Autoantibody Specificities and Type I Interferon Pathway Activation in Idiopathic Inflammatory Myopathies.

Myositis is a heterogeneous group of autoimmune diseases, with different pathogenic mechanisms contributing to the different subsets of disease. The aim of this study was to test whether the autoantibody profile in patients with myositis is associated with a type I interferon (IFN) signature, as in patients with systemic lupus erythematous (SLE). Patients with myositis were prospectively enrolled in the study and compared to healthy controls and to patients with SLE. Autoantibody status was analysed using an immunoassay system and immunoprecipitation. Type I IFN activity in whole blood was determined using direct gene expression analysis. Serum IFN-inducing activity was tested using peripheral blood cells from healthy donors. Blocking experiments were performed by neutralizing anti-IFNAR or anti-IFN-α antibodies. Patients were categorized into IFN high and IFN low based on an IFN score. Patients with autoantibodies against RNA-binding proteins had a higher IFN score compared to patients without these antibodies, and the IFN score was related to autoantibody multispecificity. Patients with dermatomyositis (DM) and inclusion body myositis (IBM) had a higher IFN score compared to the other subgroups. Serum type I IFN bioactivity was blocked by neutralizing anti-IFNAR or anti-IFN-α antibodies. To conclude, a high IFN score was not only associated with DM, as previously reported, and IBM, but also with autoantibody monospecificity against several RNA-binding proteins and with autoantibody multispecificity. These studies identify IFN-α in sera as a trigger for activation of the type I IFN pathway in peripheral blood and support IFN-α as a possible target for therapy in these patients.

Imatinib mesylate (Gleevec™) in the treatment of diffuse cutaneous systemic sclerosis: results of a 24-month open label, extension phase, single-centre trial.

OBJECTIVES: We aimed to assess the long-term safety and tolerability of imatinib in diffuse cutaneous systemic sclerosis (dcSSc). METHODS: In this open-label, single-arm, extension-phase clinical trial, patients continued imatinib for 24 months following 12 months of initial treatment. RESULTS: Seventeen patients were enrolled. Forty of 92 adverse events (AE) and 0/6 serious (S) AEs were possibly related to medication. The MRSS decreased from a median of 21 to 16, (p=0.002). CONCLUSIONS: This study demonstrates long-term safety and tolerability of imatinib in a substantial proportion of patients with dcSSc. This is important in evaluating the relevance of this therapy in a chronic disease such as SSc.

A surge in anti-dsDNA titer predicts a severe lupus flare within six months.

OBJECTIVE: Rising anti-double-stranded (ds) DNA titers have been shown by some, but not all, studies to be predictive of disease flares in systemic lupus erythematosus (SLE). We hypothesized that a rapid and substantial rise in anti-dsDNA titer (anti-dsDNA surge) would be a good predictor of a clinically important SLE flare. METHODS: A matched case-control study was conducted in an academic rheumatology practice setting. Our primary endpoint was the occurrence of a severe SELENA-SLEDAI (SS) flare within six months of an anti-dsDNA surge, and secondary endpoints were mild/moderate SS flares, as well as BILAG A and B renal flares. Cases were identified as those patients whose disease course included a surge of anti-dsDNA, defined as an increase of anti-dsDNA titer by the Crithidia luciliae immunofluorescence (CLIF) assay from 0 to 3+/4+, or from 1+ to 4+, within a period of less than 12 months. The date of the anti-dsDNA surge was defined as Day 0. Two control SLE patients were identified for each case and were matched for age, sex, race, and visit date closest to case Day 0, but without an anti-dsDNA surge. Logistic regression models were used to detect associations between anti-dsDNA surges and severe SS flares. RESULT: A higher proportion of cases, compared to controls, experienced a severe SS flare within six months of Day 0 (OR 6.3 (95% confidence intervals 2.0-19.9), p = 0.02). Associations with all flares and hospitalizations for flares were also observed. However, an anti-dsDNA surge was not predictive of a renal flare. CONCLUSION: An anti-dsDNA surge predicts the subsequent development of a severe SS flare within six months. Physicians should closely monitor such patients and treat promptly at the first sign of clinical activity.

