Demetris, AJ

Duquesnoy, RJ

Fu, F

Fung, JJ

Li, Y

Qian, S

Starzl, TE

Sun, H

Thai, NL

English

PubMed

D-Scholarship@Pitt

\'1 SO Mouse Liver Transplantation Tolerance: The Role of Hepatocytes and Nonparenchymal Cells N.L. Thai. S. Qian. F. Fu. Y. Li. H. Sun. A.J. Demetris, R.J. Duquesnoy, T.E. Starzl, and J.J. Fung MOUSE orthotopic liver allografts are spontaneously accepted without immunosuppression in many strain combinations including those crossing major and minor histoincompatibility barriers. Liver allograft accep-tance, moreover. leads to donor-specific acceptance of subsequent skin or heart allografts. Although the precise mechanisms of liver graft tolerance (LGT) are not com-pletely understood. LGT has been attributed to: (1) donor nonparenchymal cell (NPC) migration leading to subse-quent recipient/donor hematolymphoid chimerism lK~; and (2) hepatocyte (HC) release of soluble MHC molecules.3 We have used an experimental model. similar in principle to that of Rapaport et aV and used previously by Sriwa-tanawongsa et al5 to test whether the NPC or HC was the more important component in LGT by constructing chi-meric donor livers whose NPC have been replaced by allogeneic bone marrow transplantation (BMT). MATERIALS AND METHODS liver donor mice (H-2") were supralethally irradiated (9.5 Gy) and reconstituted via tail-vein injection with 1.5 x 10" syngeneic (H_2h), or fully allogeneic liver recipient type (H_2 k ). and third party (H-2") whole bone marrow cells harvested from mouse femur and tibia. Ninety days (90) days after BMT. splenocytes and peripheral blood mononuclear cells (PBMC) were phenotyped for recipient/donor cells by ftow cytometry, and liver by immunohistochemistry. A mixed leukocyte reaction was also done to assess immune status in chimeric liver donor animals. Ninety-day-old chimeric donor liver I1'Ifts were transplanted according to techniques by Oian et al2 into untreated naive H-2' recipients and challenged with H_2h and H_2d akin grafts 30 days after transplantation (Table I) or on the same day as liver transplantation (Table 2). Two combinations of mouse Itrains were analyzed: (I) The C57BUIO (H-2"), BALB/c (H_2d), and C3H (H_2k) combination, which crosses all major and minor bistoincompatibility barriers; and (2) the congenic C57BUlO, 810.D2 (H-2d ), and BIO.BR (H_2k), which cross all major loci. RESULTS AND DISCUSSION The donor preparation resulted in >99% replacement of PBMC and splenocytes with cells of bone marrow donor origin 90 days after BMT as determined by flow cytometry. Liver phenotyping by immunohistochemical staining with class I and II specific antibodies showed hepatocytes and bile duct cells to be of bone marrow recipient haplotype. while NPC to be >99% of bone marrow donor haplotype. However. rare residual NPC from the original liver per-sisted. At 90 days. chimeric animals also showed specific unresponsiveness to bone marrow donor and recipient haplotypes but not to third party as determined by the mixed leukocyte culture. suggesting immunological donor-recipient stability in these chimeras. The transplantation of chimeric donor livers with either NPC of donor (H-2"). third-party (H-2"). or liver recipient (H-2k) haplotype resulted in spontaneous acceptance of liver grafts in either of the combinations of mouse strains. However. the donor-specific tolerance normally induced for C57BUlO skin by the unaltered C57BUIO liver in this model (see Qian et all and group I of Table 1) was no longer evident when the donor livers were taken from irradiated animals that had been reconstituted with synge-neic (C57BUIO) bone marrow. This suggested that the liver NPC (or some other factor in the organ) had been changed in the first stages of these control experiments. The reduced skin graft protection was now no different than when the donor NPC were converted to those of the recipient (group 4) or to a third party (group 3). Although the skin survival From the Pittsburgh Transplantation Institute, University of Pittsburgh Medical Center. Pittsburgh, Pennsylvania. Address reprint requests to Ngoc L. Thai, E 1556 Biomedical Science Tower, Terrace and Lothrop Streets, Pittsburgh, PA 15213. © 1995 by Appleton & Lange 0041-1345/95/$3.00/ + 0 Table 1. Tolerance Induction of Donor Skin by Chimeric Livers in Mouse Liver Transplantation in C57BLl10, BALB/c. C3H Combinations Donor liver Skin Graft Survival (Days)" Group NPC HC Recipient C57BU10' BALB/c' 1 Normal C57BU10 Normal C57BU10 C3H 71,80. >100. >100. >100 16, 17. 17,23 2 C57BU10 C57BU10 C3H 29, 35, 44, 47, 86 15,17,19,20 3 BALB/c C57BU10 C3H 44, 45, 46. 48 16, 17,20 4 C3H C57BU10 C3H 34, 50, 53. 56 12.12. 14. 14 "lIoIated skin graft survival In the unaltered C3H recipient IS 9to 10 days uSing C57BU10 donor. 9 to 10 days With BALB/c. and permanent With C3H. 'Skin grafts 30 days after liver transplantation. 509 Iqff~l/anltatiEm Proceedings, Vol 27. No 1 (February), 1995: pp 509-510 510 THAI. alAN. FU ET AL Table 2. Tolerance Induction of Donor Skin by Chimeric Livera In Mouse Uver TranS!)lantation in C57BLJ10, B10.BR B10.BR, B10.D2 Combinations Donor liver Skin Graft Survival (Days)' Group NPC HC Recipient C57BU10' B10.D2' 5 Normal C57BU10 Normal C57BU10 B10.BR >100. >100. >100 6 C57BU10 C57BU10 B10.SR >100. >100. >100 28. 