Activation of nitric oxide signaling by the rheumatoid arthritis shared epitope

Abstract

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-DR␤ 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 DR␤-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 SEpositive 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.

The Rheumatoid Arthritis Shared Epitope Triggers Innate Immune Signaling via Cell Surface Calreticulin

“…Cy5-labeled streptavidin was purchased from Amersham Biosciences and monoclonal anti-GST Ab labeled with europium cryptate was from CIS Bio-International. Peptides were synthesized, purified, and immobilized on activated Sepharose as we previously described (8,9). Human and mouse fibroblast lines were maintained in culture as we previously described (8).…”

“…Peptides were synthesized, purified, and immobilized on activated Sepharose as we previously described (8,9). Human and mouse fibroblast lines were maintained in culture as we previously described (8). Murine embryonic fibroblasts (MEF) from wildtype (K41) and crt Ϫ/Ϫ (K42) mice were generated as described (12) and donated by Dr. M. Michalak (University of Alberta, Edmonton, Canada).…”

“…NO concentrations and production rates and pro-oxidative signaling were determined as we previously described (8,9).…”

“…In previous studies, we have found that the SE acts as a ligand that activates NO-mediated pro-oxidative signaling in opposite cells (8,9). For example, cells carrying SE-positive HLA-DRB1 alleles displayed increased constitutive NO production, which resulted in higher levels of reactive oxygen species and higher susceptibility to oxidative DNA damage.…”

“…It is therefore intriguing that the SE, located in the HLA-DR␤1 domain, is capable of triggering innate signaling in opposite cells (8,9), reminiscent of innate immune signaling triggered by class I ␣2 domain ligands (reviewed in Ref. 10).…”