Louis Horwitz

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.AaBu) cardiac allografts by PVG.1U (RT1.AuBu) 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.

Promiscuous recognition of major histocompatibility complex class II determinants in cyclosporine-induced syngeneic graft-versus-host disease: Specificity of cytolytic effector T cells

BACKGROUND Administration of the immunosuppressive drug cyclosporine after syngeneic/autologous bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous or syngeneic GVHD, is associated with the development of a highly restricted repertoire of cytolytic T lymphocytes that promiscuously recognizes major histocompatibility complex class II determinants, including self. METHODS Vbeta8.5+CD8+ effector lymphocytes and T-cell clones were isolated from Lewis rats with cylosporine-induced syngeneic GVHD. The specificity of the effector T cells and T-cell clones was examined in vitro. The pathogenicity of the T-cell clones was confirmed in vivo using a local graft-versus-host reaction assay. RESULTS Clonal analysis reveals that the pathogenic effector T cells recognize a peptide from the invariant chain termed CLIP in association with major histocompatibility complex class II determinants. Moreover, there appears to be an additional interaction between the N-terminal flanking region of CLIP and the Vbeta segment of the T cell receptor. CONCLUSION The results suggest that recognition of this highly conserved peptide along with the additional interaction between the flanking region and the T cell receptor may account for the promiscuous activity of the autologous/syngeneic GVHD autoreactive T cells.

Complexity of effector mechanisms in cyclosporine-induced syngeneic graft-versus-host disease.

Administration of the immunosuppressive drug cyclosporine after syngeneic or autologous bone marrow transplantation elicits a T-lymphocyte-dependent autoimmune syndrome similar to graft-versus-host disease (GVHD). The onset of this autoaggression syndrome, termed syngeneic GVHD, is associated with the development of a highly restricted repertoire of CD8+ autoreactive T cells that recognize a peptide from the invariant chain, termed CLIP, presented by major histocompatibility complex (MHC) class II molecules. Clonal analysis reveals 2 distinct subsets of autoreactive T cells defined by their activation requirement for either the N-terminal or the C-terminal flanking regions of CLIP and by their cytokine profile. The studies here reveal that the autoreactive T-cell clones requiring the N-terminal flanking region of CLIP produce type 1 cytokines (interferon [IFN]-gamma, interleukin [IL]-2, and tumor necrosis factor-alpha). In contrast, the autoreactive T-cell clones that require the C-terminal flanking region of CLIP produce type 2 cytokines (IL-4, IL-10, transforming growth factor-beta). As assessed in a local graft-versus-host reaction assay, the N-terminal flanking-restricted clones mediate changes consistent with acute GVHD, whereas the clones responsive to the C-terminal flanking region do not. Moreover, the autoreactive T-cell clones restricted by the C-terminal flanking region of CLIP ameliorate the pathogenic potential of the cells responsive to the N-terminal flanking region of CLIP. The mechanism accounting for this regulatory affect appears to be the downregulation of mRNA message for type 1 cytokines (IFN-gamma and IL-2). The C-terminal-restricted autoreactive T-cell clones, however, could manifest disease with dermal changes similar to those seen in chronic syngeneic GVHD, provided that IFN-gamma was present. Consistent with these observations was the demonstration that type 1 cytokines are preferentially detected during the acute phase of syngeneic GVHD, whereas type 2 cytokines dominate during the chronic phase. The results suggest that acute and chronic syngeneic GVHD is mediated by distinct autoreactive T cells, which are separated by their fine specificity for the CLIP-MHC class II complex and by their cytokine profiles.

Extrahepatic synthesis of C6 in the rat is sufficient for complement - Mediated hyperacute rejection of a guinea pig cardiac xenograft

The liver is the major source of complement (C) components, but extrahepatic sources of C, such as macrophages and endothelial cells, have been hypothesized to contribute to inflammation. Our experiments demonstrate that extrahepatically produced C6 can contribute to hyperacute rejection. PVG (RT1c) rats with normal C activity (PVG (C+)) reject guinea pig cardiac xenografts in 0.5 +/- 0.2 hr, but fully C6-deficient PVG (RT1c) rats (PVG (C-)) reject guinea pig cardiac xenografts in 45 +/- 9 hr. PVG (C+) rats, which received liver transplants from PVG (C-) rats and retained all extrahepatic sources of C6, rejected guinea pig cardiac xenografts in 0.6 +/- 0.03 hr (n = 3). PVG (C-) rats, which received bone marrow transplants from PVG (C+) rats, had C6 levels restored to 10% of that of the donor and rejected guinea pig cardiac xenografts in 9 +/- 3.2 hr (n = 5). Thus, extrahepatic sources of C6 can contribute to xenograft rejection.