30. 16 7 B10.02 C57BU10 S10.SR 30.20. >100. >100. >100 28.19.21.20.21 8 S10.SR C57BU10 S10.SR 24.25. >100. >100 22.21.17.17.20 'Isolated skin graft survival on the una~ered B10.BR recipient IS 12 to 14 days uSing C57BU10 donor. 12 to 14 days with BALBIc. and permanent with C3H. 'Skin grafts placed on the same day as liver transplantation. was indistinguishable. it was modestly prolonged in all compared to that of an isolated skin graft in the same strain combinations (see footnote to Table I). Similarly, the animals bearing transplanted "parked" livers rejected BALB/c skin without regard for the strain used to repopu-late the hepatic graft donor, but at a pace of rejection that again was marginally slower than that expected in these strains after a conventional isolated skin graft (see footnote to Table I). The MHC nonspecificity of these results is self-evident. The possibility that a procedural timing factor was respon-sible prompted further experiments using fully allogeneic C57BUlO ..... BlO.BR mice. With this strain combination, transplantation of an unmodified liver confers specific tolerance to concomitantly transplanted donor skin (see Qian et at2 and group I of Table I). Unlike the controls in Table I, donor-irradiated and syngeneic reconstitution did not cause loss of specific tolerance to the concomitantly engrafted skin grafts (group 2, Table I). This reaction increased the importance of the experiments in group 3 in which the C57BUlO liver donor was reconstituted with third-party NPC (BIO.D2) and group 4 in which the recon-stitution was with recipient leukocytes (BIO.BR). Skin acceptance was expected in group 4 but not group 3 if hepatocytes were potently tolerogenic. However, 40% of the skin grafts in both groups were rejected in 20 to 30 days (Table 2). In addition, C57BUIO livers reconstituted with 81O.D2 NPC did not prevent rejection of BI0.D2 skin suggesting that hepatic tolerogenicity for skin could not be attributed to NPC alone under these experimental circum-stances. These results are largely confirmatory of recent observa-tions by Caine et a15 •H in rats, differing only in that the hepatocyte effect appears to us MHC nonspecific. In com-mon with the findings of the Cambridge team, there was a dissociation of liver graft acceptance from tolerance to skin, analogous perhaps to the survival of NPC-free islets in-jected into the mouse thymus but without the systemic donor-specific tolerance that is produced by unmodified islets. As discussed before. there is room for speculation about parenchymal-non parenchymal cell collaboration in the events of tolerance or other immune eventsKRKeK~ indud-ing the cross-regulation of immunologic and growth factor networks. 10 As we have discussed earlier. I I the "parking" experimen-tal models may not be appropriate for such inquiries. The rationale for their use to investigate tolerance mechanisms has faded in the face of mounting evidence that transplan-tation tolerance is the product of a two-way immunologic transaction (graft versus host as well as host versus graft). When an organ has been parked and is stable in the.: intermediary host. both graft and recipients have exchanged NPC and have genetic composites.I.2.b.7.9.11 The organ already is tolerant to the carrier animal and vice versa. with a mixed NPC population in both that presumably has manifold changes (particularly in gene expression). Only at this time is the organ extirpated from the immunologic network into which its mini-immune system has been assimilated. Because we believe that these changes define per se the molecular secrets of tolerance, it is not surprising that the use of such an organ to study tolerance de novo in a new recipient has yielded confusing and perhaps mislead-ing information. REFERENCES 1. Starzl TE. Demetris AJ. Murase N. et al: Lancet 339:1579. 1992 2. Qian S. Fung JJ. Demetris AJ. cl al: Hcpatology 19:916, 1994 3. Kamada N. Goto S. Kobayashi E. Cl al: Transplant Proc 25:2857. 1993 4. Rapaport FT. Bachvaroff RJ. Akiyama N, et al: Transplant Proc 12:476, 1980 5. Sriwatanawongsa Y, Davies HffS. Brons IGM. et al: Trans-plant Proc 25:2865, 1993 6. Starzl TE, Demetris AJ, Murase N, et al: lmmunol Todav 14:326, 1993 . 7. Demetris AJ. Murase N. Rao AS. el al: In Touraine GL (ed): Rejection and Tolerance. Dordrecht. Kluwer. 1994. p 325 8. Caine DB. Caine R: Lancet 340:244. 1992 9. Demetris AJ. Murase N. Fujisaki S, ct al: Transplant Proc 25 :3337. 1993 Ill. Francavilla A. Haglya M. Porter KA. ct al: Hepatology 20:747. 1994 11. Starzl TE. Dcmetris AJ: Immunol Today (in press). it 1. ~DK 

Mouse liver transplantation tolerance: The role of hepatocytes and nonparenchymal cells

Qian, TS

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Mouse liver transplantation tolerance: The role of hepatocytes and nonparenchymal cells

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