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Objective: To identify new genetic associations with juvenile and adult dermatomyositis (DM)., Methods: We performed a genome-wide association study (GWAS) of adult and juvenile DM patients of European ancestry (n = 1,178) and controls (n = 4,724). To assess genetic overlap with other autoimmune disorders, we examined whether 141 single-nucleotide polymorphisms (SNPs) outside the major histocompatibility complex (MHC) locus, and previously associated with autoimmune diseases, predispose to DM., Results: Compared to controls, patients with DM had a strong signal in the MHC region consisting of GWAS-level significance (P < 5 × 10(-8)) at 80 genotyped SNPs. An analysis of 141 non-MHC SNPs previously associated with autoimmune diseases showed that 3 SNPs linked with 3 genes were associated with DM, with a false discovery rate (FDR) of <0.05. These genes were phospholipase C-like 1 (PLCL1; rs6738825, FDR = 0.00089), B lymphoid tyrosine kinase (BLK; rs2736340, FDR = 0.0031), and chemokine (C-C motif) ligand 21 (CCL21; rs951005, FDR = 0.0076). None of these genes was previously reported to be associated with DM., Conclusion: Our findings confirm the MHC as the major genetic region associated with DM and indicate that DM shares non-MHC genetic features with other autoimmune diseases, suggesting the presence of additional novel risk loci. This first identification of autoimmune disease genetic predispositions shared with DM may lead to enhanced understanding of pathogenesis and novel diagnostic and therapeutic approaches., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)

Objective: To investigate whether polymorphisms in Toll-like receptor (TLR) genes, previously reported to be associated with immune-mediated diseases, are involved in systemic sclerosis (SSc)., Methods: We genotyped 14 polymorphisms in the genes for TLRs 2, 4, 7, 8, and 9 in a discovery cohort comprising 452 SSc patients and 537 controls and a replication cohort consisting of 1,170 SSc patients and 925 controls. In addition, we analyzed 15-year followup data on 964 patients to assess the potential association of TLR variants with the development of disease complications. We analyzed the functional impact of the associated polymorphism on monocyte-derived dendritic cells., Results: In the discovery cohort, we observed that a rare functional polymorphism in TLR2 (Pro631His) was associated with antitopoisomerase (antitopo) positivity (odds ratio 2.24 [95% confidence interval 1.24-4.04], P=0.003). This observation was validated in the replication cohort (odds ratio 2.73 [95% confidence interval 1.85-4.04], P=0.0001). In addition, in the replication cohort the TLR2 variant was associated with the diffuse subtype of the disease (P=0.02) and with the development of pulmonary arterial hypertension (PAH) (Cox proportional hazards ratio 5.61 [95% confidence interval 1.53-20.58], P=0.003 by log rank test). Functional analysis revealed that monocyte-derived dendritic cells carrying the Pro63His variant produced increased levels of inflammatory mediators (tumor necrosis factor α and interleukin-6) upon TLR-2-mediated stimulation (both P<0.0001)., Conclusion: Among patients with SSc, the rare TLR2 Pro631His variant is robustly associated with antitopoisomerase positivity, the diffuse form of the disease, and the development of PAH. In addition, this variant influences TLR-2-mediated cell responses. Further research is needed to elucidate the precise role of TLR-2 in the pathogenesis of SSc., (Copyright © 2012 by the American College of Rheumatology.)

Objective: The pathogenesis of psoriatic arthritis (PsA) remains poorly understood. The underlying chronic inflammatory immune response is thought to be triggered by unknown environmental factors potentially arising from a defective immune function. We undertook this study to determine whether an impaired acute inflammatory response by dendritic cells (DCs) might compromise the clearance of bacteria and predispose to chronic inflammation., Methods: We determined cytokine production by DCs from healthy controls and from patients with rheumatoid arthritis, PsA, and psoriasis in response to Mycobacterium tuberculosis, Mycobacterium avium paratuberculosis, and a range of other bacteria and Toll-like receptor (TLR) ligands. Phenotypic differences involved in cellular responses against (myco)bacteria were determined by quantitative polymerase chain reaction and flow cytometry., Results: The secretion of proinflammatory cytokines by PsA DCs was impaired upon in vitro challenge with mycobacteria and TLR-2 ligands. This impairment was associated with elevated serum levels of C-reactive protein. The expression of TLR-2 and other receptors known to mediate mycobacterial recognition was unaltered. In contrast, the intracellular TLR inhibitors suppressor of cytokine signaling 3 and A20 were more highly expressed in DCs from PsA patients. PsA DCs further demonstrated up-regulated levels of ATG16L1, NADPH oxidase 2, and LL37, which are molecules implicated in the immune response against intracellular bacteria., Conclusion: Our findings indicate that DCs from PsA patients have a disordered immune response toward some species of (myco)bacteria. This might predispose to impaired immune responses to, and in turn impaired clearance of, these bacteria, setting the stage for the chronic inflammation of joints, entheses, skin, and the gut., (Copyright © 2011 by the American College of Rheumatology.)

