Transethnic meta-analysis identifies GSDMA and PRDM1 as susceptibility genes to systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterised by skin and systemic fibrosis culminating in organ damage. Previous genetic studies including genome-wide association studies (GWAS) have identified 12 susceptibility loci satisfying genome-wide significance. Transethnic meta-analyses have successfully expanded the list of susceptibility genes and deepened biological insights for other autoimmune diseases. METHODS: We performed transethnic meta-analysis of GWAS in the Japanese and European populations, followed by a two-staged replication study comprising a total of 4436 cases and 14 751 controls. Associations between significant single nuclear polymorphisms (SNPs) and neighbouring genes were evaluated. Enrichment analysis of H3K4Me3, a representative histone mark for active promoter was conducted with an expanded list of SSc susceptibility genes. RESULTS: We identified two significant SNP in two loci, GSDMA and PRDM1, both of which are related to immune functions and associated with other autoimmune diseases (p=1.4×10-10 and 6.6×10-10, respectively). GSDMA also showed a significant association with limited cutaneous SSc. We also replicated the associations of previously reported loci including a non-GWAS locus, TNFAIP3. PRDM1 encodes BLIMP1, a transcription factor regulating T-cell proliferation and plasma cell differentiation. The top SNP in GSDMA was a missense variant and correlated with gene expression of neighbouring genes, and this could explain the association in this locus. We found different human leukocyte antigen (HLA) association patterns between the two populations. Enrichment analysis suggested the importance of CD4-naïve primary T cell. CONCLUSIONS: GSDMA and PRDM1 are associated with SSc. These findings provide enhanced insight into the genetic and biological basis of SSc.

Vasoactive Therapy in Systemic Sclerosis: Real-life Therapeutic Practice in More Than 3000 Patients.

OBJECTIVE: Vasculopathy is a key factor in the pathophysiology of systemic sclerosis (SSc) and the main cause for Raynaud phenomenon (RP), digital ulcers (DU), and/or pulmonary arterial hypertension (PAH). It is so far unknown how patients with SSc are treated with vasoactive agents in daily practice. To determine to which extent patients with SSc were treated with different vasoactive agents, we used data from the German Network for Systemic Scleroderma registry. METHODS: The data of 3248 patients with SSc were analyzed. RESULTS: Patients were treated with vasoactive drugs in 61.1% of cases (1984/3248). Of these, 47.6% received calcium channel inhibitors, followed by 34.2% treated with angiotensin-converting enzyme (ACE) inhibitors, 21.1% treated with intravenous (IV) prostanoids, 10.1% with pentoxifylline, 8.8% with angiotensin 1 receptor antagonists (AT1RA), 8.7% with endothelin 1 receptor antagonists (ET1RA), 4.1% with phosphodiesterase type 5 (PDE5) inhibitors, and 5.3% with others. Patients with RP received vasoactive therapy in 63.3% of cases, with DU in 70.1%, and with PAH in 78.2% of cases. Logistic regression analysis revealed that patients with PAH were significantly more often treated with PDE5 inhibitors and ET1RA, and those with DU with ET1RA and IV prostanoids. In addition, 41.8% of patients were treated with ACE inhibitors and/or AT1RA. Patients registered after 2009 received significantly more often ET1RA, AT1RA, and IV prostanoids compared with patients registered prior to 2005. CONCLUSION: These data clearly indicate that many patients with SSc do not yet receive sufficient vasoactive therapy. Further, in recent years, a marked change of treatment regimens can be observed.

Defective removal of ribonucleotides from DNA promotes systemic autoimmunity.

