CCR9/CCL25Pathway Is Involved In Acute Rejection Of Mouse Skin Transplantation And Its Mechanism

Objective:Chemokines, by virtue of their ability to recruit immune cells into allografts, play critical roles in acute transplantation rejection. Targeting of chemokine and chemokine receptor remains an important trategies in acute transplantation rejection. CCR9and its ligand, CCL25, is one of the key regulators of thymocyte migration and maturation in normal and inflammatory conditions. Moreover, several studies have revealed that high expression of CCR9and CCL25participated in many kinds of diseases. However, the role of CCR9in allograft rejection is still unclear. The purpose of our experiment is to investigate the role of the CCR9/CCL25pathway in a murine skin transplantation model of acute rejection, and research the influence of blocking CCR9/CCL25pathway by intravenous injection of Anti-CCL25monoclonal antibody in the survival of grafts and the underlying mechanism of its involvement.Methods:1. We established a murine skin transplantation model of acute rejection and then collected spleen, pLN (peripheral lymph node) and skin graft from recipient mice at different time intervals during the course of rejection. HE staining was done to analyze necrosis and inflammatory cell infiltration in the skin grafts. WB, immunohistochemistry and confocal were also used to measure CCL25and CCR9expression in recipient skin grafts. Additionally, flow cytometry analysis was explored to determine CCR9expression in spleen and pLN tissues from recipient mice.2. To investigate whether blocking the interaction between CCR9and CCL25could prolong graft survival, recipient mice were injected with an Anti-CCL25mAb every other day via tail vein. The transplanted graft was scored visually daily. Additionally, HE staining was done to analyze necrosis of skin tissue.3. The effect of Anti-CCL25on proliferation and chemotactic of splenic lymphocytes from allo-transplantation recipient mice was analyzed by using mixed lymphocyte reaction and chemotaxis assays. In addition, the production of proinflammatory cytokines by these cells were measured with ELISA and data were treated with stastical analysis.Results:1. CCR9is involved in acute rejection process of skin transplantation model in mice. We have sucessully established a murine skin transplantation model of acute rejection. After that, HE staining indicated that there was a widespread inflammatory cell infiltration in the skin from allo-transplantation mice, and CCR9expression measured by immunohistochemistry and confocal was significantly elevated in the surface of the infiltrated CD3+T cells from spleen and skin grafts tissue. Similarly, allo-graft skin tissues also showed a significantly up-regulated expression of CCL25by immunohistochemistry analysis and WB when compared with iso-transplantation recipient. Additionally, our findings showed that the proportion of CCR9-expressing T cells was significantly increased in the spleen of allotransplanted mice compared with syngeneic transplantation, and the predominant CCR9-expressing population was identified within CD8+T cells. Whereas there is no significant difference of the expression of CCR9in T cells between the two groups in pLN.2. Neutralization of CCL25with Anti-CCL25mAb significantly prolonged allograft survival.We observed an obvious prolongation of skin allograft survival in Anti-CCL25mAb treatment group, which blocking the interaction between CCR9and CCL25with injection with Anti-CCL25mAb every other day. The skin grafts were subjected to H&E staining at day7post-transplantation. Widespread inflammatory cell infiltration in IgG2a isotype control group was observed whereas markedly less immune cells were seen in the Anti-CCL25treated group.3. Anti-CCL25mAb treatment decreased the expression of CCR9in allogeneic recipient mice.We analyzed expression of CCR9on splenic T cells from mice that received different treatments at day7after transplantation and a higher proportion of CCR9+T cells in splenic cells was observed in IgG2a-treated mice compared with isogeneic transplant recipient mice, whereas infiltrated CCR9+T cells were significantly reduced in Anti-CCL25mAb treatment group. Consistent with above results, a large decline in the ratio of CD8+to CD4+cells in CCR9-expressing splenic T cell was observed in the group receiving Anti-CCL25mAb compared with IgG2a. The Western blotting results also indicated that injection of Anti-CCL25significantly decreased CCR9protein expression in skin allografts.4. Anti-CCL25mAb suppressed the chemotactic and proliferative capacity of splenic T Cells coupled with Thl-type cytokine secretion.Neutralization of CCL25by intravenous injection of Anti-CCL25monoclonal antibody significantly suppressed the chemotactic ability and the proliferation of the splenic T cells in response to allogeneic antigens. Additionally, the level of IFN-γ increased progressively in the isotype-treated mice but decreased markedly in the Anti-CCL25mAb-treated mice. IL-12production was also reduced after Anti-CCL25mAb administration, while no difference was observed in the secretion of IL-4or IL-10.Conclusions:1. CCR9/CCL25pathway is involved in acute rejection process of skin transplantation model in mice.2. Neutralization of CCL25by intravenous injection of Anti-CCL25monoclonal antibody significantly prolonged skin allograft survival.3. The protective effect of Anti-CCL25blocking antibody is associated with decreased the number of splenic CCR9+CD8+T cells and simultaneously suppressed the chemotactic ability and the proliferation of the splenic T cells, and reduced production of IFN-y.