Age- and gender-specific modulation of serum osteopontin and interferon-alpha by osteopontin genotype in systemic lupus erythematosus.

Osteopontin (OPN) is a multifunctional cytokine involved in long bone remodeling and immune system signaling. Additionally, OPN is critical for interferon-alpha (IFN-alpha) production in murine plasmacytoid dendritic cells. We have previously shown that IFN-alpha is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variants of OPN have been associated with SLE susceptibility, and one study suggests that this association is particular to men. In this study, the 3' UTR SLE-risk variant of OPN (rs9138C) was associated with higher serum OPN and IFN-alpha in men (P=0.0062 and P=0.0087, respectively). In women, the association between rs9138 C and higher serum OPN and IFN-alpha was restricted to younger subjects, and risk allele carriers showed a strong age-related genetic effect of rs9138 genotype on both serum OPN and IFN-alpha (P<0.0001). In African-American subjects, the 5' region single nucleotide polymorphisms, rs11730582 and rs28357094, were associated with anti-RNP antibodies (odds ratio (OR)=2.9, P=0.0038 and OR=3.9, P=0.021, respectively). Thus, we demonstrate two distinct genetic influences of OPN on serum protein traits in SLE patients, which correspond to previously reported SLE-risk variants. This study provides a biologic relevance for OPN variants at the protein level, and suggests an influence of this gene on the IFN-alpha pathway in SLE.

Local cytokine profiles in knee osteoarthritis: elevated synovial fluid interleukin-15 differentiates early from end-stage disease.

OBJECTIVE: Much of what is known about the inflammatory response in the synovial membrane (SM) of patients with osteoarthritis (OA) comes from studies of synovial tissues from end-stage disease. In this study, we sought to better characterize the inflammatory infiltrate in symptomatic patients with early signs of knee OA, and to determine how inflammatory cell populations relate to the pattern of cytokine and degradative enzyme production. METHODS: Study populations comprised patients with degenerative meniscal tears and early cartilage thinning undergoing arthroscopic procedures (early OA) and patients undergoing total knee replacement for end-stage OA. Quantitative real-time polymerase chain reaction (PCR) was used to measure expression of SM cytokines and enzymes implicated in the pathogenesis of inflammatory arthritis and OA, as well as cell lineage-specific markers. We quantified synovial fluid (SF) cytokines and enzymes by enzyme-linked immunosorbent assay (ELISA) and SM cell populations by immunohistochemistry. RESULTS: We found increased levels of SF interleukin-15 (IL-15) protein in the early knee OA patients when compared to end-stage OA. Both SF IL-15 protein and numbers of CD8 cells within SM correlated with matrix metalloproteinase-1 (MMP-1) and three levels. TNF-alpha, IL-6 and IL-21 were also detectable in the SF of the majority of patients, and IL-15 levels were associated with IL-6 levels. CONCLUSION: IL-15 is elevated in early knee OA, suggesting activation of an innate immune response in the SM. The association of IL-15 expression with CD8 transcripts and MMPs implicates this cytokine in OA pathogenesis and as a candidate therapeutic target.

Exposure to nuclear antigens contributes to the induction of humoral autoimmunity during tumour necrosis factor alpha blockade.

