To measure interferon (IFN) inducible chemokines in plasma of patients with systemic sclerosis (SSc) and investigate their correlation with disease severity.
We examined the correlation of IFN-inducible chemokines, IFNγ-inducible protein-10 (IP-10/CXCL10), IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11), and monocyte chemoattractant protein-1 (MCP-1/CCL2) with the IFN gene expression signature. We generated an IFN-inducible chemokine score with the correlated chemokines, IP-10 and I-TAC and compared it in 266 SSc patients enrolled in the GENISOS cohort to that of 97 matched controls. Subsequently, the correlation between the baseline IFN-inducible chemokine score and markers of disease severity was assessed. Finally, the course of IFN-inducible chemokine score over time was examined.
The plasma IFN-inducible chemokine score correlated with the IFN gene expression signature and this score was higher in SSc patients. It also was associated with the absence of anti–RNA polymerase III antibodies, presence of anti–U1 ribonucleoprotein antibodies (RNP), but not with disease duration, type, or other autoantibodies. The chemokine scores correlated with concomitantly obtained muscle, skin and lung components of the Medsger Severity Index, as well as, FVC, DLco, creatine kinase. Its association with disease severity was independent of anti-RNP or other potential confounders (age, gender, ethnicity, disease duration, and treatment with immunosuppressive agents). Finally, there was not a significant change in the IFN-inducible chemokine score over time.
The IFN-inducible chemokine score is a stable serological marker of more severe subtype of SSc and may be useful for risk stratification regardless of disease type or duration.
AIM: To assess the esophageal motility in patients with irritable bowel syndrome (IBS) and to compare those with patients with autoimmune disorders.
METHODS: 15 patients with IBS, 22 with systemic lupus erythematosus (SLE) and 19 with systemic sclerosis (SSc) were prospectively selected from a total of 115 patients at a single university centre and esophageal motility was analysed using standard manometry (Mui Scientific PIP-4-8SS). All patients underwent esophago-gastro-duodenoscopy before entering the study so that only patients with normal endoscopic findings were included in the current study. All patients underwent a complete physical, blood biochemistry and urinary examination. The grade of dysphagia was determined for each patient in accordance to the intensity and frequency of the presented esophageal symptoms. Furthermore, disease activity scores (SLEDAI and modified Rodnan score) were obtained for patients with autoimmune diseases. Outcome parameter: A correlation coefficient was calculated between amplitudes, velocity and duration of the peristaltic waves throughout esophagus and patients’ dysphagia for all three groups.
RESULTS: There was no statistical difference in the standard blood biochemistry and urinary analysis in all three groups. Patients with IBS showed similar pathologic dysphagia scores compared to patients with SLE and SSc. The mean value of dysphagia score was in IBS group 7.3, in SLE group 6.73 and in SSc group 7.56 with a P-value > 0.05. However, the manometric patterns were different. IBS patients showed during esophageal manometry peristaltic amplitudes at the proximal part of esophagus greater than 60 mmHg in 46% of the patients, which was significant higher in comparison to the SLE (11.8%) and SSc-Group (0%, P = 0.003). Furthermore, IBS patients showed lower mean resting pressure of the distal esophagus sphincter (Lower esophageal sphincter, 22 mmHg) when compared with SLE (28 mmHg, P = 0.037) and SSc (26 mmHg, P = 0.052). 23.5% of patients with SLE showed amplitudes greater as 160 mmHg in the distal esophagus (IBS and SSc: 0%) whereas 29.4% amplitudes greater as 100 mmHg in the middle one (IBS: 16.7%, SSc: 5.9% respectively, P = 0.006). Patients with SSc demonstrated, as expected, in almost half of the cases reduced peristalsis or even aperistalsis in the lower two thirds of the esophagus. SSc patients demonstrated a negative correlation coefficient between dysphagia score, amplitude and velocity of peristaltic activity at middle and lower esophagus [r = -0.6, P < 0.05].
CONCLUSION: IBS patients have comparable dysphagia-scores as patients with autoimmune disorders. The different manometric patterns might allow differentiating esophageal symptoms based on IBS from other organic diseases.
Irritable bowel syndrome; Systemic lupus erythematosus; Systemic sclerosis; Esophageal manometry; Dysphagia
Purpose of review
The purpose of this study is to review recent hypothesis-driven studies that utilize global gene expression data for elucidating the molecular basis of systemic sclerosis (SSc) and its various clinical manifestations.
The longitudinal skin gene expression studies indicate that the previously identified molecular subsets are stable over time and might identify inherent subgroups of SSc patients. Skin transcript follow-up studies indicate that the Wnt/β-catenin pathway plays an important role in promotion of fibrogenesis in fibroblasts and preadipocytes. Furthermore, the transcript profile of sclerodermatous graft-versus-host disease (sclGVHD) mice resembles the skin transcriptomes of a subgroup of SSc patients with IL13/IL4-inducible skin signature wherein the profibrotic chemokine CCL2 plays a key role. The comparison of skin biopsies from SSc patients to skin lesions of patients with cutaneous lupus and dermatomyositis has provided valuable information about the interferon (IFN) signature in these autoimmune diseases. Furthermore, plasma IFN-inducible chemokines correlate with the IFN gene expression score in SSc patients, enabling researchers to examine this molecular signature in large SSc cohorts with serum or plasma collection.