Objective: Recently, a functional PTPN22 variant (R263Q; rs33996649) was found to be associated with systemic lupus erythematosus (SLE). This study was undertaken to analyze the influence of this polymorphism on the risk of rheumatoid arthritis (RA)., Methods: RA patients (n = 5,579) were recruited from outpatient clinics from 6 different countries (Spain, New Zealand, the UK, Norway, The Netherlands, and Germany). Healthy controls (n = 5,392) were recruited from the same areas. There was 100% power to detect an effect equivalent to that observed in SLE. Samples were genotyped for the PTPN22 R263Q (rs33996649) and PTPN22 R620W (rs2476601) polymorphisms using a TaqMan 5'-allele discrimination assay. The effect of the R263Q variant was analyzed in isolation and in combination with the effect of R620W, using Unphased and Stata 10 software. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined., Results: The minor allele A of PTPN22 R263Q was significantly associated with a lower risk of RA in the pooled analysis of the 6 populations (P = 0.016, Mantel-Haenszel pooled OR 0.80 [95% CI 0.67-0.96]), independent of the effect of the R620W polymorphism. Both polymorphisms had an additive effect. The more RA risk alleles carried (R263Q G allele, R620W T allele), the higher the RA risk (for 2 versus 1 risk allele P = 0.014, OR 1.28 [95% CI 1.05-1.55], for 3 versus 1 risk allele P = 6.67 × 10(-11) , OR 2.01 [1.63-2.48], and for 4 versus 1 risk allele P = 6.50 × 10(-11) , OR 3.55 [2.42-5.20])., Conclusion: Our findings indicate that the minor allele of the PTPN22 R263Q polymorphism is associated with a lower risk of RA. This association is independent of the well-established association between PTPN22 R620W and RA. Both polymorphisms have an additive effect on the risk of RA., (Copyright © 2011 by the American College of Rheumatology.)

Objective: Genome-wide association studies carried out in rheumatoid arthritis (RA) have led to the discovery of several genetic associations with this disease. Still, the current associated genetic variations can explain only part of the genetic risk involved in RA, and it is well recognized that these genome-wide association studies are likely underpowered to detect all common disease variants. This study was undertaken to explore the genomic regions showing low-significance associations in previous genome-wide association studies of RA., Methods: To reduce the false-positive signal fraction, we exploited pathway analysis to prioritize regions containing genes most likely to be implicated in RA. We hypothesized that true disease genes would be in a similar pathway. Therefore, genes from similar pathways but located in different regions were prioritized for replication using Prioritizer software. A total of 384 genetic variants selected from previous RA genome-wide association studies were tested in a Spanish case-control discovery cohort comprising 376 RA patients and 478 healthy controls for replication. Statistically significant associations were further validated in replication cohorts from Spain and The Netherlands. The study consisted of a total of 1,818 RA patients and 2,498 controls., Results: We detected a novel genetic association between RA and the MSRA gene (rs10903323) in the Spanish combined population (P = 2.91 × 10(-5) , odds ratio [OR] 1.51). This association was further tested in our independent Dutch replication cohort. Combined analysis showed an overall association of MSRA with RA (P = 3.19 × 10(-4) , OR 1.28)., Conclusion: Our findings indicate that a novel association in the MSRA gene is related to oxidative stress and support the notion of a major role for this process in RA., (Copyright © 2010 by the American College of Rheumatology.)

