Activation of nitric oxide signaling by the rheumatoid arthritis shared epitope

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–DRΒ 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 DRΒ-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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55859/1/22178_ftp.pd

The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway

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. cAMP levels, adenylyl cyclase activity, and protein kinase A 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 (human leukocyte antigen-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 five-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 an NO-mediated pro-oxidative pathway. The potential contribution of this signaling aberration to RA pathogenesis is discussed

Structure and functional properties of chimeric particles on the basis of MIR-deleted HBcAg

Summary in Latvian, English, RussianAvailable from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway

Abstract 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. cAMP levels, adenylyl cyclase activity, and protein kinase A 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 (human leukocyte antigen-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 five-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 an NO-mediated pro-oxidative pathway. The potential contribution of this signaling aberration to RA pathogenesis is discussed.http://deepblue.lib.umich.edu/bitstream/2027.42/112601/1/13075_2006_Article_1975.pd

The shared epitope (SE) accelerates endogenous reactive oxygen species (ROS) production

<p><b>Copyright information:</b></p><p>Taken from "The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway"</p><p>http://arthritis-research.com/content/9/1/R5</p><p>Arthritis Research & Therapy 2007;9(1):R5-R5.</p><p>Published online 25 Jan 2007</p><p>PMCID:PMC1865041.</p><p></p> Time-course ROS levels in representative SE-positive (red line) and SE-negative (blue line) B-lymphocyte cell lines. ROS production rates in B lymphocytes. Results are shown as the mean ± standard error of the mean fluorescent units per minute (FU/minute) during the linear late phase of the ROS curve (80 to 200 minutes). Data of the SE-positive (SE+) and SE-negative (SE-) groups were compiled from three to five consecutive experiments in each cell line. The values in parentheses represent the number of donors in each group. ROS levels in M1 cells stimulated with 2.5 μg/ml of either SE-positive hepatitis B core (HBc) capsids (HBc*0401, red line) or SE-negative HBc capsids (HBc*0402, blue line). ROS levels in M1 cells stimulated with 100 μg/ml of either an SE-positive 15 mer peptide (65–79*0401, red circles) or an SE-negative 15 mer peptide (65–79*0402, blue circles). Calculations of values in and were based on paired test analysis. DCF, dichlorofluorescein

The shared epitope (SE) attenuates adenosine-induced protection against oxidative DNA damage

<p><b>Copyright information:</b></p><p>Taken from "The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti-oxidative pathway"</p><p>http://arthritis-research.com/content/9/1/R5</p><p>Arthritis Research & Therapy 2007;9(1):R5-R5.</p><p>Published online 25 Jan 2007</p><p>PMCID:PMC1865041.</p><p></p> The potency of adenosine-mediated anti-oxidative signaling was determined in L-cell transfectants expressing cDNA corresponding to (L565.5), (L514.3), or (L259.3). Human M1 fibroblasts were incubated for 1 hour with or without 24 μg/ml of SE-positive (T-DRB1*0401) or SE-negative (T-DRB1*0401) tetrameric molecules. At the end of incubation, the extent of adenosine-induced protection against oxidative DNA damage was determined as above. M1 human fibroblasts were pre-incubated overnight in the absence or presence of 2.5 μg/ml of either HBc*0401 or HBc*0402 multimeric proteins. At the end of incubation, the extent of adenosine-induced protection against oxidative DNA damage was determined. *< 0.05