Global gene expression profiling in skin and peripheral blood can contribute to a better understanding of SSc pathogenesis and identify novel biomarkers and therapeutic targets.
gene expression profiling; pathogenesis; systemic sclerosis
To identify differentially expressed genes in peripheral blood cells
(PBC) of patients with ankylosing spondylitis (AS) relative to healthy
controls and controls with systemic inflammation.
We investigated PBC samples of 16 patients with AS and 14 matched
controls, in addition to systemic lupus erythematosus (SLE) and systemic
sclerosis (SSc) samples utilizing Illumina Human Ref-8 BeadChips. Candidate genes were confirmed using
quantitative PCR. Subsequently, these genes were also validated in a
separate sample of 27 patients with AS [before and after antitumor necrosis
factor (anti-TNF) treatment] and 27 matched controls.
We identified 83 differentially expressed transcripts between AS
patients and controls. This gene list was filtered through the lists of
differentially expressed transcripts in SLE and SSc, which resulted in
identification of 52 uniquely dysregulated transcripts in AS. Many of the
differentially expressed genes belonged to Toll-like receptor (TLR) and
related pathways. TLR4 and TLR5 were the
only dysregulated TLR subtypes among AS patients. We confirmed the
overexpression of TLR4 and TLR5 in AS
patients in comparison to controls (p = 0.012 and p = 0.006, respectively)
and SLE (p = 0.002, p = 0.008) using quantitative PCR in the same sample.
Similarly, TLR4 (p = 0.007) and TLR5 (p =
0.012) were significantly upregulated among the AS patients before anti-TNF
treatment in the confirmatory sample. TLR4 (p = 0.002) and
TLR5 (p = 0.025) decreased significantly after anti-TNF
PBC gene expression profiling in AS shows an upregulation of
TLR4 and TLR5. This supports the
importance of TLR subtypes in the pathogenesis of AS that are responsible
for the immune response to Gram-negative bacteria.
ANKYLOSING SPONDYLITIS; TOLL-LIKE RECEPTORS; IMMUNE SYSTEM; AUTOIMMUNITY; BACTERIA; GENE EXPRESSION PROFILING
Systemic Sclerosis (Scleroderma, SSc) is an autoimmune disease characterized by vasculopathy, inflammation, and fibrosis that can lead to loss of organ function. Type I interferons (IFNs) are family of cytokines that mitigate the deleterious effects of viral and bacterial infections in the innate immunity system. Past several years, research efforts have been focused on the role of type I IFN and IFN-inducible genes in the pathogenesis of SSc. Polymorphisms in the Interferon regulatory factor (IRF)-5, IRF7, and IRF8 are associated with SSc, Similarly, polymorphism of Signal Transducer and Activator of Transcription (STAT)-4, has been established as a genetic risk factor of SSc. IRFs and STAT4 proteins are key activators of type I IFN signaling pathways. An IFN signature (increased expression and activation of IFN-regulated genes) has been observed in the peripheral blood and skin biopsy samples of patients with SSc. Furthermore, a plasma IFN-inducible chemokine score correlated with markers of disease severity and autoantibody subtypes in SSc. In this review, we summarize our current knowledge of the role of type I IFNs and IFN-inducible genes in the pathogenesis of SSc and their potential role as biomarkers and therapeutic targets.
systemic sclerosis; innate immunity; type 1 IFN; interferon regulatory factor; IFN-inducible cytokines and chemokines
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are potent autoantigenic targets in systemic autoimmune rheumatic diseases (SARD). Loss of tolerance to the RA33 complex consisting of hnRNP A2 and its alternatively spliced variants B1 and B2 has been the interest of rheumatologists. A novel ELISA for the detection of anti-hnRNP B1 autoantibodies has been developed to investigate the prevalence thereof in 397 patients with SARD, including patients with rheumatoid arthritis (RA), spondyloarthropathy (SPA), juvenile chronic arthritis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjögren's syndrome (SS), in comparison to 174 controls. Anti-hnRNP B1 autoantibodies were significantly more prevalent in patients with SARD than controls (47/397, 11.8% versus 2/174, 1.1%; P < 0.001). In particular, anti-hnRNP B1 were found more frequently in the disease cohorts than in the controls and were present in 24/165 (14.5%) patients with RA, 6/58 (10.3%) SPA, 11/65 (16.9%) SSc, and 4/50 (8.0%) SLE. In RA patients, anti-hnRNP B1 autoantibodies correlated significantly with C-reactive protein levels and erythrocyte sedimentation rate, while in patients with SSc it was associated with features of arterial wall stiffness and presence of hypertension. Anti-hnRNP B1 autoantibodies occur in SARD and seem to be correlated with distinct clinical characteristics in patients with RA and SSc.
