Institute of Bone and Joint Surgery

We have recently demonstrated that the rheumatoid arthritis (RA) shared epitope (SE) acts as a ligand that triggers nitric oxide (NO) signaling in opposite cells. Given the known pro-oxidative effect of NO and the proposed role of oxidative stress in the pathogenesis of RA, this study explores whether SE-triggered signaling can increase cellular oxidative stress. Cyclic AMP (cAMP) levels, adenylyl cyclase (AC) activity and protein kinase A (PKA) activity were measured using commercial kits. Generation of reactive oxygen species (ROS) was quantified using the fluorochrome dichlorofluorescein diacetate. Oxidative DNA damage was quantified using the single cell electrophoresis technique. Here we report that cells exposed to cell surface SE-positive HLA-DR molecules, to cell-free recombinant proteins genetically engineered to express the SE motif, or to SE-positive synthetic peptide, showed diminished cAMP-dependent signaling, increased ROS levels and higher vulnerability to oxidative DNA damage. Introduction of single amino acid substitutions into SE-positive peptides revealed a consensus 5 amino acid sequence motif of Q/R-K/R-X-X-A that is necessary and sufficient for SE-triggered signaling. The pro-oxidative effect of the SE could be reversed by inhibiting NO production. We conclude that the SE acts as a signaling ligand that activates a NO-mediated pro-oxidative pathway. The potential contribution of this signaling aberration to RA pathogenesis is discussed.