Characterization of the natural suppressor cell population in adult rat bone marrow.

Natural suppressor cell activity (NSCA) has been ascribed to a subset of cells present in human and murine hematopoietic tissues which can suppress a variety of lymphocyte responses without MHC restriction. We investigated NSCA in lymphocyte‐depleted rat bone marrow (BM) which is used as a model for prevention of graft vs host disease (GVHD) following allogeneic BM transplantation (BMT). The T‐cell depleted fraction obtained after elutriation contained higher levels of NSCA than the unseparated BM. Further separation of this graft fraction by discontinuous Percoll gradient centrifugation revealed high levels of radiosensitive NSCA in the low density (< 1.070) fraction which represented 0.5% of the original BM population. These cells were of blast morphology, stained intensely with a dansylated derivative of cyclosporine A (dans CsA) and weakly expressed macrophage/granulocyte antigens and non‐specific esterase (NSE). These cells were initially non‐adherent but proliferated in culture to produce intensely NSE positive, adherent, phagocytic cells of macrophage morphology. We conclude that the highly suppresses, radiosensitive cell present in rat BM may be of early progenitor or monocyte lineage. The grafting of natural suppressor (NS) cells and progenitor cells may affect graft/host immunoregulation and their characterization may provide insight into GVH biology and graft rejection.

The role of cyclosporine-induced autoreactive T lymphocytes in solid organ allograft survival and chronic rejection.

Cyclosporine (CsA) has profound but paradoxical effects on the immune system. CsA can facilitate the induction of transplantation tolerance in some animal systems but it inhibits the clonal deletion of MHC class II autoreactive T cells. The present studies evaluated whether the autoreactive T cells participate in the induction of facilitated graft acceptance after CsA treatment by recognizing and eliminating activated allograft responsive T cells that express MHC class II determinants. Transfer of autoreactive T cells into naive Lewis rats pretreated with cyclophosphamide significantly prolonged the survival of heterotopic cardiac allografts from MHC-disparate BN strain donors. Following transfer of the autoreactive T cells, there was a marked reduction in the frequency of alloreactive T lymphocytes responsive to donor alloantigens. The role of MHC class II autoreactive CD8+ V beta 8.5+ T cells in facilitated graft acceptance was also supported by the findings that (1) treatment with anti-MHC class II antibody abrogated prolonged allograft survival after CsA therapy and (2) V beta 8.5+ lymphocytes infiltrate the allograft during CsA therapy but are absent in the graft in non-CsA-treated control animals. Although these data are consistent with the hypothesis that autoreactive T cells prolong cardiac allograft survival after CsA treatment, the autoaggressive cells failed to inhibit the development of chronic rejection of both heart and skin allografts. These data suggest either that the autoreactive T cells do not inhibit immune mechanisms responsible for chronic graft rejection or that the autoaggressive lymphocytes may participate in and exacerbate chronic rejection of allografts. Taken together, the induction of MHC class II autoreactive T cells may provide a common fundamental mechanism explaining the paradoxical effects of CsA.

Effect of cyclosporine on T lymphocyte development: Relationship to syngeneic graft-versus-host disease

This report investigates the effects of cyclosporine on the reconstitution of T lymphocytes after syngeneic bone marrow transplantation and its role in the development of a novel T cell-mediated autoimmune disease, syngeneic graft versus host disease. We analyzed the effect of CsA treatment on T lymphocyte differentiation during reconstitution after bone marrow transplantation and correlated the maturation of CD4+ and CD8+ T cell subsets with the onset of syngeneic GVHD. Administration of CsA following syngeneic bone marrow transplantation leads to a developmental arrest of mature CD4+ and CD8+ T lymphocytes in the thymus and a marked reduction in cells expressing the alpha beta T cell receptor. The reduction of CD4+ and CD8+ T cell subsets is also reflected in the peripheral lymphoid compartment with an altered CD4/CD8 ratio. Functional assessment of the cells revealed that CD8+ cells respond normally to mitogenic signalling whereas CD4+ cells exhibit marginal proliferative responses. Both subsets of T lymphocytes respond to syngeneic B lymphoblasts, comparable to the response of T lymphocytes from non-CsA-treated syngeneic BMT recipients, suggesting that autoreactive cells are produced despite CsA treatment. Following discontinuation of CsA, T cell differentiation in the thymus is rapidly restored to normal. However, concurrent with the onset of syngeneic GVHD, a compensatory insurgence of CD4+ T helper cells is observed.

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.