Effect Of Mycophenolic Acid On Liver Resident NK Cells Activation

Background and AimsIn the past decade, organ transplantation has become an almost routine life-saving procedure for patients with organ failure. And with the development of immunosuppressant, the danger of rejection of the transplant which resulting in allograft loss has been greatly declined. But after long period of being treated with immunosuppressant, the immunity to resist infection would be weakened. Many patients after liver transplantation yet reinfect HBV. It may be related with immunosuppression of the liver. The liver resident Natural killer (NK) cells were first discovered on 1998, and in hepatic lymphocytes the NK cells above 50%. NK cells constitute the first line of host defense against invading pathogens. And atpresent, it is confirmed that the liver resident NK cells are very important on resisting HBV infection. So HBV reinfection after liver transplantation might be induced by immunosuppressant inhibiting the liver resident NK cells activity. Mycophenolate Mofetil (MMF), a new immunosuppressant, is the morpholinoethyl ester prodrug of its active metabolite mycophenolic acid (MPA). In this task, we study the effect of MPA/MMF on liver resident NK cells activation.Materials and Methods1. Mice: Six- to 8-wk-old male C57BL/6 mice and HBV-transgene mice, 16 to 20g weights, male,were housed according to institutional regulations.2. Methods!1) In vivo, MMFSOmg/kg.d'1 i.p. for 10 days2) Preparation of Intrahepatic lymphocytes and Purifying liver resident NK cells by magnetic microbead separation.3) In vitro, the NK cells were purified to be cultured with different concentration MPA for 24 hours4) NK cells were tested for cytolytic activity in a [3H]-TdR-release assay.5) Secretions of IFN- Y > IL-2 and IL-10 were measured by ELISA.6) Expression of NK cell receptors Ly49A> NKG2A and NKG2D was quantitated by flow cytometry.7) Expression of NK cell receptor NKp46 was measured by RT-PCR, and the expression of some ligand was also measured by RT-PCR.Results:1. After cultured with MPA, the liver resident NK cells cytotoxicity was impaired, more MPA more impaired, the secretion of IFN- Y and IL-2 was decreased while the secretion of IL-10 was increased, and the expression of the NK cell surface molecules NKG2D and Ly49A was down-regulated, but that of NK.G2A was up-regulated. In high concentration MPA group(0.1 u mol/ml), those were most significant(/><0.001).2. The liver resident NK cells cytotoxicity was impaired in MMF group, and there was significant difference from that in Control group (PO.01). In MMF group the expression of the liver resident NK cells receptors NKG2D% NKp46 and Ly49A was down-regulated but that of NKG2A was up-regulated. And the expression of IL-15mRNA and the ligand Rae-lmRNA was down- regulated in MMF group.3. The liver resident NK cells cytotoxicity was impaired in HBV-transgene mice group, and there were significant differences from that in Control group (PO.01). The expression of the liver resident NK cells receptors NKG2D was down-regulated in HBV-transgene mice group^O.OS.VS Control group), but the expression of NKG2An Ly49A and NKp46 was no difference from that in Control group(P>0.05). After MMF30mg/kg.d'1 i.p. for 1 month, the liver resident NK cells cytotoxicity was impaired in the MMF-HBVTg group, and there were significant differences from that in HBVTg group(P<0.05), but the level of HBsAg was no diffenrence between MMF-HBVTg group and HBVTg group(P>0.05).Conclusions:1. MPA inhibits the activity of the liver resident NK cells and inhibits the secretionof IFN- Y and IL-2 while promotes the secretion of IL-10 maybe through down-regulating the expression of the NK cells receptors NKG2D and NKp46 while up-regulating that of NKG2A, and above effects beyond the effect of down-regulating the receptor Ly49A. IL-15 can reverse the inhibiting effect of MPA on the activity of the liver resident NK cells maybe through up-regulating the expression of NKG2D receptor.2. MMF inhibits the activity of the liver resident NK cells maybe through down-regulating the expression of the NK cells receptors NKG2D and NKp46 while up-regulating that of NKG2A. And above effects beyond the effect of down-regulating the receptor Ly49A. MMF also down-regulates the expression of the ligand Rae-1 and IL-15 mRNA to impact the expression of NKG2D.3. HBV inhibits the activity of the liver resident NK cells maybe through down-regulating the expression of the NK cells receptor NKG2D. MMF can inhibits the activity of the liver resident NK cells in HBV-transgene mice, but no influence on the replication of HBV.