OBJECTIVE: Type I interferons and apoptotic particles contribute to antinuclear autoimmunity in experimental models. This study assessed whether similar mechanisms contribute to break peripheral B-cell tolerance in humans by studying the induction of antinuclear antibodies by tumour necrosis factor blockade in spondyloarthritis. METHODS: 40 spondyloarthritis patients treated with infliximab or etanercept and 20 renal cell carcinoma patients treated with sorafenib were studied. Serum antinucleosome IgM and nucleosomes were measured by ELISA. Type I interferon serum activity was measured using a functional reporter cell assay. Synovial apoptosis was assessed by terminal transferase nick end-labelling (TUNEL) assay and anti-active caspase-3 immunostaining. Complement was measured by nephelometry. RESULTS: Despite a similar clinical improvement and reduction of synovial inflammation, antinucleosome IgM were induced by infliximab but not etanercept. This induction did not correlate with type I interferon activity, which was transiently downmodulated by infliximab but persistently upregulated by etanercept. In contrast, antinucleosome IgM levels did correlate with serum nucleosome levels, which were significantly upregulated by infliximab but not by etanercept treatment. This increase in serum nucleosome levels was not directly related to massive cell death, but rather to a decrease of complement 3 and 4 serum levels during infliximab treatment. CONCLUSION: Infliximab and etanercept have a differential effect on both type I interferon activity and nucleosome levels. Only elevated serum nucleosomes relate to the induction of antinucleosome antibodies after infliximab treatment.

Lack of association of the TP53 Arg72Pro SNP and the MDM2 SNP309 with systemic lupus erythematosus in Caucasian, African American, and Asian children and adults.

The p53 tumour suppressor is the central regulator of apoptosis. Previously, the functional TP53 Arg72Pro polymorphism was found to be associated with systemic lupus erythematosus (SLE) in Koreans but not Spaniards. MDM2 is the major negative regulator of p53. An intronic polymorphism in MDM2, the SNP309, attenuates p53 activity and is associated with accelerated tumour development in premenopausal women. Polymorphic variation in MDM2 has never been studied in SLE. The aim of this study is to further assess the contribution of p53-pathway genetic variation to SLE by testing the association of the TP53 Arg72Pro polymorphism and the MDM2 SNP309 with SLE in a well-characterised and ethnically diverse cohort of patients with both childhood- and adult-onset SLE (n = 314). No association was found between the TP53 Arg72Pro polymorphism and SLE in patients of European descent, Asian descent or in African Americans, nor was an association found between the MDM2 SNP309 and SLE in patients of European descent or in African Americans. In addition, there was no correlation between either variant and early-onset disease or nephritis, an index of severe disease. It is concluded that neither the TP53 Arg72Pro polymorphism nor the MDM2 SNP309 contributes significantly to either susceptibility or disease severity in SLE.

A high incidence of disease flares in an open pilot study of infliximab in patients with refractory inflammatory myopathies.

OBJECTIVE: To investigate the effect of the tumour necrosis factor (TNF) blocking agent infliximab in patients with treatment-resistant inflammatory myopathies. METHODS: A total of 13 patients with refractory polymyositis (PM), dermatomyositis (DM), or inclusion body myositis (IBM) were treated with 4 infliximab infusions (5 mg/kg body weight) over 14 weeks. Outcome measures included myositis disease activity score with improvement defined according to The International Myositis Assessment and Clinical Studies Group (IMACS), and MRI. Repeated muscles biopsies were investigated for cellular infiltrates, major histocompatibility complex (MHC) class I and II, TNF, interleukin (IL)1alpha, IL6, high mobility group box chromosomal protein 1 (HMGB-1), interferon gamma (IFNgamma), myxovirus resistance protein A (MxA) and membrane attack complex (MAC) expression. Type I IFN activity was analysed in sera. RESULTS: Nine patients completed the study. Three patients discontinued due to adverse events and one due to a discovered malignancy. Three of the completers improved by >or=20% in three or more variables of the disease activity core set, four were unchanged and two worsened >or=30%. No patient improved in muscle strength by manual muscle test. At baseline, two completers had signs of muscle inflammation by MRI, and five at follow-up. T lymphocytes, macrophages, cytokine expression and MAC deposition in muscle biopsies were still evident after treatment. Type I IFN activity was increased after treatment. CONCLUSIONS: Infliximab treatment was not effective in refractory inflammatory myopathies. In view of radiological and clinical worsening, and activation of the type I IFN system in several cases, infliximab is not an alternative treatment in patients with treatment-resistant myositis.