Objective: To investigate the possible role of the FAS -670A>G functional polymorphism in the genetic predisposition to systemic sclerosis (SSc) susceptibility or clinical phenotype., Methods: A total of 2,900 SSc patients and 3,186 healthy controls were included in this study. We analyzed the genotype and allele frequencies of the FAS -670A>G polymorphism in 9 distinct ethnic cohorts, including 6 cohorts of European ancestry (a Spanish cohort of 228 SSc patients and 265 controls, a Dutch cohort of 203 SSc patients and 277 controls, a German cohort of 313 SSc patients and 247 controls, an Italian cohort of 323 SSc cases and 89 controls, a British cohort of 269 SSc patients, and a Swedish cohort of 182 patients) and 3 distinct ethnic cohorts from the US (a cohort of 1,047 white patients and 692 controls, a cohort of 159 Hispanic patients and 137 controls, and a cohort of 176 black SSc patients and 194 controls). Genotyping was performed using a TaqMan 5' allelic discrimination assay., Results: In the British, Italian, and American white cohorts we observed an association of the FAS -670G allele with limited cutaneous SSc (lcSSc) (odds ratios [ORs] 1.25, 1.43, and 1.18, respectively). A meta-analysis comprising all 9 cohorts revealed an association of both the FAS -670G allele (OR 1.10) and the FAS -670GG genotype (OR 1.13) with the lcSSc phenotype. In a meta-analysis including only white subjects, both the FAS -670G allele and the FAS -670GG genotype remained associated with lcSSc (allele OR 1.12; genotype OR 1.16). In addition, a recessive model of the -670GG genotype exhibited a strong association with SSc, lcSSc, and anticentromere antibody-positive lcSSc (OR 1.23, OR 1.33, and OR 1.45, respectively)., Conclusion: Our data show that the FAS -670A>G polymorphism plays a role in lcSSc susceptibility. A similar trend has been observed in other autoimmune diseases.

Objective: The guanine-thymidine (GT)n repeat in the HMOX1 promoter determines the level of induction of the heme-degrading enzyme heme oxygenase 1 (HO-1), which protects against inflammatory and oxidative stress. In individuals with short (GT)n repeats (where n < 25; SS genotype), higher levels of HO-1 activity are induced more rapidly than in those with long (GT)n repeats (where n > or = 25; LL genotype). Recently, it was demonstrated that HO-1 activity protects against the onset of rheumatoid arthritis (RA). The aim of this study was to determine whether the (GT)n-repeat length within the HMOX1 promoter region is associated with RA disease severity and radiographic joint damage., Methods: A cohort of 325 well-characterized RA patients and 273 controls was investigated by DNA fragment-length analysis for the association of (GT)n repeats in the HMOX1 promoter region with RA disease susceptibility and severity., Results: Although no significant differences in genotype or allele frequency were found between controls and RA patients, the odds ratios corresponded well to those in the previously described cohort. Among patients, those carrying the SS genotype had a more favorable radiographic outcome over 9 years than those carrying the LL genotype. This was unexpected since no differences in disease activity were found between the genotypes or alleles., Conclusion: Patients with the SS genotype have a better long-term radiographic outcome despite poor prognostic markers at baseline and despite disease activity at followup similar to that of patients with the LL genotype. This suggests that the HMOX1/HO-1 system is involved in the uncoupling of disease activity and joint damage and may provide a novel target for the treatment of RA.

Objective: This study was undertaken to investigate the previously reported association of the STAT4 polymorphism rs7574865 with rheumatoid arthritis (RA) in 3 different European populations from Spain, Sweden, and The Netherlands, comprising a total of 2,072 patients and 2,474 controls., Methods: Three different cohorts were included in the study: 923 RA patients and 1,296 healthy controls from Spain, 273 RA patients and 285 healthy controls from Sweden, and 876 RA patients and 893 healthy controls from The Netherlands. DNA from patients and controls was obtained from peripheral blood. Samples were genotyped for the STAT4 single-nucleotide polymorphism rs7574865 using a TaqMan 5'-allele discrimination assay. The chi-square test was performed to compare allele and genotype distributions. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated., Results: We observed a significantly increased frequency of the minor T allele in RA patients compared with healthy controls in the Spanish population (24.8% versus 20.8%; P = 0.001, OR 1.26 [95% CI 1.09-1.45]). This association was confirmed in both the Swedish population (P = 0.03, OR 1.35 [95% CI 1.03-1.77]) and the Dutch population (P = 0.03, OR 1.45 [95% CI 1.21-1.73]). The overall P value for all 3 populations was 9.79 x 10(-6) (OR 1.25 [95% CI 1.13-1.37]). No association between rs7574865 and the presence of rheumatoid factor or anti-cyclic citrullinated peptide autoantibodies was observed. A meta-analysis of all published STAT4 associations revealed an OR of 1.25 (95% CI 1.19-1.33) (P = 1 x 10(-5))., Conclusion: Our findings indicate an association between the STAT4 polymorphism rs7574865 and RA in 3 different populations, from Spain, Sweden, and The Netherlands, thereby confirming previous data.

