OR13 Mitogen-activated protein kinase P38 and C-JUN NH2-terminal kinase regulate hla class II antibody-stimulated activation of human aortic endothelial cells

Abstract

Aim Transplant recipients developing donor specific HLA antibodies (DSA) are at a higher risk for acute and chronic antibody (Ab) mediated rejection (AMR). HLA Ab contribute to the process of AMR by binding to the HLA molecules on endothelial cells (EC) and triggering signal transduction pathways leading to EC activation, adhesion molecule expression, proliferation and migration. DSA against HLA class I appear earlier, while class II DSA tends to prevail late post transplantation and associate with chronic graft dysfunction. Ligation of HLA-I with Ab on EC and smooth muscle cells have been shown to contribute to the process of transplant vasculopathy. However, the mechanism(s) underlying how HLA-II Ab contribute to chronic rejection remain unknown. Method Primary human aortic EC were infected with recombinant adenovirus pAd/PL-DEST encoding CIITA (Ad-CIITA) an HLA-II transactivator, or pretreated with TNF-alpha/IFN-gamma. HLA-II expression was determined by flow cytometry. Ad-CIITA infected or cytokine-treated EC were stimulated with anti-HLA-II Ab, phosphorylation was detected by Western Blot, EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Infection of EC with Ad-CIITA or pretreatment of EC with TNF-alpha and IFN-gamma induced a marked increase in HLA-II antigen expression and CIITA protein presentation. Ligation of HLA-II antigens on Ad-CIITA infected EC or TNF-alpha/IFN-gamma pretreated EC stimulated a significant increase in phosphorylation of MAPK p38 and JNK. Ligation of HLA on Ad-CIITA infected EC or TNF-alpha/IFN-gamma pretreated EC with class II mAb also stimulated cell proliferation. Pretreatment of class II positive EC with pharmacological inhibitors of p38 (SB203580) or JNK (SP600125) or siRNA inhibited HLA-II Ab-stimulated EC proliferation. Conclusions In this study we found that Ad-CIITA infection or TNF-alpha/IFN-gamma treatment can induce HLA-II expression on EC. Ligation of class II with Ab on EC triggers intracellular signal transduction promoting cell proliferation via a MAPK dependent pathway. Our results provide information on novel mechanisms of HLA-II Ab-mediated activation of EC. Disrupting activation of MAPK p38 or JNK may provide new therapeutic targets to prevent AMR.