Nitric Oxide Signaling Triggered by the Rheumatoid Arthritis–Shared Epitope

Many immune-mediated diseases are associated with particular MHC class I or class II alleles. In rheumatoid arthritis (RA-shared), the vast majority of patients possess HLA-DRB1 alleles encoding a shared epitope, which is a five–amino acid sequence motif in positions 70–74 of the HLA-DRΒ chain. The mechanistic basis for this association is unknown. Here we discuss recent evidence suggesting that the shared epitope may act as an allele-specific ligand that triggers increased nitric oxide (NO) production in opposite cells with resultant immune dysregulation. We propose that by doing that, the RA-shared shared epitope may form an unintended bridge between the innate and adaptive immune systems, thereby allowing aberrant signaling events that could trigger disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72272/1/annals.1423.009.pd

New insights into the functional role of the rheumatoid arthritis shared epitope

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116315/1/feb2s0014579311001980.pd

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

Shared Epitope–Antagonistic Ligands: A New Therapeutic Strategy in Mice With Erosive Arthritis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112271/1/art39158.pd

Sphingosine kinase 1–mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells

Objective It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas-mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls. Methods Cell death was determined by the MTS assay, and apoptosis was detected by the TUNEL assay and DNA laddering. Proteolytic activation of caspase 3 was determined by immunoblotting, and its enzymatic activity was determined by a fluorometric technique. Messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction (PCR) analysis. The functional role of sphingosine kinase (SPHK) was determined by measuring its enzymatic activity, by quantifying the levels of its product, sphingosine 1-phosphate (S1P), and by investigating the ability of the SPHK inhibitor N , N -dimethylsphingosine and isozyme-specific small interfering RNA (siRNA) oligonucleotides to reverse signaling aberrations. Results LCLs from patients with RA displayed disease-specific Fas-mediated signal transduction impairment with consequent resistance to cell death. RA LCLs displayed high constitutive SPHK activity and increased levels of S1P. Real-time PCR analysis showed higher SPHK-1 mRNA expression levels in RA patients compared with paired controls. Increased SPHK-1 (but not SPHK-2) mRNA levels were observed in synovial tissue from RA patients. Competitive inhibitors of SPHK reversed the resistance of RA LCLs to Fas-induced apoptosis. Additionally, resistance to Fas-mediated signaling was reversed by siRNA oligonucleotides specific for SPHK-1 but not by oligonucleotides specific for SPHK-2. Conclusion These findings demonstrate disease-specific resistance to Fas-mediated death signaling in patients with RA and implicate increased SPHK-1 activity as the cause of this aberration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49513/1/21635_ftp.pd

Activation of human T cell clones through the UM4D4/CDw60 surface antigen

UM4D4 is a recently defined antigen that is expressed on ~25% of peripheral blood T cells, but on the majority of T cells in inflammatory synovial fluid. Anti-UM4D4 activates peripheral blood T cells in the presence of accessory cells and/or phorbol ester. UM4D4 has been assigned to a new antigen cluster termed CDw60. The present study examined the ability of anti-UM4D4 to activate T cell clones derived from the synovial fluid of patients with rheumatoid arthritis. UM4D4 was expressed at varying levels on both lectin-generated and antigen-specific clones, including clones of CD4+, CD8+, and CD4-CD8- phenotypes. Anti-UM4D4 used in soluble form as a single stimulus was typically mitogenic for the CD4+ and some of the CD8+ clones, but not for the CD4-CD8- clones. Phorbol ester boosted the response to anti-UM4D4 in some clones, had no effect in others, and diminished the responses in some cases. In contrast to anti-UM4D4, anti-CD3 was generally not mitogenic in soluble form, although it was mitogenic when conjugated to beads. The data show that T cell clones derived from an inflammatory T cell infiltrate can be readily activated through the UM4D4/CDw60 antigen.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28512/1/0000309.pd

Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

Background: The rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRb chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of- RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT. Principal Findings: Surface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217–224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu 217 and Glu 223- and to a lesser extent residue Asp 220- in cell-free SPR-based binding and signal transduction assays. Significance: We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive an

Potential role of [gamma][delta] T cells in autoimmune diseases

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28910/1/0000747.pd