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Disease progression in systemic sclerosis-overlap syndrome is significantly different from limited and diffuse cutaneous systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc)-overlap syndromes are a very heterogeneous and remarkable subgroup of SSc-patients, who present at least two connective tissue diseases (CTD) at the same time, usually with a specific autoantibody status. OBJECTIVES: To determine whether patients, classified as overlap syndromes, show a disease course different from patients with limited SSc (lcSSc) or diffuse cutaneous SSc (dcSSc). METHODS: The data of 3240 prospectively included patients, registered in the database of the German Network for Systemic Scleroderma and followed between 2003 and 2013, were analysed. RESULTS: Among 3240 registered patients, 10% were diagnosed as SSc-overlap syndrome. Of these, 82.5% were female. SSc-overlap patients had a mean age of 48±1.2 years and carried significantly more often 'other antibodies' (68.0%; p<0.0001), including anti-U1RNP, -PmScl, -Ro, -La, as well as anti-Jo-1 and -Ku antibodies. These patients developed musculoskeletal involvement earlier and more frequently (62.5%) than patients diagnosed as lcSSc (32.2%) or dcSSc (43.3%) (p<0.0001). The onset of lung fibrosis and heart involvement in SSc-overlap patients was significantly earlier than in patients with lcSSc and occurred later than in patients with dcSSc. Oesophagus, kidney and PH progression was similar to lcSSc patients, whereas dcSSc patients had a significantly earlier onset. CONCLUSIONS: These data support the concept that SSc-overlap syndromes should be regarded as a separate SSc subset, distinct from lcSSc and dcSSc, due to a different progression of the disease, different proportional distribution of specific autoantibodies, and of different organ involvement.

A regulatory variant in CCR6 is associated with susceptibility to antitopoisomerase-positive systemic sclerosis.

OBJECTIVE: Recognition of the well-known pleiotropism of autoimmune genes supports the concept of a shared pathogenesis across autoimmune diseases such as rheumatoid arthritis (RA) and systemic sclerosis (SSc). Studies have reproducibly demonstrated an association between susceptibility to RA and polymorphisms of the CCR6 gene, a surface marker for Th17 cells, and the causal variant was recently identified. The present study was thus undertaken to investigate whether CCR6 polymorphisms could also be associated with susceptibility to SSc. METHODS: Twelve tag single-nucleotide polymorphisms (SNPs) of CCR6, including the known RA-associated SNP rs3093023, were genotyped in a total of 2,411 SSc patients and 7,084 healthy individuals from 3 European populations (France, Italy, and Germany). Meta-analyses of the data were performed to assess whether an association exists between CCR6 polymorphisms and susceptibility to SSc or its main subtypes. Direct sequencing of DNA was performed to ascertain whether the functional dinucleotide polymorphism of CCR6 previously identified in RA (CCR6DNP) was also present in SSc. RESULTS: Combined analyses revealed an association between the rs10946216 SNP and SSc susceptibility (odds ratio [OR] 1.13, 95% confidence interval [95% CI] 1.05-1.21, adjusted P [P(adj)] = 0.026). The rs3093023 A allele and rs10946216 T allele were in high linkage disequilibrium, and both were found to confer disease susceptibility in the antitopoisomerase-positive subset of SSc patients (OR 1.27, 95% CI 1.13-1.42, P(adj) = 1.5 × 10(-3) and OR 1.32, 95% CI 1.17-1.48, P(adj) = 9.0 × 10(-5), respectively, relative to healthy controls). Direct sequencing of the DNA of 78 individuals supported the hypothesis that the regulatory dinucleotide CCR6DNP could be the causal variant in SSc. CONCLUSION: The results of this study establish CCR6 as a new susceptibility factor for antitopoisomerase-positive SSc, as demonstrated in 3 European Caucasian populations, confirming the notion that SSc and RA could conceivably share autoimmune risk alleles. The results also suggest a potential role of the interleukin-17 pathway in SSc.

TGFß receptor gene variants in systemic sclerosis-related pulmonary arterial hypertension: results from a multicentre EUSTAR study of European Caucasian patients.

