into a rat 2 months after hamster liver transplantation. Furthermore. this protection was so robust that injection of specific hyperimmune serum was unable to reverse this process. We attributed these observations to a switch of the recipient's complement (C) to that of the donor type. shielding the subsequently transplanted heart from the deleterious effects of antibody- and complement-mediated injury. fK~ In the present study. we assessed whether this protective effect would also manifest itself in the early postliver transplant period. when the titer of xenospecific antibodies is ex-tremely high in the recipient and the transformation of the C from the recipient to that of the donor type is underway. MATERIALS AND METHODS Animals Syrian Golden hamsters (100 to 120 g) and Lewis rats (250 to 270 g) were used as donors and recipients. respectively. Transplants Liver was placed orthotopically according to the cutI technique' Heart was transplanted heterotopically into the abdominal cavill on day 1 after liver grafting. Immunosuppression was with FK 50h (1 mg/kg). which was given daily intramuscularly (1M) for 30 days. The experimental design is described in Table 1. Complement-Dependent Cytotoxicity Assay Cytotoxic antibody titers in the recipients were determined using hamster lymphocytes as targets and baby rabbit serum as the source of C. From the Pittsburgh Transplantation Institute, University ('. Pittsburgh Health Science Center, Pittsburgh, Pennsylvania. Address reprint requests to T.E. Starzl. MD. PhD. Departmen' of Surgery. University of Pittsburgh, 3601 Fifth Ave, Falk CliniC 5C, Pittsburgh, PA 15213. © 1995 by Appleton & Lange 0041-1345/95/$3.00/ + 0 Table 1. Survival of Hamster Hearts Transplanted Into Lewis Rats on Day 1 After Receiving a Rat or a Hamster Liver Xenograft OlTX OlTX (Treatment) (Survival Days) 1. None 2. Lew ..... Lew >100 x 5 (none) 3. Lew ..... Lew >100 x 5 (FK 506") 4. Ham ..... lew 7x5 (none) 5. Ham ..... Lew 16.34. 51, 51 (FK 506") 'FK 506 (1 mglkgld) was given daily 1M for 30 days. "Mean survival time (days). OL T = orthotopIC liver transplantatIOn: HTX = hean transplantation. 300 HTX MST"" (Survival Days) MST'" 3x5 3.0 >100 3.3.3.3.4 3.2:: 0.4 >100 3x5 3.0 7 6x5 6.0 38.2 :!: 16.6 15,33.50.50 37.2:: 16.6 Transplantation Proceedings, Vol 27, No 1 (February). 1995: pp 300-302 rs I "lIne. 11\.'\.,' '11 i Ie It • G PROTECTION FROM HUMORAL REJECTION Radial Immunodiffusion Assay Goat anti rat C3 was used for the quantitatlon of Cl in the serum of liver reclplcnb. This polvclonal antibody crossrcacts with hamster 0. EJectrophoresls and Immunofixation These techniques were used to follow temporallv the transtorma-tion of thc recipient C to that of the donor type. Plasma sample, from liver xcnogratt recIpients on postopcratlvc days I. 2. and .' were tested bv a method described elsewhere.' RESULTS As depicted in Table 1. there was no difference in the lurvival of hamster hearts transplanted into Lewis rats in JrOups 1. 2. and 3. indicating that a syngeneic liver trans-plantation with or without FK 506 treatment does not Influence the rejection of subsequentlv transplanted ham-Iter hearts. On the contrary. the survival of hamster hearts was signiticantly prolonged in groups ~ and 5. in which the rats had previouslv received a hamster liver: all recipients. however. died with a beating heart graft. Furthermore. it is Interesting to note that the recipients in group 4 died due to aevere hepatic reJection with minimal histopathological changes in the secondary cardiac xenotransplants. The gel electrophoresis of plasma obtained after liver lenotransplantation showed the coexistence of recipient and donor C3 on days I and 2. with almost a complete IWitchover to donor-type C3 hy day 3 after transplantation (Fig I). Figure 2 shows the levels of C3 in the serum of liver recipients. On the first postoperative day. the level of circulating ('3 in the serum was SWr of that found in DOnnal rats: however. by day 5 after transplantation. it had reverted hack to the baseline. The determination of antibody titers in the rat recipients 01 either hamster liver alone or combined liver and heart transplants showed llO statisticallv significant difference between the two groups. with the p~ak titer of the antibod-... being observed at the end of the first postoperative week . (data not shown). ''1¥ . : DISCUSSION ::~:; We had previously demonstrated that the almost complete .. Il~; replacement of recipient C with that of the donor type after ·iDIDDDy~·lher transplantation plays an important role in the preven-tion of humoral rejection of secondarilv transplanted hearts . ···.Iracn the same donor species but not from others.2 Further-1IOrc. in the present experIments we have shown that after ··lher transplantation. 72 hours were required for a complete rsion of (. III donor type. The eventual loss of g rat C might he attributed to the orthotopic ",Mac:cnlent of Lewis liver with that of hamster. thus the maIn source of C synthesis. and to the of activated C by liver grafts. leading to precipi-depletion of recipient type C. Although on postoper-day 3 the levels of circulating ('3 were markedly 301 DONOR-RECIPIENT SWITCH OF C3 oltx day 1 oltx day 2 -4. oltx day 3 HAM +C3 Fig 1. Gel electrophoresIs test of sera obtained from rats on days 1. 2. and 3 after hamster liver transplantation. This Indi-cates that by day 3. C had SWitched to that of the donor type. HAM: hamster. reduced in recipients of hamster liver. \\e were ahle to detect their presence on the vasculature 01 thl.: liver and heart xenografts. Taken together. these ohservations sug-gest that the apparent inability of a Le\\ i, rat rl.:cipient of a 100 I -- -90 jl ..... ~ - ---~ M 80 \ I U '=" 70 C E ::::l 60 '-' ... v ~ 50 \ /1 \ _A \/f' ... -40 30 -o 48 Post-transplant days Fig 2. Amount of CirculatIng C3 (radial Immunodiffusion) In liver reCIpients after transplantation. The value obtained from normal rat sera was conSidered 100%. 302 hamster liver to mount a humoral rejection of a subse-quentlv transplanted hamster heart hinges on the presence of C of the xenodonor type. This underscores their inherent deficiency to form an effective membrane attack complex due to the presence of species-specific regulators of C activation on the endothelium of the xenotransplanted hamster tissue.' Furthermore. this study has unequivocally established that a xenografted hamster liver protects a subsequently transplanted hamster heart during the first week after liver placement. when the antibody titer in the recipient is extremely high. CELLI, VALDIVIA. KELLY ET AL REFERENCES I. Valdivia LA. Demetris AI. Fung JJ. et al: Transplantation 55:659. 1993 2. Valdivia LA. Fung J1. Demelris AI. et al: Transplantation 57:918. 1994 3. Valdivia LA. Monden M. GOloh M. et al: Transplantallol' 44:759. 1987 4. Celli S. Valdivia LA. Kelly RH. et al: Xenotransplantation (Ill press) 5. Zhao J. Rollins SA. Maher SE. et al: J Bioi Chern 266: 1341 H. 1991 P A tv' a' ] h a n (1 a {' c 

Mechanisms of protection from humoral rejection by a xenografted liver

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Mechanisms of protection from humoral rejection by a xenografted liver

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