OBJECTIVE: Susceptibility to rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles encoding a shared epitope (SE) in positions 70-74 of the HLA-DRbeta chain. The mechanistic basis for this association is unknown. Given the proposed pathogenic role of nitric oxide (NO) in RA, this study was undertaken to examine whether the SE can trigger NO signaling events. METHODS: The intracellular levels of NO were measured with the fluorescent NO probe 4,5-diaminofluorescein diacetate and by the 2,3-diaminonaphthalene method. NO synthase activity was determined by measuring the rate of conversion of radioactive arginine to citrulline. Levels of cGMP were measured with a commercial enzyme-linked immunosorbent assay, and the cytolytic activity of T cells was measured using a standard (51)Cr release assay. RESULTS: Lymphoblastoid B cell lines carrying SE-positive HLA-DR alleles displayed a higher rate of spontaneous NO production compared with SE-negative cells. L cell transfectants expressing SE-positive DR molecules on their surface also generated higher levels of NO. Tetrameric HLA-DR molecules containing a DRbeta-chain encoded by the SE-positive DRB1*0401 allele stimulated fibroblast cells to produce higher levels of NO compared with cells stimulated with a control HLA-DR tetramer. Multimeric hepatitis B core proteins engineered to express region 65-79 encoded by the DRB1*0401 allele, but not the same region encoded by the control allele DRB1*0402, stimulated NO production in fibroblasts. Similarly, synthetic 15-mer peptides corresponding to the region 65-79 encoded by SE-positive alleles triggered increased NO levels when incubated with class II major histocompatibility complex-negative cells. The signaling pathway was found to involve NO synthase activation, followed by increased production of cGMP. SE-triggered increased NO levels inhibited cytolytic elimination of target cells. CONCLUSION: The SE can trigger NO-mediated signaling events in opposite cells, and may thereby contribute to RA pathogenesis.