INTRODUCTION: Systemic sclerosis (SSc)-related pulmonary arterial hypertension (PAH) has emerged as a major mortality prognostic factor. Mutations of transforming growth factor beta (TGFß) receptor genes strongly contribute to idiopathic and familial PAH. OBJECTIVE: To explore the genetic bases of SSc-PAH, we combined direct sequencing and genotyping of candidate genes encoding TGFß receptor family members. MATERIALS AND METHODS: TGFß receptor genes, BMPR2, ALK1, TGFR2 and ENG, were sequenced in 10 SSc-PAH patients, nine SSc and seven controls. In addition, 22 single-nucleotide polymorphisms (SNP) of these four candidate genes were tested for association in a first set of 824 French Caucasian SSc patients (including 54 SSc-PAH) and 939 controls. The replication set consisted of 1516 European SSc (including 219 SSc-PAH) and 3129 controls from the European League Against Rheumatism Scleroderma Trials and Research group network. RESULTS: No mutation was identified by direct sequencing. However, two repertoried SNP, ENG rs35400405 and ALK1 rs2277382, were found in SSc-PAH patients only. The genotyping of 22 SNP including the latter showed that only rs2277382 was associated with SSc-PAH (p=0.0066, OR 2.13, 95% CI 1.24 to 3.65). Nevertheless, this was not replicated with the following result in combined analysis: p=0.123, OR 0.79, 95% CI 0.59 to 1.07. CONCLUSIONS: This study demonstrates the lack of association between these TGFß receptor gene polymorphisms and SSc-PAH using both sequencing and genotyping methods.

A trans-ethnic genetic study of rheumatoid arthritis identified FCGR2A as a candidate common risk factor in Japanese and European populations.

Rheumatoid arthritis (RA) is a common systemic autoimmune disease and its onset and prognosis are controlled by genetic, immunological, and environmental factors. The HLA locus, particularly HLA-DRB1, is its strongest genetic risk determinant across ethnicities. Several other genes, including PTPN22 and PADI4, show modest association with RA. However, they cover only a part of its genetic components and their relative contribution is different between populations. To identify novel genetic determinants, we took a candidate gene approach in a trans-ethnic manner. After critical selection of 169 genes based on their immunological function, we performed SNP discovery of these genes by the resequencing of exons and surrounding areas using European and Japanese DNAs. We then generated a panel of 1,509 SNPs for case-control association study in both populations. The DerSimonian-Laird test for meta-analysis, using the combined results of the two populations, identified rs7551957 at the 5'-flanking region of the low-affinity Fc-gamma receptor IIa (FCGR2A) gene as the strongest candidate for the association (p = 8.6 × 10(-5), odds ratio = 1.58 with 95%CI 1.25-1.99). Suggestive signals were also obtained for three SNPs in the dihydropyrimidine dehydrogenase (DPYD) gene (rs6685859; p = 1.3 × 10(-4), rs7550959; p = 1.5 × 10(-4) and rs7531138; p = 1.7 × 10(-4)) and an intronic SNP, rs2269310, of the erythrocytic spectrin beta (SPTB) gene (p = 7.9 × 10(-4)).

Frequency of disease-associated and other nuclear autoantibodies in patients of the German Network for Systemic Scleroderma: correlation with characteristic clinical features.

INTRODUCTION: In the present study, we analysed in detail nuclear autoantibodies and their associations in systemic sclerosis (SSc) patients included in the German Network for Systemic Scleroderma Registry. METHODS: Sera of 863 patients were analysed according to a standardised protocol including immunofluorescence, immunoprecipitation, line immunoassay and immunodiffusion. RESULTS: Antinuclear antibodies (ANA) were detected in 94.2% of patients. In 81.6%, at least one of the autoantibodies highly associated with SSc or with overlap syndromes with scleroderma features was detected, that is, anti-centromere (35.9%) or anti-topoisomerase I (30.1%), followed in markedly lower frequency by antibodies to PM-Scl (4.9%), U1-ribonucleoprotein (U1-RNP) (4.8%), RNA polymerases (RNAPs) (3.8%), fibrillarin (1.4%), Ku (1.2%), aminoacyl-transfer RNA synthetases (0.5%), To (0.2%) and U11-RNP (0.1%). We found that the simultaneous presence of SSc-associated autoantibodies was rare (1.6%). Furthermore, additional autoantibodies were detected in 55.4% of the patients with SSc, of which anti-Ro/anti-La, anti-mitochondrial and anti-p25/p23 antibodies were most frequent. The coexistence of SSc-associated and other autoantibodies was common (43% of patients). SSc-associated autoantibodies disclosed characteristic associations with clinical features of patients, some of which were previously not acknowledged. CONCLUSIONS: This study shows that five autoantigens (that is, centromere, topoisomerase I, PM-Scl, U1-RNP and RNAP) detected more than 95% of the known SSc-associated antibody responses in ANA-positive SSc patients and characterise around 79% of all SSc patients in a central European cohort. These data confirm and extend previous data underlining the central role of the determination of ANAs in defining the diagnosis, subset allocation and prognosis of SSc patients.

