The inhibitor of differentiation-2 promotes synovial fibroblast-dependent osteoclastogenesis in rheumatoid arthritis

Objectives: Despite indirect evidence suggesting that low oxygen levels might occur in the rheumatoid arthritis (RA) synovium, direct proof of the presence of hypoxia in the arthritic synovium as well as the relevance of low oxygen levels for joint destruction is lacking. The aim of this study was to analyse the distribution of hypoxia in arthritic joints and to evaluate the molecular effects of the hypoxic environment on the phenotype of RA synovial fibroblasts (SF).<p></p> Methods: The hypoxia marker EF-5 was applied in mice with the collagen-induced arthritis (CIA). Expression profile analysis with hypoxic and normoxic SF was performed using subtractive hybridization and microarray. The expression of the inhibitor of differentiation-2 (Id-2), CD68 (macrophage marker) and prolyl hydroxylase (fibroblast marker) was evaluated by immunohistochemistry on synovial tissues from RA, osteoarthritis patients and CIA mice. To evaluate the function of Id-2 in SF, cells were transfected with the pcDNA3.1 containing cDNA for Id-2 or Id-2-specific siRNA or mock controls. The expression of Id-2 and genes regulated by Id-2 in transfected SF was evaluated by SYBR Green real-time PCR and western blot. SF stably transfected with Id-2 were cocultured with bone marrow cells in a transwell system. The expression of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin were measured by real-time PCR. The development of osteoclasts was evaluated by visualization of the activity of tartrate-resistant acid phosphatase.<p></p> Results: Using the hypoxia marker EF-5 we found that in mice with CIA, synovial cells invading bone and cartilage are exposed to reduced oxygen levels. Expression profile studies identified Id-2 as being upregulated under low oxygen conditions. In addition, IL-1beta stimulation increased the expression of Id-2 in these cells. Histological studies of RA synovium and CIA synovium showed strong expression of Id-2 in SF at sites of synovial invasion into bone. Overproduction of Id-2 in SF by stable transfection triggered the expression of several genes promoting osteoclastogenesis, including BMP-2, PTHrP, Wnt5a and vascular endothelial growth factor. Conversely, the suppression of endogenous Id-2 led to the downregulation of the expression of these molecules. Consistent with these findings coculture of Id-2 transfected SF with bone marrow cells increased the expression of the osteoclast differentiation factor RANKL, and decreased the expression of the osteoclast inhibitory factor osteoprotegerin in bone marrow stromal cells, which was followed by an increase in the number of osteoclasts.<p></p> Conclusion: Taken together, our data provide evidence that hypoxia is present at sites of synovial invasion in RA and that Id-2 induced by hypoxia contributes at these sites to joint destruction by promoting SF-dependent osteoclastogenesis

Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Objective Citrullinated proteins are immunogenic in rheumatoid arthritis (RA), particularly in patients who carry shared epitope (SE)–coding HLA–DRB1 alleles. The mechanism underlying this association is unknown. We have previously identified the SE as a ligand that interacts with cell surface calreticulin (CRT) and activates immune dysregulation. This study was undertaken to determine the effect of CRT citrullination on SE signaling. Methods CRT–SE binding affinity was measured by surface plasmon resonance. The role of individual CRT arginine residues was determined by site‐directed mutagenesis, and nitric oxide levels were measured using a fluorochrome‐based assay. CRT citrullination in synovial tissue samples and cell cultures was determined by 2‐dimensional gel electrophoresis, immunoblotting, and mass spectrometry techniques. Results Synovial tissue and fibroblast‐like synoviocytes from RA patients were found to express a higher abundance of citrullinated CRT than samples from osteoarthritis patients. Citrullinated CRT showed more robust interaction with the SE ligand, and transduced SE signaling at a 10,000‐fold higher potency, compared to noncitrullinated CRT. Site‐directed mutation analysis identified Arg 205 , which is spatially adjacent to the SE binding site in the CRT P‐domain, as a dominant inhibitor of SE–CRT interaction and signaling, while a more remote arginine residue, Arg 261 , was found to enhance these SE functions. Conclusion Our findings indicate that citrullinated CRT is overabundant in the RA synovium and potentiates SE‐activated signaling in vitro. These findings could introduce a new mechanistic model of gene–environment interaction in RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96655/1/37814_ftp.pd