Background: Systemic sclerosis (SSc) is accompanied by abnormalities in humoral and cellular immune systems.
Objective: To determine the genes specifically expressed in the immune system in SSc by analysis of the gene expression profile of peripheral blood mononuclear cells (PBMC) from patients with SSc, including those treated with haematopoietic stem cell transplantation (HSCT). Additionally, to investigate the clinical significance of the up regulation of tumour necrosis factor α (TNFα) converting enzyme (TACE).
Methods: PBMC from patients with SSc (n = 23) and other autoimmune diseases (systemic lupus erythematosus (SLE, n = 16), rheumatoid arthritis (RA, n = 29)), and from disease-free controls (n = 36) were examined. Complementary DNA arrays were used to evaluate gene expression of PBMC, in combination with real time quantitative polymerase chain reactions. TACE protein expression in PBMC was examined by fluorescence activated cell sorter (FACS).
Results: In patients with SSc 118 genes were down regulated after HSCT. Subsequent comparative analysis of SSc without HSCT and healthy controls indicated SSc-specific up regulation for three genes: monocyte chemoattractant protein-3 (p = 0.0015), macrophage inflammatory protein 3α (p = 0.0339), and TACE (p = 0.0251). In the FACS analysis, TACE protein was mainly expressed on CD14+ monocytes both in patients with SSc and controls. TACE expression on CD14+ cells was significantly increased in patients with early SSc (p = 0.0096), but not in those with chronic SSc, SLE, or RA. TACE protein levels in SSc monocytes correlated with the intracellular CD68 levels (p = 0.0016).
Conclusions: Up regulation of TACE expression was a unique profile in early SSc, and may affect the function of TNFα and other immunoregulatory molecules.
Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P = 1.34×10−8, OR = 1.22, CI 95% = 1.14–1.30; rs2004640: P = 4.60×10−7, OR = 0.84, CI 95% = 0.78–0.90; rs10488631: P = 7.53×10−20, OR = 1.63, CI 95% = 1.47–1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P = 9.04×10−22, OR = 1.75, CI 95% = 1.56–1.97) better explained the observed association (likelihood P-value = 1.48×10−4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.
To explore the relationship between biomarkers of pulmonary arterial hypertension (PAH), interferon (IFN)–regulated gene expression, and the alternative activation pathway in systemic sclerosis (SSc).
Peripheral blood mononuclear cells (PBMCs) were purified from healthy controls, patients with idiopathic PAH, and SSc patients (classified as having diffuse cutaneous SSc, limited cutaneous SSc [lcSSc] without PAH, and lcSSc with PAH). IFN-regulated and “PAH biomarker” genes were compared after supervised hierarchical clustering. Messenger RNA levels of selected IFN-regulated genes (Siglec1 and MX1), biomarker genes (IL13RA1, CCR1, and JAK2), and the alternative activation marker gene (MRC1) were analyzed on PBMCs and on CD14− and CD14+ cell populations. Interleukin-13 (IL-13) and IL-4 concentrations were measured in plasma by immunoassay. CD14, MRC1, and IL13RA1 surface expression was analyzed by flow cytometry.
Increased PBMC expression of both IFN-regulated and biomarker genes distinguished SSc patients from healthy controls. Expression of genes in the biomarker cluster, but not in the IFN-regulated cluster, distinguished lcSSc with PAH from lcSSc without PAH. The genes CCR1 (P < 0.001) and JAK2 (P < 0.001) were expressed more highly in lcSSc patients with PAH compared with controls and mainly by CD14+ cells. MRC1 expression was increased exclusively in lcSSc patients with PAH (P < 0.001) and correlated strongly with pulmonary artery pressure (r = 0.52, P = 0.03) and higher mortality (P = 0.02). MRC1 expression was higher in CD14+ cells and was greatly increased by stimulation with IL-13. IL-13 concentrations in plasma were most highly increased in lcSSc patients with PAH (P < 0.001).
IFN-regulated and biomarker genes represent distinct, although related, clusters in lcSSc patients with PAH. MRC1, a marker for the effect of IL-13 on alternative monocyte/macrophage activation, is associated with this severe complication and is related to mortality.
To determine aggregation of autoimmune diseases in the first degree relatives (FDR) of patients with systemic sclerosis (SSc) and to investigate frequencies of antinuclear antibodies (ANA) and other autoantibodies in the FDRs and spouses of patients with SSc.
Information on FDRs including history of autoimmune disease was obtained from unrelated SSc probands in the Scleroderma Family Registry and DNA Repository. FDRs were contacted to verify any reported autoimmune diseases. The prevalence of autoimmune disease in probands’ families was compared with the corresponding prevalence in controls’ families as reported in the literature. Furthermore, sera from probands’ FDRs and spouses in addition to unrelated controls were investigated for the presence of autoantibodies (ANA).