Genome-wide scan identifies TNIP1, PSORS1C1, and RHOB as novel risk loci for systemic sclerosis.

Systemic sclerosis (SSc) is an orphan, complex, inflammatory disease affecting the immune system and connective tissue. SSc stands out as a severely incapacitating and life-threatening inflammatory rheumatic disease, with a largely unknown pathogenesis. We have designed a two-stage genome-wide association study of SSc using case-control samples from France, Italy, Germany, and Northern Europe. The initial genome-wide scan was conducted in a French post quality-control sample of 564 cases and 1,776 controls, using almost 500 K SNPs. Two SNPs from the MHC region, together with the 6 loci outside MHC having at least one SNP with a P<10(-5) were selected for follow-up analysis. These markers were genotyped in a post-QC replication sample of 1,682 SSc cases and 3,926 controls. The three top SNPs are in strong linkage disequilibrium and located on 6p21, in the HLA-DQB1 gene: rs9275224, P = 9.18×10(-8), OR = 0.69, 95% CI [0.60-0.79]; rs6457617, P = 1.14×10(-7) and rs9275245, P = 1.39×10(-7). Within the MHC region, the next most associated SNP (rs3130573, P = 1.86×10(-5), OR = 1.36 [1.18-1.56]) is located in the PSORS1C1 gene. Outside the MHC region, our GWAS analysis revealed 7 top SNPs (P<10(-5)) that spanned 6 independent genomic regions. Follow-up of the 17 top SNPs in an independent sample of 1,682 SSc and 3,926 controls showed associations at PSORS1C1 (overall P = 5.70×10(-10), OR:1.25), TNIP1 (P = 4.68×10(-9), OR:1.31), and RHOB loci (P = 3.17×10(-6), OR:1.21). Because of its biological relevance, and previous reports of genetic association at this locus with connective tissue disorders, we investigated TNIP1 expression. A markedly reduced expression of the TNIP1 gene and also its protein product were observed both in lesional skin tissue and in cultured dermal fibroblasts from SSc patients. Furthermore, TNIP1 showed in vitro inhibitory effects on inflammatory cytokine-induced collagen production. The genetic signal of association with TNIP1 variants, together with tissular and cellular investigations, suggests that this pathway has a critical role in regulating autoimmunity and SSc pathogenesis.

Myelin basic protein as a novel genetic risk factor in rheumatoid arthritis--a genome-wide study combined with immunological analyses.

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10(-4) by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10(-8), OR 1.23, 95% CI: 1.14-1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

The human AIRE gene at chromosome 21q22 is a genetic determinant for the predisposition to rheumatoid arthritis in Japanese population.

Rheumatoid arthritis (RA) is a typical complex trait and the major cause of chronic inflammation worldwide. Although multiple genetic loci have been shown for their association with the onset of RA, they cover only a part of its genetic components and are largely ethnicity-specific. To identify novel genetic factors related to the predisposition and prognosis of RA in Japanese, we conducted a large-scale genome-wide association (GWA) study. We performed a GWA analysis by scanning the genome of 1247 RA cases and 1486 controls for 277 420 single nucleotide polymorphisms (SNPs), followed by replication analysis using two independent sample sets consisting of 1865 cases and 1623 controls, and 2303 cases and 3380 controls. We identified two SNPs, rs2075876 and rs760426, in intron of the autoimmune regulator AIRE gene at chromosome 21q22 that showed strong associations with the disease (P= 3.6 × 10(-9) and P= 4.4 × 10(-8), respectively). Rs1800250, in exon7 of AIRE, was in strong linkage disequilibrium (r(2)= 0.94) with rs2075876 and introduced an amino acid alteration (S278R) in the SAND domain of the AIRE protein. In silico analysis showed the decreased transcription of AIRE by the risk allele of rs2075876 compared with the alternative allele (P= 6.8 × 10(-5)). No correlation was observed between the rs2075876 genotype and quantitative traits reflecting the progression of RA. As AIRE is a key molecule which regulates the expression and presentation of self-antigens in thymic negative selection, its downregulation by genetic polymorphisms may result in the survival of auto-reactive T cells to trigger auto-inflammation in RA.