Type I interferon in systemic lupus erythematosus.

Studies of the immunopathogenesis of systemic lupus erythematosus (SLE) have traditionally focused on the mechanisms of generation of the characteristic autoantibodies reactive with nucleic acid-containing intracellular particles and the contribution of autoantibody-autoantigen immune complexes to the inflammation and tissue damage that result in the clinical manifestations of lupus. The recent recognition of the central role of type I interferons (IFN) in this classic autoimmune disease has led to new understanding of the significant role of the innate immune system in the predisposition to and amplification of autoimmunity and tissue damage. Ongoing studies are defining the genetic factors, immune stimuli, and molecular pathways that contribute to production of IFN and induction of its downstream targets in SLE. Investigations of lupus patients and murine lupus models suggest a primary role for type I IFNs in systemic autoimmunity and support the case for therapeutic inhibition of the IFN pathway in lupus and possibly other systemic autoimmune diseases.

High serum IFN-alpha activity is a heritable risk factor for systemic lupus erythematosus.

Interferon alpha (IFN-alpha) levels are elevated in many patients with systemic lupus erythematosus (SLE); however it is not known whether high serum IFN-alpha activity is a cause or a result of the disease. We studied 266 SLE patients and 405 of their healthy relatives, and frequently found high serum IFN-alpha activity in both patients and healthy relatives as compared to healthy unrelated individuals. High IFN-alpha activity was clustered in specific families in both SLE patients and their healthy first-degree relatives, suggesting a heritable trait. Heritability was also supported by quantitative familial correlation of IFN-alpha activity, concordance in affected sib pairs and frequent transmission of the high IFN-alpha activity trait from parents to offspring. Autoantibodies to RNA-binding proteins and double-stranded DNA were associated with high IFN-alpha activity in SLE patients; however these autoantibodies were very uncommon in healthy family members and did not explain the observed familial correlations. The frequency of high IFN-alpha activity was similar across all studied ethnic backgrounds. These data suggest that high serum IFN-alpha activity is a complex heritable trait, which plays a primary role in SLE pathogenesis.

Elevated high-sensitivity C-reactive protein levels are associated with local inflammatory findings in patients with osteoarthritis.

OBJECTIVE: C-reactive protein (CRP) has been associated with disease progression in patients with osteoarthritis (OA), but the reasons for this remain unclear. We hypothesized that higher CRP would be related to local inflammatory findings in the joints of patients with OA. METHODS: Plasma and synovial membrane specimens from 54 OA patients undergoing total hip or knee arthroplasty or arthroscopy were obtained. Synovial fluid was obtained from 25 of these patients. Hematoxylin and eosin stained synovial membrane sections were scored for degree of inflammatory cell infiltration. Plasma high-sensitivity CRP (hsCRP) levels, and serum and synovial fluid interleukin (IL)-6 and IL-1beta levels were measured by enzyme-linked immunosorbent assay. RESULTS: Fifty-seven percent of patients with idiopathic OA had inflammatory infiltrates within the synovial membrane. The mean hsCRP level in patients with inflammatory infiltrates was significantly higher than those without inflammation (4.7 +/- 5.0 mg/L vs 1.7 +/- 3.6 mg/L, P = 0.003). There were significant correlations between hsCRP levels and synovial fluid IL-6 (r = 0.64, P = 0.0006), degree of synovial inflammatory infiltration (r = 0.43, P = 0.002), and body mass index (r = 0.31, P = 0.02). Multivariate analysis indicated that only degree of inflammatory infiltrate was significantly associated with hsCRP level (P = 0.026). CONCLUSION: These results suggest that systemic hsCRP levels reflect synovial inflammation in OA patients, perhaps by means of synovial IL-6 production. Future studies are needed to clarify how these infiltrates and their products may contribute to disease pathogenesis.

Studies of autologous T cell activation in the Kunkel laboratory.