We investigated 4612 FDRs of 1071 SSc probands. SSc probands with anti-centromere antibodies (ACA) and limited disease type were more likely to report familial autoimmunity (p = 0.022 and p = 0.041, respectively). The four most prevalent autoimmune diseases among SSc probands’ FDRs were hypothyroidism (4%), Rheumatoid arthritis (1.5%), hyperthyroidism (1.3%) and systemic lupus erythematosus-SLE (0.4%). Compared to control families, SLE, hypothyroidism and hyperthyroidism were more common in SSc probands’ families. The most striking increase for familial prevalence was observed in SLE (OR = 16.98, 95% CI = 1.02–227.82, p = 0.004). ANA was present in 14.2% of probands’ FDR’s and 8.6% of spouses and did not differ from the prevalence of ANA among controls (p = 0.124 and p = 0.477, respectively). Only two FDRs of probands had ACA while none had anti-topoisomerase antibodies.
Our study implies varying degrees of risk for familial autoimmunity among subtypes of SSc and provides further support for common genetic and potentially environmental factors leading to SSc and SLE.
Systemic sclerosis; Scleroderma; First degree relatives; ANA; Autoimmune disease
Anti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc). ACA are found in 20 to 40% of SSc patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. The objective of this study was to evaluate a novel CENP-A peptide ELISA.
Sera collected from SSc patients (n = 334) and various other diseases (n = 619) and from healthy controls (n = 175) were tested for anti-CENP-A antibodies by the novel CENP-A enzyme linked immunosorbent assay (ELISA). Furthermore, ACA were determined in the disease cohorts by IIF (ImmunoConcepts, Sacramento, CA, USA), CENP-B ELISA (Dr. Fooke), EliA® CENP (Phadia, Freiburg, Germany) and line-immunoassay (LIA, Mikrogen, Neuried, Germany). Serological and clinical associations of anti-CENP-A with other autoantibodies were conducted in one participating centre. Inhibition experiments with either the CENP-A peptide or recombinant CENP-B were carried out to analyse the specificity of anti-CENP-A and -B antibodies.
The CENP-A ELISA results were in good agreement with other ACA detection methods. According to the kappa method, the qualitative agreements were: 0.73 (vs. IIF), 0.81 (vs. LIA), 0.86 (vs. CENP-B ELISA) and 0.97 (vs. EliA® CENP). The quantitative comparison between CENP-A and CENP-B ELISA using 265 samples revealed a correlation value of rho = 0.5 (by Spearman equation). The receiver operating characteristic analysis indicated that the discrimination between SSc patients (n = 131) and various controls (n = 134) was significantly better using the CENP-A as compared to CENP-B ELISA (P < 0.0001). Modified Rodnan skin score was significantly lower in the CENP-A negative group compared to the positive patients (P = 0.013). Inhibition experiments revealed no significant cross reactivity of anti-CENP-A and anti-CENP-B antibodies. Statistically relevant differences for gender ratio (P = 0.0103), specific joint involvement (Jaccoud) (P = 0.0006) and anti-phospholipid syndrome (P = 0.0157) between ACA positive SLE patients and the entire SLE cohort were observed.
Anti-CENP-A antibodies as determined by peptide ELISA represent a sensitive, specific and independent marker for the detection of ACA and are useful biomarkers for the diagnosis of SSc. Our data suggest that anti-CENP-A antibodies are a more specific biomarker for SSc than antibodies to CENP-B. Furthers studies are required to verify these findings.
To investigate the relationship of the polymorphic enhancer HS1,2 central to the 3′ enhancer complex regulatory region (IgH3′EC) of the immunoglobulin heavy chain genes with systemic sclerosis (SSc) disease and compare it with HLA‐DR and DQ associations.
A total of 116 patients with SSc were classified as diffuse (dSSc) or limited (lSSc), and as carriers of antitopoisomerase I (anti‐Scl70) or anticentromere (ACA) antibodies. Allele and genotype frequencies were assessed in the population as a whole and in the two major subsets, dSSc and lSSc. The concentration of peripheral blood immunoglobulin levels was also determined and analysed according to the genotypes.
The analysis of genotypes for the four alleles of the HS1,2A enhancer showed an increased frequency of allele *2 in the SSc cohort highly significant versus controls (57% vs. 40%, p<0.0001). Considering the autoantibody pattern, we found that the frequency of the 2/2 genotype was increased in ACA+ patients (42%) and anti‐Scl70+ patients (31%) compared with the control group (15%). The differences of allelic frequencies among dSSc versus lSSc or ACA+ versus anti‐Scl70+ patients were not significant, although highly significant when comparing each subgroup with the control group. HLA‐DRB1*11 and DQB1*03 associated with SSc. No association was seen between HS1,2A enhancer polymorphism and HLA alleles.
These data confirm there was an increased risk of having SSc in carriers of allele *2, suggesting an intriguing function of this polymorphism for B‐cell regulation.