Association of the X-chromosomal genes TIMP1 and IL9R with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory joint disease with features of an autoimmune disease with female predominance. Candidate genes located on the X-chromosome were selected for a family trio-based association study. METHODS: A total of 1452 individuals belonging to 3 different sample sets were genotyped for 16 single-nucleotide polymorphisms (SNP) in 7 genes. The first 2 sets consisted of 100 family trios, each of French Caucasian origin, and the third of 284 additional family trios of European Caucasian origin. Subgroups were analyzed according to sex of patient and presence of anti-cyclic citrullinated peptide (anti-CCP) autoantibodies. RESULTS: Four SNP were associated with RA in the first sample set and were genotyped in the second set. In combined analysis of sets 1 and 2, evidence remained for association of 3 SNP in the genes UBA1, TIMP1, and IL9R. These were again genotyped in the third sample set. Two SNP were associated with RA in the joint analysis of all samples: rs6520278 (TIMP1) was associated with RA in general (p = 0.035) and rs3093457 (IL9R) with anti-CCP-positive RA patients (p = 0.037) and male RA patients (p = 0.010). A comparison of the results with data from whole-genome association studies further supports an association of RA with TIMPL The sex-specific association of rs3093457 (IL9R) was supported by the observation that men homozygous for rs3093457-CC are at a significantly higher risk to develop RA than women (risk ratio male/female = 2.98; p = 0.048). CONCLUSION: We provide evidence for an association of at least 2 X-chromosomal genes with RA: TIMP1 (rs6520278) and IL9R (rs3093457).

High frequency of corticosteroid and immunosuppressive therapy in patients with systemic sclerosis despite limited evidence for efficacy.

INTRODUCTION: In systemic sclerosis (SSc) little evidence for the effectiveness of anti-inflammatory and immunosuppressive therapy exists. The objective of this study was to determine the extent to which SSc patients are treated with corticosteroids and immunosuppressive agents. METHODS: Data on duration and dosage of corticosteroids and on the type of immunosuppressive agent were analyzed from 1,729 patients who were registered in the German Network for Systemic Scleroderma (DNSS). RESULTS: A total 41.3% of all registered SSc patients was treated with corticosteroids. Corticosteroid use was reported in 49.1% of patients with diffuse cutaneous SSc and 31.3% of patients with limited cutaneous SSc (P < 0.0001). Among patients with overlap disease characteristics, 63.5% received corticosteroids (P < 0.0001 vs. limited cutaneous SSc). A total 16.1% of the patients received corticosteroids with a daily dose >or= 15 mg prednisone equivalent. Immunosuppressive therapy was prescribed in 35.8% of patients. Again, among those patients with overlap symptoms, a much higher proportion (64.1%) was treated with immunosuppressive agents, compared with 46.4% of those with diffuse cutaneous SSc sclerosis and 22.2% of those with limited cutaneous SSc (P < 0.0001). The most commonly prescribed drugs were methotrexate (30.5%), cyclophosphamide (22.2%), azathioprine (21.8%) and (hydroxy)chloroquine (7.2%). The use of these compounds varied significantly between medical subspecialties. CONCLUSIONS: Despite limited evidence for the effectiveness of corticosteroids and immunosuppressive agents in SSc, these potentially harmful drugs are frequently prescribed to patients with all forms of SSc. Therefore, this study indicates the need to develop and communicate adequate treatment recommendations.

No association between systemic sclerosis and C77G polymorphism in the human PTPRC (CD45) gene.

OBJECTIVE: The functional variant C77G (rs17612648) of PTPRC (CD45) was described to confer risk for systemic sclerosis (SSc) in German Caucasians. We analyzed this association in an independent, larger German cohort. METHODS: We genotyped 171 cases and 179 controls. Cases were subgrouped according to sex, autoantibody profiles, or clinical subsets. RESULTS: No association of SSc with C77G was detected in the whole dataset, in subgroups, or in combined analyses with a previous study. CONCLUSION: The results do not confirm PTPRC C77G as a general and independent risk factor for development of SSc.