T helper cells and their antigen receptors were topics of keen interest in Henry Kunkel's laboratory during the early 1980s. The activation of human T cells by foreign antigen, allogeneic cells and autologous non-T cells had been established, but the most effective stimulator cells in those responses had not yet been identified. Dendritic cells, along with activated B cells, were demonstrated to be important stimulators of autologous T cells, and studies of peripheral blood from patients with SLE supported the conclusion that the non-T cells in those patients were deficient in their capacity to stimulate an autologous mixed lymphocyte reaction (AMLR). Subsequent studies have defined the role of apoptotic cells processed by dendritic cells in autologous T cell activation. In view of recent data demonstrating depletion of dendritic cell subsets in SLE peripheral blood and recruitment of those cells to sites of immune system activity, it is proposed that SLE T cells are indeed capable of self-reactivity and that the impaired in vitro proliferative response to autologous non-T cells as assessed in the AMLR may reflect the shift of dendritic cells, with their antigen presenting activity augmented by adjuvant-like factors, from peripheral blood to peripheral lymphoid organs and sites of disease.

Regulation of CD40 ligand expression in systemic lupus erythematosus.

Production of pathogenic autoantibodies in systemic lupus erythematosus (SLE) requires T cell help, along with ligation of the B cell surface immunoglobulin receptor by antigen. It is likely that macrophages, dendritic cells, and endothelial cells are also activated by interactions with T cells and contribute to lupus pathology. CD40 ligand (CD40L, CD154), a member of the tumor necrosis factor family of cell surface molecules, mediates these contact dependent signals delivered by CD4 + T helper cells to CD40 + target cells. Recent data from SLE patients and murine lupus models have demonstrated prolonged expression of CD40L on lupus T cells and its capacity to mediate excessive B cell activation. This review summarizes the current information regarding transcriptional and post-transcriptional regulation of CD40L expression in normal and SLE T cells. More complete characterization of the mechanisms that regulate the magnitude and duration of CD40L expression should suggest new approaches to modulate this promising therapeutic target.

Segmental amplification of 11q23 region identified by fluorescence in situ hybridization in four patients with myeloid disorders: a review.

Four cases with a segmental amplification of 11q23 region were detected by FISH. The amplification was either contiguous amplification on chromosome 11, or multiple markers involving the 11q23 region. The markers were derivative chromosomes, or isochromosomes. Amplification of 11q23 region was associated with complex karyotypes at the time of diagnosis or following treatment in secondary leukemias. Three were AML cases belonging to either AML-M5a or AML-1 subtypes and one was a myeloproliferative disorder. These cases were resistant to treatment. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) studies using MLL, 11 painting, or 11 centromere probes ascertained the segmental amplification. Since the patients did not respond to treatment the amplification of gene or genes that map to 11q23 may be responsible for the unfavorable prognosis. Hence, this type of amplifications could have clinical significance.

Protein aggregation mediated by cysteine oxidation during the stacking phase of discontinuous buffer SDS-PAGE.

The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel, followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C chemokine with four cysteines, aggregated during the stacking phase of SDS-PAGE and generated a band with an apparent molecular mass of 18 kDa. This aggregation depended on the presence of reduced sulfhydryl residues on IP-10, on the amount of loaded protein, and on the concentration of the ammonium persulfate used to polymerize the stacking gel. The aggregation of IP-10 could be prevented by reduction of its sulfhydryls with dithiothreitol followed by irreversible blockade with iodoacetamide. These methods may be useful in the prevention of aggregation of sulfhydryl-containing proteins during SDS-PAGE, especially when large quantities are analyzed to assess their purity.

Functional properties of lymphocytes in idiopathic thrombocytopenic purpura.