Our goal was to study the prevalence of systemic sclerosis (SSc) subtypes, autoantibody profile, and pulmonary fibrosis in a large group of Han Chinese. Chinese SSc patients (n=419) were recruited from a multicenter study including hospitals and outpatient clinics in China. All patients met the American College of Rheumatology classification criteria for SSc. Anti-topoisomerase (ATA), anti-centromere (ACA), anti- RNA polymerase III (anti-RNAP3), and anti-U1- ribonucleoprotein (anti-U1RNP) were detected utilizing commercially available kits. The clinical and autoantibody information in Chinese patients was compared to that in the US Caucasian patients (n=834), recruited from the Genetics versus Environment in Scleroderma Outcome Study and Scleroderma Family Registry. Chi-square test was utilized for the abovementioned comparisons. Chinese patients showed 40.3 % limited (lcSSc) and 59.7 % diffuse (dcSSc) forms of SSc. ATA was found in 59.9 %, ACA in 13.4 %, anti-RNAP3 in 1.3 %, and anti-U1RNP in 18 % of Chinese SSc patients. Compared to US patients (65.1 % lcSSc, 34.9 % dcSSc, ATA in 18.7 %, ACA in 32.4 %, anti-RNAP3 in 17.4 %, and anti-U1RNP in 2.8 %), Chinese SSc patients are significantly higher in dcSSc and the frequencies of ATA and anti-U1RNP, but lower in ACA and anti-RNAP3. In addition, pulmonary fibrosis was observed in 78 % Chinese SSc patients and was strongly associated with the presence of ATA. The present study represents the first report of SSc features in a large group of Chinese patients. Clinical subtypes and the frequencies of SSc-related autoantibodies in Chinese SSc patients are significantly different from those in SSc patients of the US Caucasian descent.
Anti-centromere antibody; Anti-topoisomerase antibody; Autoantibodies; Pulmonary fibrosis; Systemic sclerosis
Independent replication with large cohorts and metaanalysis of genetic associations are necessary to validate genetic susceptibility factors. The known tumor necrosis factor (ligand) superfamily, member 4 gene (TNFSF4) systemic lupus erythematosus (SLE) risk locus has been found to be associated with systemic sclerosis (SSc) in 2 studies, but with discrepancies between them for genotype-phenotype correlation. Our objective was to validate TNFSF4 association with SSc and determine the subset with the higher risk.
Known SLE and SSc TNFSF4 susceptibility variants (rs2205960, rs1234317, rs12039904, rs10912580, and rs844648) were genotyped in 1031 patients with SSc and 1014 controls of French white ancestry. Genotype-phenotype association analysis and metaanalysis of available data were performed, providing a population study of 4989 patients with SSc and 4661 controls, all of European white ancestry.
Allelic and genotypic associations were observed for the 5 single-nucleotide polymorphisms (SNP) with the subset of patients with SSc who are positive for anticentromere antibodies (ACA) and only a trend for association with SSc and limited cutaneous SSc. Rs2205960 exhibited the strongest allelic association in ACA+ patients with SSc [p = 0.0015; OR 1.37 (1.12–1.66)], with significant intracohort association when compared to patients with SSc positive for ACA. Metaanalysis confirmed overall association with SSc but also raised preferential association with the ACA+ subset and strongest effect with rs2205960 [T allele p = 0.00013; OR 1.33 (1.15–1.54) and TT genotype p = 0.00046; OR 2.02 (1.36–2.98)].
We confirm TNFSF4 as an SSc susceptibility gene and rs2205960 as a putative causal variant with preferential association in the ACA+ SSc subphenotype. (First Release March 15 2012; J Rheumatol 2012;39:997–1003; doi:10.3899/jrheum.111270)
SYSTEMIC SCLEROSIS; TNFSF4; AUTOIMMUNITY; AUTOANTIBODIES
To estimate the frequency of work disability (WD) in a cohort of patients with Systemic Sclerosis (SSc) vs an internal control group of patients with rheumatoid arthritis (RA) with a known high frequency of WD; and to investigate the association between WD and other factors including Health Assessment Questionnaire Disability Index (HAQ-DI) scores, HAQ pain, age, sex, disease duration and education level.
Cross-sectional data on WD status were obtained from a questionnaire sent to all SSc (n = 35 limited [lcSSc], 26 diffuse [dcSSc]) and a subset of RA patients (n=104) from a rheumatology practice. WD data, HAQ-DI scores, and demographic/clinical features (age, sex, high school education, disease duration and SSc disease subtype [dcSSc vs lcSSc]) were recorded.
The proportion with WD was 0.56 in SSc (95% CI: 0.43-0.68) vs 0.35 in RA (95% CI: 0.25-0.44), p= 0.009. HAQ-DI scores were significantly higher in work-disabled SSc and RA patients vs those who were employed (p=0.0001, and p <0.0001). Multivariate logistic regression analysis demonstrated that higher HAQ-DI scores (β=1.78, p <0.001), disease type (dcSSc, lcSSc, RA) (β=1.32 for dcSSc, p=0.032), and self-reported disease duration (β=0.04, p=0.042) were significantly associated with WD (R2=0.311). Adding a work-related factor (self-reported physically demanding work) improved the regression model (R2=0.346) and strengthened the HAQ-DI (β=1.86, p <0.001) and lcSSc (β=1.24, p=0.024) coefficients.