Sequence analysis of TNFRSF13b, encoding TACI, in patients with systemic lupus erythematosus.

B cell activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL), and their receptors BAFF receptor (BAFFR), B cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI) are involved in the regulation of B cell homeostasis and differentiation. BAFF overexpression leads to systemic lupus erythematosus (SLE) in mice and elevated BAFF levels have been observed in human SLE and mouse models for SLE. Furthermore, genetic inactivation of TACI in mice results in a SLE-like phenotype. Based on our recent finding that TACI is mutated in patients with common variable immunodeficiency, of whom more than 30% suffer from autoimmune conditions, we analyzed TACI in humans with SLE. Sequence analysis of TNFRSF13b/TACI in 119 unrelated SLE patients revealed four variants: R20C in exon 1, R72H in exon 3, the silent variation c.327 G > A in exon 3, and A181E in exon 4. No significant association with any of these variants was found, when compared to the frequencies of the variants in a healthy control cohort. Furthermore, the mutated alleles R20C and R72H did not segregate with the SLE phenotype in familial cases of SLE. Thus, our evaluation of the coding region of TNFRSF13b/TACI did not reveal any deleterious or disease-associated mutations.

IL-10-producing monocytes differentiate to alternatively activated macrophages and are increased in atopic patients.

BACKGROUND: Recently the immune regulatory role of T cell-derived IL-10 in allergic disease has been extensively studied. In contrast, there is mounting evidence that IL-10 might also have a role in the perpetuation of allergic inflammation and fibrotic remodeling. It has been reported that alternatively (IL-4) activated macrophages (aaMPhi) produce large quantities of IL-10 and lack IL-12 production. OBJECTIVE: Bearing this in mind, we hypothesized whether functionally different properties of IL-10-producing monocytes could be identified. METHODS: Intracellular cytokine expression of IL-10, IL-12, and IL-6 in peripheral blood CD14(+) monocytes was measured in 19 atopic patients and 18 healthy control subjects by means of flow cytometry. In addition, IL-10-secreting monocytes were sorted by means of flow cytometry. Capabilities of these cells regarding further differentiation, accessory cell capacity, and surface molecule expression were analyzed. RESULTS: Our data show a dichotomous expression pattern of either IL-10 or IL-12p40/p70 in peripheral blood monocytes after LPS stimulation. Compared with healthy control subjects, the percentage of IL-10-producing monocytes was significantly increased in atopic patients. IL-10-secreting monocytes were isolated by using an IL-10 secretion assay, and functional analysis of these sorted cells revealed that IL-10-secreting monocytes preferentially differentiate into suppressor of cytokine signaling 3 expressing aaMPhi, which perpetuate T(H)2 immune response. CONCLUSION: Our study shows the existence of an IL-10-producing monocyte subset, which is increased in atopic disease and which might facilitate allergic inflammation and fibrotic remodeling by differentiation into aaMPhi. CLINICAL IMPLICATIONS: Controlling aaMPhi in T(H)2-driven inflammatory processes might be a novel target for intervention strategies.

The -786C/T single-nucleotide polymorphism in the promoter of the gene for endothelial nitric oxide synthase: insensitivity to physiologic stimuli as a risk factor for rheumatoid arthritis.

