Effects And Mechenisms Of Mif On Skin Allograft Rejection In Mice

Background and ObjectivesAUo-transplantation initiates complex inflammatory responses that are mediated by cytokines and that, in the absence of immunosuppression, usually results in graft rejection. Thus, the effect of cytokines in transplantation has been pursued with great interest.The protein known as macrophage migration inhibitory factor (MIF) was one of the first soluble cytokines to be discovered in 1966, to be produced by many kinds of cells such as monocytes/macrophages, T cells and others. A much broader role for MIF has emerged recently. MIF was reported not only be associated with various macrophage functions including adherence, migration, and tumoricidal activity but also was important for vital function and act to modulate inflammatory and immune responses. MIF acts to stimulate macrophage to release inflammatory cytokine such as IL-1, TNF-a, and NO. Recent research showed that the cytokine MIF was a major constituent of secretary granules located within the isotropic cells of the anterior pituitary gland, participating in inflammatory and immune responses. MIF can override or counter-regulate glucocorticoid inhibition of cytokine production, participate in the development of the glomerulonephritis and arthritis. Based on the immuno-modulatory properties of MIF and the postulated role of macrophages and DTH responses as indirect mediators of allograft rejection, MIF may play an important role in allograft rejection.However, few reports have demonstrated the relationship between MIF and allograft rejection. To investigate this relationship and to elucidate the mechanism of MIF on this reaction, here we constructed murine allograft rejection models, examined therelative expression amount of MIF mRNA and MIF protein before and after the transplantation. Blocking with anti-MIF monoclonal antibody (III.D.9), we studied the roles of MIF on the survival time of allograft in the models of CD4+ mediated allo-transplantation rejection model, and the effect of anti-MIF on lymphocytes proliferation, killing activity, memorial response, IL-2/IL-4 production and the expression of cellular surface markers. Also we studied the mechanisms of anti-MIF during the graft rejection by indirective pathway.In order to study the bioactivities further, we isolate cDNA of mouse MIF from the spleen, expressed cloned cDNA in E. coli and obtaited rMIF. The purpose of this paper is to study the roles and mechanism of MIF on allograft rejection so as to provide the theoretical and experimental data for the therapy to allograft rejection and for prolonged graft survival rate.Methods and Results1. Expression of MIF mRNA and protein during allograft rejection Using female B6 mice as donors, female BALB/c mice as recipients, we constructed skin allograft model and observed daily for rejection. In selected animals, allograft rejection was confirmed histological. The allograft rejection period is 11~17days after transplantation. At the day 10-14, severe rejection happened with a dense macrophage and T cell infiltration within graft.Extracted total RNA from the skin graft and spleen tissue in marine allograft models at different times before and after the transplantation compared the relative expression amount of MIF in the product of RT-PCR for each sample. The relative expression amount of MIF in the skin graft markedly changed after the transplantation, that is, it increased on the day 1, 7, 14 post-transplantation and a peak appeared at the top period of the rejection, and then it decreased after the completely rejection. The relative expression amount in the spleen cells did not change obviously.Anti-MIF Abs was radioiodinated by Na125I, and isolated /purified by Sephadex -G25 agarose. Harvest the labeled protein; the radiochemical purity was analyzed by paper chromatography. Labeling rate is 76.67%, specific activity is 29.53 TBq /mmol, radiochemical purity is 95.13%. The immunoactivity was tested with ELISA. The uptake of radioactivity in tissues in the allo-transplantaion mice was measured on day 1, 7, 10, 14, 20 after transplantation (peritoneal injection of 125I-anti-MIF antibody 48 hours ahead). Mice receiving allograft exhibited severe rejection on day 10 as shown by high level of radioactivity, decreased greatly at the day 20 (the graft rejected fully). Radioactivity of graft of the B6-BALB/c group was higher obviously than that of the BALB/c- BALB/c group. 2. The roles of anti MIF during allograft rejectionWith 3H-TdR incorporation assays, JAM'S assay and MTT method, we studied the effect of blocking MIF on the lymphocyte proliferation, killing activity and memorial response in vitro. Using ELISPOT Assay and radioimmunoassay (RIA), we examined cytokine profiles in experimental model and the IL-2 concentration in marine serum and culture supernatant of spleen cells after the transplantation. The effect of MIF on the expression of CDllb and FasL was detected by flow cytometry before /after the transplantation. The results as following:Treated with anti-MIF in vivo, the graft survival time in the allograft model with both directive and indirective pathways was prolonged and the DTH reaction was inhibited obviously. Compared with control antibody, the proliferation of T cells treated with anti-MIF was inhibited significantly on the day 1, 14 and day 20 after allo-transplantation (P<0.05). The ELISPOT Assay showed that Thl type cytokine was predominant in allo-transplantation mice. There was an obvious increasing of serum IL-2 level on the day 1, 7, 14 by turns and a peak on the day 14 and then a descent afterwards. Compared with control antibody, anti—MIF Abs inhibited the IL-2 production. Both anti—MIF Abs and control Abs treated spleen cells made the most IL-4 production, no obviously difference between both groups. The killing activity of NK cells and T cells from transplanted mice was markedly enhanced. Compared with the non-transplanted group, FasL positive spleen cells of the transplanted group increased nearly 3 times. Anti—MIF Abs can enhanced the expression of FasL in rejected group. Compared with the non-transplanted group, the expression of CDllb on marine macrophages was higher in transplanted group, there was more expression in III.D9 treated group than HB49 treated group inrejected group.3. The roles of anti-MIF Abs during allograft rejection through indirective pathway Constructed indirective allograft rejection models with B6 MHC Class II KO mice and BALB/c SCIDs mice, studied the effect mechanisms of MIF on the allo-rejection through indiretive pathways, The results showed that anti-MIF treated in vivo prolonged the graft survival time, and has no effect on the production of IFNg and IL-2 by spleen cells treated with anti-MIF, but it inhibited the DTH reactions of those mice treated with anti-MIF in vivo. And immunohistochemical analysis showed that obviously infiltration of macrophage and CD3+ T cells in locus in transplanted mice, treated with anti-MIF lowered this infiltration of macrophages, but no effect on the accumulation of T cells.4. Cloning, expression, and purification of the marine MIFThe cDNA of the MIF was cloned from the spleen of the BALB/c mice.The resulting RT-PCR product was purified and inserted into pMD18-T vector. The recombinant plasmid pMD18-MIF was then transformed to DH5a. The positive clone were screened by enzyme digestion analysis and identified with DNA sequencing. The result showed that sequence of MIF cDNA that we cloned completely matched with that of MIF cDNA in the Gene Bank. The MIF cDNA fragment treated with EcoRI and BamHI was inserted into pQE-31 expression vector which was digested with the same restriction endonucleases; recombinant vector was then transformed to E.coli Ml5.The positive clones were identified with enzyme digestion. In order to obtain MIF protein, the positive clone pGE-31-MIF was induced by IPTG, and the SDS-PAGE analysis showed that a 15.4kD protein was expressed. The immune activity of MIF was identified by Western blot. The recombinant MIF was purified from positive clone pQE-31-MIF/M15 by the Ni-NTA affinity gel. And biological activity was analysis through macrophage migration inhibition test.CONCLUSION1. The expression of MIF mRNA and MIF protein in the graft was enhanced during the allograft rejection, and positively related with graft rejection. MIF expression in...