The frequency of WD in SSc was high and was greater than in RA. SSc (and dcSSc) had significantly more WD than RA. The HAQ-DI was strongly associated with WD in SSc
Scleroderma; systemic sclerosis; work disability; health assessment questionnaire
It is increasingly being appreciated that multiple autoimmune diseases share common susceptibility genes. The tumour necrosis factor ligand superfamily member 4 gene (TNFSF4, OX40L), which encodes for the T cell costimulatory molecule OX40 ligand, has been identified as a susceptibility gene for the development of systemic lupus erythematosus (SLE). Accordingly, the aim of the current study was to investigate the possible association of the TNFSF4 gene region with systemic sclerosis (SSc), an autoimmune disease that leads to the development of cutaneous and visceral fibrosis.
A total of 9 single nucleotide polymorphisms (SNPs) in the TNFSF4 gene region, previously associated with susceptibility to SLE, were tested for association with SSc in a collection of 1059 patients with SSc and 698 controls.
Case-control comparisons revealed a significant association between susceptibility to SSc and the minor alleles at SNPs rs1234314 (OR 1.20, 95% CI 1.04 to 1.4, pFDR=0.019), rs2205960 (OR 1.24, 95% CI 1.10 to 1.50, pFDR=0.019) and rs844648 (OR 1.16, 95% CI 1.01 to 1.30, pFDR=0.032). The minor allele at rs844644 was protective (OR 0.84, 95% CI 0.70 to 0.97, pFDR=0.038). Analysis of subsets of patients with SSc demonstrated significant associations of the TNFSF4 SNPs with limited and diffuse SSc as well as specific SNPs that were associated with SSc-associated autoantibodies. Finally, the analyses suggest a potential interaction between two TNFSF4 SNPs, rs2205960 and rs844648, with regards to SSc susceptibility.
Polymorphisms in the TNFSF4 gene region are associated with susceptibility to SSc and its clinical and autoantibody subsets. TNFSF4 may be another gene that confers risk to multiple autoimmune diseases.
We assessed the profile and frequency of malignancy subtypes in a large single-centre UK cohort for patients with scleroderma (systemic sclerosis; SSc). We evaluated the cancer risk among SSc patients with different antibody reactivities and explored the temporal association of cancer with the duration between SSc onset and cancer diagnosis.
We conducted a retrospective study of a well-characterised cohort of SSc patients attending a large tertiary referral centre, with clinical data collected from our clinical database and by review of patient records. We evaluated development of all cancers in this cohort, and comparison was assessed with the SSc cohort without cancer. The effect of demographics and clinical details, including antibody reactivities, were explored to find associations relevant to the risk for development of cancer in SSc patients.
Among 2,177 patients with SSc, 7.1% had a history of cancer, 26% were positive for anticentromere antibodies (ACAs), 18.2% were positive for anti-Scl-70 antibodies and 26.6% were positive for anti-RNA polymerase III (anti-RNAP) antibody. The major malignancy cancer subtypes were breast (42.2%), haematological (12.3%), gastrointestinal (11.0%) and gynaecological (11.0%). The frequency of cancers among patients with RNAP (14.2%) was significantly increased compared with those with anti-Scl-70 antibodies (6.3%) and ACAs (6.8%) (P < 0.0001 and P < 0.001, respectively). Among the patients, who were diagnosed with cancer within 36 months of the clinical onset of SSc, there were more patients with RNAP (55.3%) than those with other autoantibody specificities (ACA = 23.5%, P < 0.008; and anti-Scl-70 antibodies = 13.6%, P < 0.002, respectively). Breast cancers were temporally associated with onset of SSc among patients with anti-RNAP, and SSc patients with anti-RNAP had a twofold increased hazard ratio for cancers compared to patients with ACAs (P < 0.0001).
Our study independently confirms, in what is to the best of our knowledge the largest population examined to date, that there is an association with cancer among SSc patients with anti-RNAP antibodies in close temporal relationship to onset of SSc, which supports the paraneoplastic phenomenon in this subset of SSc cases. An index of cautious suspicion should be maintained in these cases, and investigations for underlying malignancy should be considered when clinically appropriate.
The pathogenesis and etiology of systemic sclerosis (SSc) are complex and poorly understood. To date, several studies have demonstrated that the activation of the immune system undoubtedly plays a pivotal role in SSc pathogenesis. Activated immune effector T cells contribute to the release of various pro-inflammatory cytokines and drive the SSc-specific autoantibody responses. This, and a profibrotic environment, are all-important components of abnormal active immune responses that can lead to pathological disorders of SSc. CD11a is essential to inflammatory and immune responses, regulating adhesive and co-stimulatory interactions between CD4+ T cells and other cells. Although CD11a is overexpressed in SSc patients, the mechanisms leading to this overexpression and its consequences remain unclear. DNA methylation, a main epigenetic modification, plays an important role in the regulation of gene expression and is involved in the pathogenesis of autoimmune diseases. This work aims to investigate the effect of DNA demethylation on CD11a expression in SSc CD4+ T cells and to determine its functional significance. CD11a expression was measured using RT-PCR and flow cytometry. Bisulfite sequencing was used to determine the methylation status of the CD11a regulatory region. CD4+ T cells were co-cultured with antigen-presenting cells, B cells, or fibroblasts with and without anti-CD11a, and proliferation of CD4+ T cells, IgG production by B cells, and expression levels of COL1A2 mRNA by fibroblasts were evaluated.