OBJECTIVE: Shear stress is the main physiologic stimulus for the expression of NOS3, the gene for human endothelial nitric oxide synthase. Interestingly, a promoter variant of the NOS3 gene, the -786C variant, is insensitive to shear stress, and individuals homozygous for this single-nucleotide polymorphism (SNP) have an increased risk of developing coronary artery disease. The cytokine interleukin-10 (IL-10) is also capable of up-regulating endothelial NOS3 expression through binding of the transcription factor STAT-3 to a nearby promoter sequence. The aim of this study was to explore the possibility that the -786C variant of the NOS3 gene is also insensitive to IL-10 and that individuals with the -786C/C genotype are more prone to developing rheumatoid arthritis (RA). METHODS: Endothelial cells were isolated from human umbilical cord veins, clonally expanded, and analyzed for NOS3 and IL-12 expression by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Umbilical cord arteries and blood samples from RA patients were genotyped for the -786C/T SNP of the NOS3gene. RESULTS: In contrast to cells of other genotypes, endothelial cells of the -786C/C genotype did not reveal an increase in NOS3 expression upon exposure to IL-10, and the cytokine failed to suppress IL-12 expression upon stimulation of CD40. Preincubation of these cells with a 16-mer C-type decoy oligonucleotide fully reconstituted the defective IL-10-induced suppression of IL-12 synthesis. The frequency of the -786C/C genotype was significantly higher in the 596 RA patients than in the general population (19.1% versus 12.1%; P < 0.0001). CONCLUSION: Individuals with the -786C/C genotype have an increased risk of developing RA. This may be explained by the IL-10 insensitivity of the C-type NOS3 gene promoter and the resulting failure to subdue CD40-mediated proinflammatory gene expression.

Continuous enhanced expression of Hsc70 but not Hsp70 in rheumatoid arthritis synovial tissue.

OBJECTIVE: To investigate the expression of constitutive and inducible members of the Hsp70 protein family in synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: Frozen sections of synovial tissue and isolated synovial adherent cells obtained from 17 RA patients, 5 OA patients, and 1 patient with carpal tunnel syndrome (CTS) were analyzed with specific monoclonal antibodies, by immunohistochemistry, immunocytochemistry, and immunoblotting. RESULTS: Expression of the constitutive chaperone Hsc70 was increased in synovial tissue from 9 of 9 patients with RA, but was faint or undetectable in 3 of 3 samples from patients with OA. In RA samples, cells mainly of the synovial lining stained intensely for Hsc70 as well as for HLA-DR, CD14, and CD68. Also, in vitro-cultured synovial adherent cells from 8 of 9 RA patients overexpressed Hsc70 (specimens from 1 RA patient were used in both the immunochemistry and the in vitro culture studies). On immunoblots of protein extracts, the synovial and HeLa cell molecules appeared identical in size. The inducible chaperone Hsp70 was not detected in samples from any of the same 17 RA patients, except for rare, isolated cells in 3. Samples from 4 of 5 OA patients also were negative for the inducible chaperone Hsp70, and the fifth was very weakly positive. In addition, tissue from 1 patient with CTS was analyzed 10 months before diagnosis of RA. Synovial tissue from this patient showed extreme overexpression of both Hsc70 and Hsp70. CONCLUSION: In RA, synovial lining cells continuously overexpress Hsc70 but not Hsp70. Hsc70 may be up-regulated due to the high activity of these cells in several respects, including antigen processing and presentation.

Major differences in antigen-processing correlate with a single Arg71<-->Lys substitution in HLA-DR molecules predisposing to rheumatoid arthritis and with their selective interactions with 70-kDa heat shock protein chaperones.

Several HLA-DR alleles are genetically associated with rheumatoid arthritis. DRB1*0401 predominates in Northern Europe and has a characteristic (70)QKRAA motif. This sequence contacts bound peptides and the TCR. Further interactions have been suggested with additional proteins during Ag loading. We explored the much stronger processing/presentation of full-length recombinant human acetylcholine receptor alpha subunit to a specific T cell clone by APC from DRB1*0401+ than *0408+ donors. Using DR*04 transfectants, we show that this difference results largely from the single Lys71<-->Arg interchange (0401<-->0408), which scarcely affects epitope binding, rather than from any other associated polymorphism. Furthermore, we proved our recombinant polypeptides to contain the Escherichia coli 70-kDa heat shock protein molecule DnaK and its requirement for efficient processing and presentation of the epitope by DRB1*0401+ cells. According to a recent report, 70-kDa heat shock protein chaperones preferentially bind to the QKRAA, rather than the QRRAA, motif. Variations between the shared epitope motifs QKRAA and QRRAA are emphasized by underlining. We propose that such interactions enhance the intracellular epitope loading of *0401 molecules. They may thus broaden immune responses to pathogens and at least partially explain the distinct contributions of DRB1*0401 and other alleles to disease predisposition.