Idiopathic or immune thrombocytopenic purpura (ITP) is characterized by antibody-mediated destruction of platelets. The etiology is unknown. We postulated that increased autoantibody production in ITP might be attributable to either increased or prolonged expression of CD40 ligand (CD40L, CD154) in T or B lymphocytes, as has been previously observed in systemic lupus erythematosus (SLE). In addition, we hypothesized that ITP is characterized by increased levels of interleukin 4 (IL-4), a prototypic Th2 cytokine which, along with CD40 ligation, is required for B cell differentiation and production of several IgG subclasses. Cell surface CD154 expression was measured in freshly-isolated and in vitro-activated peripheral blood lymphocytes of sixteen ITP patients and eight healthy volunteers. Plasma levels of IL-4 and the prototypic Th1 cytokine interferon-gamma (IFNgamma) were determined. We observed that CD154 expression in unstimulated and in vitro-activated lymphocytes did not differ between ITP patients and healthy controls. Plasma levels of the Th2 cytokine IL-4 were significantly higher in the ITP patients. These studies indicate that overexpression of CD154 in lymphocytes is unlikely to be a primary pathophysiological defect in most patients with ITP. The data support that in addition to cell membrane antigens such as CD154, soluble cytokines such as IL-4 should be considered as potential targets for therapy in this disease.

Induction of Fas ligand-mediated apoptosis by interferon-alpha.

Interferon-alpha (IFN-alpha) was among the first cytokines studied and the earliest to be used in clinical medicine for the treatment of viral infections and malignancies. Although the capacity of IFN-alpha to augment NK cell cytotoxicity against virus-infected target cells or tumor cells is well established, the mechanism has not been fully elucidated. Here we report that IFN-alpha stimulation of PBMC from healthy donors induces Fas (CD95) ligand (FasL) transcription and leads to increased cell surface FasL expression exclusively on the NK cell fraction. Furthermore, IFN-alpha augments the FasL-mediated cytotoxicity of normal PBMC against Fas-sensitive lymphoid tumor cells. In the context of innate immunity, induction of FasL by IFN-alpha can be viewed as an efficient mechanism to potentiate NK cell cytotoxicity in the presence of harmful targets, such as virally infected or transformed cells.

Remission of juvenile rheumatoid arthritis after infection with parvovirus B19.

A 22-year-old Caucasian woman with a 6 year history of persistently active, systemic onset juvenile rheumatoid arthritis (JRA) developed symptoms of headache, dry cough, nausea, vomiting, abdominal pain, diarrhea, and dehydration associated with a high fever, elevated liver enzymes, and lymphopenia. Subsequent investigation revealed acute infection with parvovirus B19. Following clinical improvement over 10-14 days solely with supportive care, her underlying disease remained in remission for about 7 months.

Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera.

OBJECTIVE: To measure soluble CD40 ligand (sCD40L) in sera from patients with systemic lupus erythematosus (SLE) and to study the functional capacity of sCD40L in mediating B cell activation. METHODS: A 2-site enzyme-linked immunosorbent assay (ELISA) was used to measure sCD40L in the sera of 66 SLE patients, 30 disease control patients, and 23 healthy subjects. Induction of B cell activation antigen expression was used to assess the functional capacity of sCD40L in SLE sera. RESULTS: The mean concentration of sCD40L was statistically significantly higher (P < 0.0001) in SLE patients than in disease controls or healthy subjects, and segregation of SLE patients by severe, moderate, or mild extent of disease showed a relationship between disease severity and sCD40L concentration. Western blot analysis demonstrated the presence of the 18-kd band of sCD40L in SLE sera, and the results of a 1-site ELISA protocol suggested that some of the product in SLE sera was present in dimer or trimer form. Functional studies showed that 10 ng/ml of recombinant CD40L, a level present in some SLE sera, induced increased expression of CD95 on B cells. Several SLE sera also induced CD95 or CD86 on Ramos B cells, a result that was inhibited by anti-CD40L monoclonal antibodies. CONCLUSION: The soluble form of CD40L is present in the sera of most patients with SLE and may have the capacity to mediate B cell activation. Aberrant expression of CD40L might be predicted to result in activation of bystander B cells, including those that have encountered self antigens, and to contribute to autoantibody secretion.