Elevated CD11a expression levels were observed in CD4+ T cells from SSc patients; these levels were found to be positively correlated with disease activity. The methylation levels of the CD11a regulatory sequences were lower in SSc patients than in controls and inversely correlated with CD11a mRNA expression. Treatment of CD4+ T cells with 5-azacytidine (5-azaC) decreased CD11a promoter methylation and caused CD11a overexpression. SSc CD4+ T cells and 5-azaC-treated CD4+ T cells showed increased proliferation of CD4+ T cells, increased production of IgG by co-cultured B cells, and induced expression of COL1A2 mRNA by co-cultured fibroblasts. These stimulatory effects were abrogated by anti-CD11a.
Demethylation of CD11a regulatory elements and subsequent CD11a overexpression in CD4+ T cells may mediate immunological abnormalities and fibrotic processes in SSc.
CD11a; CD4+ T cells; COL1A2; DNA methylation; Systemic sclerosis
Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are related chronic autoimmune diseases of complex aetiology in which the interferon (IFN) pathway plays a key role. Recent studies have reported an association between IRF7 and SLE which confers a risk to autoantibody production. A study was undertaken to investigate whether the IRF7 genomic region is also involved in susceptibility to SSc and the main clinical features.
Two case-control sets of Caucasian origin from the USA and Spain, comprising a total of 2316 cases of SSc and 2347 healthy controls, were included in the study. Five single nucleotide polymorphisms (SNPs) in the PHRF1-IRF7-CDHR5 locus were genotyped using TaqMan allelic discrimination technology. A meta-analysis was performed to test the overall effect of these genetic variants on SSc.
Four out of five analysed SNPs were Significantly associated with the presence of anticentromere autoantibodies (ACA) in the patients with SSc in the combined analysis (rs1131665: pFDR=6.14 × 10−4, OR=0.78; rs4963128: pFDR=6.14 × 10−4, OR=0.79; rs702966: pFDR=3.83 × 10−3, OR=0.82; and rs2246614: pFDR=3.83 × 10−3, OR=0.83). Significant p values were also obtained when the disease was tested globally; however, the statistical significance was lost when the ACA-positive patients were excluded from the study, suggesting that these associations rely on ACA positivity. Conditional logistic regression and allelic combination analyses suggested that the functional IRF7 SNP rs1131665 is the most likely causal variant.
The results show that variation in the IRF7 genomic region is associated with the presence of ACA in patients with SSc, supporting other evidence that this locus represents a common risk factor for autoantibody production in autoimmune diseases.
BACKGROUND—Excess tissue matrix accumulates in systemic sclerosis (SSc), accounting for both visceral and dermal fibrosis. It is suggested that decreased serum levels of matrix metalloproteinases (MMPs) or increased levels of tissue inhibitors of matrix metalloproteinases (TIMPs) may account for this matrix accumulation.
OBJECTIVE—To measure serum levels of tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and collagenase-1 (MMP-1), in patients with diffuse cutaneous systemic sclerosis (dcSSc), limited cutaneous systemic sclerosis (lcSSc), primary Raynaud's phenomenon (RP), and in normal controls.
METHODS—Serum samples from patients with dcSSc (n=83), lcSSc (n=87), RP (n=80), and normal controls (n=98) were analysed using enzyme linked immunosorbent assays (ELISAs) for total TIMP-1, TIMP-2, and MMP-1. Results from each assay were analysed by the Kruskal-Wallis test. Dunn's multiple comparison post-test was then applied between groups.
RESULTS—TIMP-1 levels were significantly raised in dcSSc and lcSSc groups compared with the RP group and normal controls (p<0.01 to p<0.001). In the dcSSc group, TIMP-1 levels were significantly higher in early disease (<2 years) than in late stage disease (>4 years) (p<0.05). This was not found for the lcSSc group. Serum TIMP-2 and MMP-1 levels in dcSSc and lcSSc did not differ significantly from those in normal controls. Increased levels of TIMPs were not convincingly associated with organ disease. No assay result correlated with autoantibody status (anti-topoisomerase 1 (anti-Scl-70), anticentromere antibody, or anti-RNA polymerase). No significant differences in serum TIMP-1, TIMP-2, or MMP-1 levels were shown in the RP group compared with normal controls.
CONCLUSIONS—Raised TIMP-1 levels in the SSc groups support the hypothesis that matrix accumulation occurs in SSc at least in part owing to decreased degradation. Moreover, the variation in TIMP-1 levels between the early and late disease stages of dcSSc seems to reflect the early progressive course of dermal fibrosis seen clinically. The expected reduction in serum MMP-1 levels in the SSc groups was not found. This suggests that tissue matrix accumulation is due to increased inhibitors rather than to decreased MMPs.
