Transcriptional Regulation Of Human Granzyme B Gene And The Role Of Perforin/granzyme B Pathway In EAU Disease

Perforin/granzyme B mediated apoptosis pathway plays an essential role in bothinnate and adaptive immune system. This pathway is especially important for defenseagainst viral and bacterial infection and immunosurveillance of tumors. Evidence forthe importance of perforin is indicated by the finding that perforin deficient mice haveprofound defects in their clearance of viral infections, delayed type hypersensitivity,and tumor rejection. Similarly, NK cells, LAK cells and CTL from granzymeB-deficient mice showed a rapid decrease in induction of DNA fragmentation andapoptosis on their target cells. Perforin and granzymes are mainly expressed bynatural killer cells, CTL cells and LAK cells.However, the transcriptional regulation of granzyme B gene is not wellunderstood, though there are many reports on perforin gene's transcription regulation.In this paper, we demonstrate in human NK cells and T cells that NF-κB signalingpathway is involved in such control. Furthermore, a novel downstream humangranzyme B gene sequence (GGAGATTCCC) was identified for NF-κB binding.EMSA, luciferase and chromatin immunoprecipitation assays in vitro and in vivoindicated that this NF-κB binding site is functional in an NK cell line and its primarycounterpart. Our data also demonstrate that this binding site is functional in Jurkat Tcells. Taken together, we identified a novel NF-κB binding site, which plays a pivotalrole in controlling human granzyme B gene transcription.Recently, more and more works indicate that apoptosis is involved in themechanism of autoimmune diseases. So we also investigated the role ofperforin/granzymes pathway on an autoimmune disease model — EAU. Butunexpectedly, the incidence and mean severity of EAU were significantly higher inPKO mice than in WT mice. Furthermore, proliferation to IRBP of primedlymphocytes was considerably higher in the PKO mice than in the WT controls. Theselymphocytes from PKO mice also produced more cytokines than cells from controlmice. When na?ve CD4+ T cells were isolated from PKO mice and stimulated withplate-bound anti-CD3 antibody, they also displayed an enhanced proliferation inresponse to anti-CD3. Flow cytometric analyses revealed that the loss of perforinenhances the cell cycle entry of T cells activated through the TCR. So it is the firstreport to our knowledge that perforin negatively regulates T cell proliferation and cellcycle entry. And it also explained why perforin deficiency exacerbates EAU disease.