S.8.1 An immunochip-based interrogation of scleroderma susceptibility variants

Introduction. Understanding the genetic architecture of scleroderma (SSc) susceptibility is vital both in gene discovery and in determining the influence of previously identified susceptibility variants. It is particularly important in understanding disease mechanism in a disease with few therapies and great morbidity and mortality. Methods. We selected 557 cases from the Australian Scleroderma Cohort Study (ASCS), for genotyping with the Immunochip, a custom Illumina Infinium genotyping array containing 196 524 rare and common variants shown to be important in a wide variety of autoimmune disorders. A total of 4537 controls were taken from the 1958 British Birth cohort. Genotype data were analysed with PLINK. Samples and SNPs with low call rates were excluded, as were SNPs in Hardy-Weinberg disequilibrium or with less than two occurrences of the minor allele. Eigenstrat was used to analyse population structure. The final data set consisted of 505 cases, 4491 controls and 146 867 SNPs. Allelic association analyses were conducted using Fisher's exact test. Genotype clusters were manually examined for all associations of P < 10−5 since calling is difficult for some rare variants. Results. Significant and suggestive associations were detected at seven loci. Several of these have been previously implicated in scleroderma susceptibility (HLA-DRB1 and STAT4) and several are novel associations, including SNPs near PXK (P = 4.4 × 10−6) and CFDP1(P = 2.6 × 10−6). The strongest associations were with SNPs in the Class II region of the MHC. One of the most strongly associated SNPs [rs4639334; P = 1.6 × 10−8; odds ratio (OR) = 1.8] is in linkage disequilibrium (r2 = 0.46) with the Class II allele HLA-DRB1*11:01. This allele has been associated with SSc. Another strongly associated SNP is rs2857130 (P = 1.6 × 10−8; OR = 0.67), which lies in the promoter region of HLA-DRB1, but is not in LD with any classical MHC alleles. Outside the MHC, there were six regions of association with P < 10−5,including the confirmed SSc locus at STAT4. Several SNPs implicate a locus at PXK, which has been previously associated with SLE but not with SSc. The remaining associations are novel for both SSc and SLE and require replication. Of particular interest is a rare variant located within a non-coding RNA on chromosome 6q21 which was ∼20 times more frequent in cases than controls. We are currently dissecting the potential biological implications of this locus. Conclusions. This pilot study has confirmed previously reported SSc associations, revealed further genetic overlap between SSc and SLE, and identified putative novel SSc susceptibility loci including a rare allele with major effect siz

CXCL16-mediated cell recruitment to rheumatoid arthritis synovial tissue and murine lymph nodes is dependent upon the MAPK pathway

Objective Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor Α (TNFΑ), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process. Methods Using a human RA ST–SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo. Results CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFΑ. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFΑ in vitro, suggesting that CXCL16 protein may function in large part independently of TNFΑ. Conclusion Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49514/1/21662_ftp.pd

Ectopic Lymphoid Structures: Powerhouse of Autoimmunity

Arthritis Research UK (grant 20089 to MB and grant 20858 to EC)

Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy

Subantimicrobial Dose Doxycycline Worsens Chronic Arthritis-Induced Bone Microarchitectural Alterations in a Mouse Model: Role of Matrix Metalloproteinases?

Background: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease hallmarked by irreversible damage of cartilage and bone. Matrix metalloproteinases (MMPs) involved in connective tissue remodeling play an important role in this process. Numerous MMPs have been examined in humans and animals, but their functions are still not fully understood. Therefore, we investigated the role of MMPs in the K/BxN serum-transfer model of RA with the broad-spectrum MMP inhibitor subantimicrobial dose doxycycline (SDD) using complex in vivo and in vitro methodolgy. Methods: Chronic arthritis was induced by repetitive i.p. injections of K/BxN serum in C57BL/6J mice. SDD was administered daily in acidified drinking water (0.5 mg/mL, 80 mg/kg) during the 30 days experimental period. Mechanonociceptive threshold of the paw was evaluated by aesthesiometry, grasping ability by grid test, arthritis severity by scoring, neutrophil myeloperoxidase activity by luminescence, vascular hyperpermeability and MMP activity by fluorescence in vivo imaging and the latter also by gelatin zymography, bone structure by micro-computed tomography (micro-CT). Plasma concentrations of doxycycline were determined by liquid chromatography-mass spectrometry analysis. Results: K/BxN serum induced significant inflammatory signs, mechanical hyperalgesia, joint function impairment, increased myeloperoxidase activity and vascular hyperpermeability. Significant increase of MMP activity was also observed both in vivo and ex vivo with elevation of the 57-60, 75, and 92 kDa gelatinolytic isoforms in the arthritic ankle joints, but neither MMP activity nor any above described functional parameters were influenced by SDD. Most importantly, SDD significantly reduced bone mineral density in the distal tibia and enhanced the Euler number in the ankle. Arthritis-induced microarchitectural alterations demonstrating increased irregularity and cancellous bone remodeling, such as increased Euler number was significantly elevated by SDD in both regions. Conclusion: We showed increase of various MMP activities in the joints by in vivo fluorescence imaging together with ex vivo zymography, and investigated their functional significance using the broad-spectrum MMP inhibitor SDD in the translational RA model. This is the first demonstration that SDD worsens arthritis-induced bone microarchitectural alterations, but it appears to be independent of MMP inhibition