Simple measures of type I interferon (IFN) activity constitute highly attractive biomarkers in systemic lupus erythematosus (SLE). We explore galectin-3-binding protein (G3BP) as a novel measure of type I IFN activity and serum/plasma biomarker in large independent cohorts of patients with SLE and controls.
Serum and plasma G3BP concentrations were quantified using ELISA. Type I IFN activity was assessed by Mx1 reporter gene expression assays and correlated to serum G3BP concentrations (SLE-IFN-α, n=26 and healthy controls (HCs), n=10). Plasma G3BP concentrations in the SLE-Denmark (DK) (n=70) and SLE-Sweden (SE) (n=68) cohorts were compared with the HC-DK (n=47) and HC-SE (n=50) cohorts and patients with systemic sclerosis (n=111). In 15 patients with SLE, serum G3BP in consecutive samples was correlated to disease activity. Correlation analysis between G3BP, clinical parameters including disease activity in the four SLE cohorts was performed.
G3BP concentrations correlated significantly with the IFN-α reporter gene assay (r=0.56, p=0.0005) and with IFN-α gene expression scores (r=0.54, p=0.0002). Plasma concentrations were significantly increased in the SLE-DK and SLE-SE cohorts compared with HCs and patients with systemic sclerosis (p<0.0001 and p=0.0009). G3BP concentrations correlated with disease activity measures in the SLE-DK- and SLE-IFN-α cohorts (p=0.0004 and p=0.05) but not in the SLE-SE cohort (p=0.98). Markedly temporal variation was observed in G3BP levels in the consecutive SLE-samples and was significantly associated with changes in disease activity (r=0.44, p=0.014).
G3BP plasma levels reflect type I IFN activity and are increased in SLE. Associations with disease activity or clinical manifestations are uncertain. This study highlights G3BP as a convenient measure of type I IFN-dependent gene activation.
Systemic Lupus Erythematosus; Systemic Sclerosis; Interferon; Lupus Nephritis; Autoantibodies
Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.
Immunohistology and enzyme-linked immunosorbent assays (ELISAs) were used to determine chemokine and chemokine receptor expression in the skin and serum, respectively, of SSc and normal patients. Endothelial cells (ECs) were isolated from SSc skin biopsies and chemokine and chemokine receptor expression was determined by quantitative PCR and immunofluorescence staining.
Antiangiogenic IP-10/CXCL10 and MIG/CXCL9 were elevated in SSc serum and highly expressed in SSc skin. However, CXCR3, the receptor for these chemokines, was decreased on ECs in SSc vs. normal skin. CXCL16 was elevated in SSc serum and increased in SSc patients with early disease, pulmonary arterial hypertension, and those that died during the 36 months of the study. In addition, its receptor CXCR6 was overexpressed on ECs in SSc skin. At the mRNA and protein levels, CXCR3 was decreased while CXCR6 was increased on SSc ECs vs. human microvascular endothelial cells (HMVECs).
These results show that while the expression of MIG/CXCL9 and IP-10/CXCL10 are elevated in SSc serum, the expression of CXCR3 is downregulated on SSc dermal ECs. In contrast, CXCL16 and CXCR6 are elevated in SSc serum and on SSc dermal ECs, respectively. In all, these findings suggest angiogenic chemokine receptor expression is likely regulated in an effort to promote angiogenesis in SSc skin.
Overexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity.
Peripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNβ were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNβ (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNβ induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC.
These findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.
To identify potential pharmacodynamic biomarkers to guide dose selection in clinical trials using anti-interferon-alpha (IFN-α) monoclonal antibody (mAb)
therapy for systemic lupus erythematosus (SLE), we used an Affymetrix human genome array platform and identified 110 IFN-α/β-inducible transcripts significantly upregulated in whole blood (WB) of 41 SLE patients. The overexpression of these genes was confirmed prospectively in 54 additional SLE patients and allowed for the categorization of the SLE patients into groups of high, moderate, and weak overexpressers of IFN-α/β-inducible genes. This approach could potentially allow for an accurate assessment of drug target neutralization in early trials of anti-IFN-α mAb therapy for SLE. Furthermore, ex vivo stimulation of healthy donor peripheral blood mononuclear cells with SLE patient serum and subsequent neutralization with anti-IFN-α mAb or anti-IFN-α receptor mAb showed that anti-IFN-α mAb has comparable effects of neutralizing the overexpression of type I IFN-inducible genes as that of anti-IFNAR mAb. These results suggest that IFN-α, and not other members of type I IFN family in SLE patients, is mainly responsible for the induction of type I IFN-inducible genes in WB of SLE patients. Taken together, these data strengthen the view of IFN-α as a therapeutic target for SLE.
In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the two immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17.
Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity.
Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI ≥3 (Fisher exact test, P = 0.045) or less restrictive DAI≥2.5 (P = 0.009).
ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lc SSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance.
Systemic sclerosis; CENP-A; peptide; FOXE-3; disease activity index