Objective: Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll-like receptor 4 (TLR-4). The objective of this study was to investigate the involvement of TLR-4 activation in the development and progression of autoimmune destructive arthritis., Methods: A naturally occurring TLR-4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen-induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency were treated with TLR-4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL-1 in joints were evaluated after treatment., Results: The TLR-4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR-4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR-4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti-type II collagen immunity crucial for this model. Treatment with TLR-4 antagonist strongly reduced IL-1beta expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL-1-mediated autoimmune arthritis in IL-1Ra(-/-) mice and protected the mice against cartilage and bone pathology., Conclusion: In the present study, we demonstrate for the first time that inhibition of TLR-4 suppresses the severity of experimental arthritis and results in lower IL-1 expression in arthritic joints. Our data suggest that TLR-4 might be a novel target in the treatment of rheumatoid arthritis.

Objective: Fcgamma receptors (FcgammaRs) recognize immune complexes (ICs) and coordinate the immune response by modulating the functions of dendritic cells (DCs). The purpose of this study was to unravel the role of the inhibitory FcgammaRIIb in rheumatoid arthritis (RA) by studying the effect of the FCGR2B 695T>C polymorphism on susceptibility to RA, severity of the disease, and DC function., Methods: Genotyping was performed in RA patients (n = 246) and healthy blood donors (n = 269). The patients' demographic data, disease severity, and disease progression were assessed over a followup of 6 years. DCs were cultured for flow cytometry to determine the expression of FcgammaRs. For detection of FcgammaRIIb (CD32B), a unique anti-FcgammaRIIb antibody (2B6-fluorescein isothiocyanate [FITC]) was used. The capacity for antigen uptake by DCs was studied by assessing the uptake of FITC-labeled ICs. Levels of cytokine production by DCs were measured during lipopolysaccharide-mediated cell activation in the presence and absence of ICs., Results: Although no role of the FCGR2B variant in RA susceptibility was demonstrated, this variant was associated with a nearly doubled rate of radiologic joint damage during the first 6 years of RA. Multiple regression analysis showed that FCGR2B was by far the strongest predictor of joint damage identified to date. DCs from patients carrying this variant failed to display the inhibitory phenotype normally observed upon IC-mediated triggering of inflammation and displayed diminished FcgammaRII-mediated antigen uptake compared with wild-type DCs. However, the levels of FcgammaRs were not affected, suggesting that the FCGR2B variant alters the function rather than regulation of proteins., Conclusion: This study is the first to show that a single genetic variant, the FCGR2B 695T>C polymorphism, is a critical determinant of disease severity in RA and radically changes DC behavior. Our results underscore the key role of DCs in the progression of RA and reveal FcgammaRIIb as an important potential therapeutic target in RA and other autoimmune conditions.

