An Experimental Study Of Macrophages On Heterogeneous Immunotolerance Iduced By Mixed Xenogeneic Chimera

Objectives:In this study, we investigate whether macrophages mediate strong rejection of guinea pig bone marrow cells in rat. Then, we explore the mechanisms of immunotolerance in xenotransplantation induced by establishing the guinea pig to rat mixed chimeras model with a non-myeloabalative preparative regimen.Methods: Recipient SD rats were conditioned with sublethal whole body irradiation (WBI) ,divided randomly into three groups, followed by infusion of 0.8ml guinea pig bone marrow cells(2x108/ml )within 4 h . Group A was infused guinea pig bone marrow cells (BMC) only; Group B was injected with blank liposomes,followed by the intravenous infusion of guinea pig bone marrow cells; Group C was treated with clodronate-encapsulated liposomes before guinea pig bone marrow cells were infused. Then all recipients were administered cytoxan(CTX) by intraperitoneal injection. Rat spleen and liver were collected to examine macrophage depletion by immunohistologic analyses. For 24-h experiments, guinea pig bone marrow cells were labeled with carboxymethylfluorescein succinimidyl ester (CFSE) before i.v. injection. Blood samples were taken at 1, 6,12 and 24 h post-injection for flow cytometric analysis. Bone marrow and spleen were collected at 24 h. The percentages of infused labeled guinea pig bone marrow cells in the rat BM and spleen were analyzed by flow cytometry.The level of guinea pig bone marrow cells chimerism in the peripheral blood lymphocyte of recipient rats was detected on 21 day and 35 day by flow cytometry. To explore tolerance mechanisms by performing mixed lymphocyte reaction (MLR). The flow cytometry was used to check the changes of subgroup of lymphocytes.Donors (guinea pigs) and recipients (SD rats) were divided ranmomly into 6 groups: group I(control,n=5),group II(CVF, n=5),group III(CVF+ clodronate-encapsulated liposomes, n=5),group IV(CVF+ BMC, n=5),group V(CVF+ BMC + blank liposomes, n=5),group V(ICVF+ BMC + clodronate-encapsulated liposomes, n=5).The survive time of xenograft was measured and the histopathologic observation was carried out after the graft arrest. The changes of IgG were detected before heart transplantation and graft arrest by flow cytometry.Results: Clodronate-encapsulated liposomes treatment resulted in complete depletion of both liver Kupffer cells and splenic macrophages (red pulp and marginal macrophages). Kupffer cells in the liver and red pulp macrophages in the spleen recovered rapidly in treated rats and became indistinguishable from those of control rats by 21 days. Macrophages depletion increase the percentages of infused labeled guinea pig bone marrow cells in the rat blood samples taken at 1, 6,12 and 24 h post-injection. The percentages of infused labeled guinea pig bone marrow cells in the macrophages depletion rat BM and spleen were also increased. Macrophages depletion also improved guinea pig chimerism in rat. Recipient SD rats were specifically tolerant to the guinea pig in MLR assay. MLR in macrophages depletion group was siginificantly decreased comparared with other groups.The lymphocyte's subset CD3+,CD4+ and CD8+ count,especially the rate of CD3 T cell in peripheral leucocytes, showed a significant decrease after treatment, but the ratios of CD4/CD8 was indiscriminating among every group.Pathology: Group I showed hyper acute rejection (HAR) after cardiac transplantation and others showed acute vascular rejection( AVR ). The mean survival time of cardiac xenograft was :306±45min in group II,314±51min in group III,366±35min in group IV,372±25min in group V,424±32min in group VI. The level of IgG was increased slightly in every group after heat transplantation, but the increase of group VI was slower significantly than other groups.Conclusions: (1)Clodronate-encapsulated liposomes could be used to deplete macrophages in vivo in rat; (2) Macrophages are primarily responsible for the rapid loss of guinea pig bone marrow cells in rat. Depletion of macrophages with clodronate-encapsulated liposomes can improve guinea pig chimerism in sublethally irradiated rats.(3) Mixed xenogeneic chimera can't influence the percent of CD4+ and CD8+ T cells in peripheral blood, but can induce T cells immune suppression. It also can inhibit the entrain of antibody IgG. (4) Mixed xenogeneic chimera can significantly prolong discordant xenograft survival time after using Chinese Cobra Venom Factor (CVF) in guinea pig to rat model.