Passive transfer of alloantibodies restores acute cardiac rejection in IgKO mice.

BACKGROUND: Alloantibody is an intrinsic component of the immune response to organ transplants. Although alloantibodies have been correlated with decreased graft survival, the mechanisms of alloantibody-mediated injury remain largely undefined in vivo. In the present study, we have established a model of alloantibody-mediated graft injury using B10.A (H-2a) hearts transplanted to wild type (WT) or immunoglobulin knock out (IgKO) C57BL-Igh-6 (H-2b) mice. METHODS: Alloantibodies were measured in the circulation and graft by flow cytometry and in immunofluorescence staining, respectively. Intragraft cytokine mRNA expression was evaluated using a competitive template reverse transcriptase polymerase chain reaction (RT-PCR) technique. P-selectin and von Willebrand factor expression were localized by immunoperoxidase staining. The capacity of alloantibodies to restore acute cardiac allograft rejection was tested by passive transfer of monoclonal antibodies (mAbs) against donor major histocompatibility complex (MHC) class I antigens to IgKO recipients. RESULTS: B10.A cardiac allografts are rejected acutely by WT C57BL/6 recipients, but over 50% of the cardiac allografts survived more than 50 days after transplantation in IgKO mice. Competitive template RT-PCR on the cardiac transplants demonstrated similar levels of IL-1-alpha, IL-12 (p40), TNF-alpha, IL-2, IFN-gamma, IL-4, and IL-10 mRNA in WT and IgKO recipients 8-10 days after transplantation, indicating that macrophage- and T-cell-dependent immune responses were intact in IgKO recipients. The rejection of B10.A hearts in WT recipients was characterized by interstitial and perivascular cellular infiltration; IgG, IgM, and complement (C3) deposition; vascular cell injury and intravascular platelet aggregation; and release of von Willebrand factor and P-selectin. In IgKO recipients the lower degree of vascular injury in the absence of alloantibody responses was reflected by the lack of release of von Willebrand factor and P-selectin, which remained confined to cytoplasmic storage granules of endothelial cells and platelets. Acute rejection of cardiac allografts was restored to IgKO recipients by passive transfer of proinflammatory IgG2b mAbs against donor MHC; recipients injected with isotype-matched control mAbs did not reject. In contrast, passive transfer of IgG1 mAbs against donor MHC failed to restore acute rejection of cardiac allografts to IgKO recipients. Passive transfer of IgG2b, but not IgG1 mAbs was associated with endothelial cell activation and plate. let aggregation together with the release of preformed von Willebrand factor and P-selectin from storage granules. CONCLUSIONS: Acute rejection of cardiac allografts can be reconstituted in IgKO recipients by passive transfer of IgG2b, but not IgG1 antibody. This model allows the mechanism of alloantibody-mediate graft injury to be dissected in vivo.

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.