Objective: To study whether genetic variants of macrophage migration inhibitory factor (MIF), the MIF -173G>C and CATT(5-8) alleles, are associated with disease severity and levels of circulating MIF in patients with rheumatoid arthritis (RA)., Methods: Genotyping was performed in patients with early RA and in healthy controls. Demographic data, disease activity, and outcome measurements were compared between patients with and without the MIF variants. MIF -173G>C and CATT(5-8) polymorphisms were genotyped, and a newly developed enzyme-linked immunosorbent assay for human MIF was used. Allele and genotype distributions of the MIF -173G>C and CATT(5-8) polymorphisms were compared between patients and controls by chi-square test. Multiple regression analysis was performed to assess the independence of the MIF functional genetic variants as risk factors for radiologic joint damage., Results: Genotyping of the -173G>C and CATT(5-8) polymorphisms of MIF in RA patients and healthy individuals (n = 277 each) revealed similar frequencies of genotypes and haplotypes in both groups. No significant differences in demographic or clinical features were observed between RA patients carrying the MIF -173C allele or the MIF CATT7 allele or both and non-carrier RA patients. Radiologic joint damage was significantly higher in patients carrying risk alleles of the MIF -173G>C or the MIF CATT(5-8) functional variants. No synergistic effects between both genetic variants were observed. Multivariate regression analysis revealed that presence of the MIF -173C/C and MIF CATT(7/7) genotypes and having 1 MIF -173C allele were independent prognostic variables. Carriership of the MIF -173C allele (P = 0.002) or MIF CATT7 allele (P = 0.004) was associated with significantly higher circulating MIF levels compared with those in subjects having none of the risk-conferring alleles, and greater circulating MIF levels correlated with more severe radiologic joint damage (r = 0.64, P = 0.001)., Conclusion: The MIF polymorphisms are not associated with RA susceptibility but are associated with high levels of radiologic joint damage. High circulating MIF levels were shown to correlate strongly with radiologic joint damage, suggesting that MIF expression is genetically determined and can be used as a novel prognostic tool in RA.

Objective: To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls., Methods: Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients., Results: TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients., Conclusion: TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.

Objective: Directional migration of leukocytes is orchestrated by the regulated expression of chemokine receptors and their ligands. The receptor CXCR6 is abundantly expressed by Th1-polarized effector/memory lymphocytes accumulating at inflammatory sites. This study was undertaken to examine the presence of CXCR6+ T cells and of CXCL16, the only ligand for CXCR6, in the joints of patients with rheumatoid arthritis (RA)., Methods: Flow cytometry analysis of the expression of CXCR6 by peripheral blood and synovial fluid (SF) T cells. In addition, by performing conventional and real-time reverse transcriptase-polymerase chain reaction, immunohistochemistry, and enzyme-linked immunosorbent assay, we determined the expression of CXCL16 and its protease ADAM-10 within synovium and by cultured macrophages. SF T cell migration was studied with the Transwell system., Results: Accumulation of CXCR6+ T cells within RA SF coincided with highly elevated levels of CXCL16+ macrophages. In vitro studies revealed that monocytes started to express CXCL16 upon differentiation into macrophages, and that RA SF and tumor necrosis factor (TNF) enhanced CXCL16 expression. Moreover, RA patients responding to anti-TNF therapy showed a strongly decreased CXCL16 expression, whereas nonresponding patients did not. Interestingly, ADAM-10, a recently identified protease of CXCL16, was abundantly expressed by CXCL16+ macrophages in vitro and in RA in vivo, which resulted in increased levels of cleaved CXCL16 in RA SF relative to controls. Finally, CXCR6+ T cells from RA SF were attracted by CXCL16., Conclusion: These data provide evidence that enhanced production of CXCL16 in RA synovia leads to recruitment of CXCR6+ memory T cells, thereby contributing to the inflammatory cascade associated with RA pathology.

Objective: To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals., Methods: Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor alpha [TNFalpha]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNFalpha and IL-6 was measured using Luminex bead array technology., Results: In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4., Conclusion: TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.

Objective: To evaluate Fcgamma receptor (FcgammaR) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), IL-12, and matrix metalloproteinase 1 (MMP-1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of FcgammaRI, FcgammaRII, and FcgammaRIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation., Methods: Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. FcgammaR I, II, and III were detected, as well as the proinflammatory mediators IL-1, TNFalpha, IL-12, and MMP-1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony-stimulating factor to obtain macrophages. Using fluorescence-activated cell sorting, the expression of FcgammaRI, FcgammaRII, and FcgammaRIII was determined. On day 7, macrophages were stimulated with heat-aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme-linked immunosorbent assay, and production of gelatinases/collagenases was measured by degradation of fluorescent gelatin., Results: Immunohistochemistry showed higher FcgammaRII and FcgammaRIII expression in RA synovium than in controls. FcgammaRII and FcgammaRIII, but not FcgammaRI, were highly correlated with the number of synovial macrophages. Consistent with this, TNFalpha expression correlated positively with FcgammaRIII expression. Moreover, MMP-1 expression strongly correlated with FcgammaR I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of FcgammaRII and FcgammaRIII compared with controls. Twenty-four hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNFalpha and gelatinase/collagenase was measured., Conclusion: RA synovium and mature RA macrophages express significantly elevated levels of FcgammaRII and FcgammaRIII, resulting in much higher production of TNFalpha and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of FcgammaR on mature synovial macrophages is involved in the pathology of RA.

Objective: To examine the expression patterns of interkeukin-18 (IL-18) in synovial biopsy tissue of patients with rheumatoid arthritis (RA), and to determine whether expression of this primary cytokine is related to the expression of other cytokines and adhesion molecules and related to the degree of joint inflammation., Methods: Biopsy specimens of knee synovial tissue either without synovitis (n = 6) or with moderate or severe synovitis (n = 11 and n = 12, respectively) were obtained from 29 patients with active RA. Paraffin-embedded, snap-frozen sections were used for immunohistochemical detection of IL-18, tumor necrosis factor alpha (TNFalpha), IL-1beta, IL-12, and IL-17. Furthermore, adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, and cell markers CD3, CD14, and CD68 were stained., Results: IL-18 staining was detectable in 80% of the RA patients, in both the lining and sublining of the knee synovial tissue. IL-18 expression in the synovial tissue was strongly correlated with the expression of IL-1beta (in the sublining r = 0.72, in the lining r = 0.71; both P < 0.0001) and TNFalpha (in the sublining r = 0.59, P < 0.0007, and in the lining r = 0.68, P < 0.0001). In addition, IL-18 expression in the sublining correlated with macrophage infiltration (r = 0.64, P < 0.0007) and microscopic inflammation scores (r = 0.78, P < 0.0001), and with the acute-phase reaction as measured by the erythrocyte sedimentation rate (r = 0.61, P < 0.0004). Interestingly, RA synovial tissue that coexpressed IL-18 and IL-12 demonstrated enhanced levels of the Th1-associated cytokine IL-17., Conclusion: Our results show that expression of IL-18 is associated with that of IL-1beta and TNFalpha and with local inflammation in the synovial tissue of patients with RA. In addition, synovial IL-18 expression correlates with the acute-phase response. These data indicate that IL-18 is a primary proinflammatory cytokine in RA that drives the local production of IL-1beta and TNFalpha.

Objective: It has been proposed that noninherited maternal antigens (NIMA) (HLA-DR antigens) might play a role in susceptibility to rheumatoid arthritis (RA), especially in patients who are not genetically predisposed, such as those who are HLA-DR4 and/or shared epitope (SE) negative. The present study was undertaken to test the NIMA hypothesis in a large cohort of European RA patients assembled by the European Consortium on RA Families (ECRAF)., Methods: HLA-DRB1 oligotyping was performed in families of European RA patients for whom both parents were alive. These families were consecutively recruited by the ECRAF between 1996 and 1998, for association studies. The frequencies of HLA-DR NIMA were compared with those of the noninherited paternal antigens (NIPA) after stratification for HLA-DR*04, *0401 and/or *0404, and SE status. NIMA or NIPA that coincided with inherited HLA-DR antigens were considered redundant and excluded from analysis. Calculations concerning the whole group and restricted to patients lacking parental RA were performed., Results: One hundred seventy families from France (n = 81), Belgium (n = 23), Spain (n = 24), Italy (n = 19), Portugal (n = 14), and The Netherlands (n = 9) were oligotyped. The group of probands was predominantly female (88%), positive for rheumatoid factor, DR*04, and SE (71%, 58%, and 75%, respectively), and had erosive disease (75%). Parental RA was reported in 21 families. Using the NIPA as control, the frequency of HLA-DRB1*04, *0401 and/or *0404-, or SE-positive NIMA was not found to be increased in patients lacking these susceptibility alleles. The same was true when the 21 probands with parental RA were excluded from analysis. In DRB1*04-positive patients, we found no evidence of a relevant effect of HLA-DR3 or DR6 in the NIMA., Conclusion: Our results do not support the notion that noninherited maternal antigens have a role in susceptibility to RA in the offspring.