Dysfunction of endothelial progenitor cells is associated with the type I IFN pathway in patients with polymyositis and dermatomyositis.

OBJECTIVE: Alterations in phenotype and function of endothelial progenitor cells (EPCs) have been associated with poor vascular outcomes and impaired vascular repair in various conditions. Our hypothesis was that patients with PM and DM have dysregulation of EPCs driven by type I IFN and IL-18 similar to other autoimmune diseases. METHODS: Quantification of circulating EPCs was performed by flow cytometry in patients with PM/DM and matched healthy controls. The ability of EPCs to differentiate into mature endothelial cells was investigated by light and fluorescence microscopy quantification in the presence or absence of PM/DM or control serum, neutralizing antibodies to type I IFN receptor or IL-18. Serum type I IFN activity was quantified by induction of type I IFN-inducible genes in HeLa cells. Circulating IL-18 concentrations were assessed by ELISA. RESULTS: Circulating EPCs were significantly lower in PM/DM patients compared with controls. PM/DM EPCs displayed a decreased capacity to differentiate into mature endothelial cells and PM/DM serum significantly inhibited differentiation of control EPCs. This effect was reversed in the majority of samples with neutralizing antibodies to IL-18 or to type I IFN receptor or by a combination of these antibodies. Patients with associated impairments in EPC function had higher type I IFN serum activity. CONCLUSION: PM/DM is associated with dysregulation of EPC phenotype and function that may be attributed, at least in part, to aberrant IL-18 and type I IFN pathways. The implication of these vasculopathic findings for disease prognosis and complications remains to be determined.

Expression of BAFF receptors in muscle tissue of myositis patients with anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies.

INTRODUCTION: Anti-Jo-1 and anti-Ro52 autoantibodies are common in patients with myositis, but the mechanisms behind their production are not known. Survival of autoantibody-producing cells is dependent on B-cell-activating factor of the tumour necrosis factor family (BAFF). BAFF levels are elevated in serum of anti-Jo-1-positive myositis patients and are influenced by type-I interferon (IFN). IFN-producing cells and BAFF mRNA expression are present in myositis muscle. We investigated expression of the receptors for BAFF in muscle tissue in relation to anti-Jo-1 and anti-Ro52/anti-Ro60 autoantibodies and type-I IFN markers. METHODS: Muscle biopsies from 23 patients with myositis selected based on autoantibody profile and 7 healthy controls were investigated for expression of BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Nineteen samples were assessed for plasma (CD138) and B-cell (CD19) markers. The numbers of positive cells per area were compared with the expression of plasmacytoid dendritic cell (pDC) marker blood dendritic cell antigen-2 (BDCA-2) and IFNα/ß-inducible myxovirus resistance-1 protein (MX-1). RESULTS: BAFF-R, BCMA and TACI were expressed in five, seven and seven patients, respectively, and more frequently in anti-Jo-1-positive and/or anti-Ro52/anti-Ro60-positive patients compared to controls and to patients without these autoantibodies (P = BAFF-R: 0.007, BCMA: 0.03 and TACI: 0.07). A local association of receptors with B and plasma cells was confirmed by confocal microscopy. The numbers of CD138-positive and BCMA-positive cells were correlated (r = 0.79; P = 0.001). Expression of BDCA-2 correlated with numbers of CD138-positive cells and marginally with BCMA-positive cells (r = 0.54 and 0.42, respectively; P = 0.04 and 0.06, respectively). There was a borderline correlation between the numbers of positively stained TACI cells and MX-1 areas (r = 0.38, P = 0.08). CONCLUSIONS: The expression pattern of receptors for BAFF on B and plasma cells in muscle suggests a local role for BAFF in autoantibody production in muscle tissues of patients with myositis who have anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies. BAFF production could be influenced by type-I IFN produced by pDCs. Thus, B-cell-related molecular pathways may participate in the pathogenesis of myositis in this subset of patients.

Anakinra treatment in patients with refractory inflammatory myopathies and possible predictive response biomarkers: a mechanistic study with 12 months follow-up.

OBJECTIVE: To perform a mechanistic study on the effect of interleukin (IL)-1 blockade by anakinra in patients with refractory myositis and to explore possible predictive biomarkers. METHODS: Fifteen patients with refractory myositis were treated with anakinra for 12 months. Clinical response was assessed by the six-item core set measures of disease activity International Myositis Assessment and Clinical Studies (IMACS) and functional index (FI). Repeated muscle biopsies were investigated for cellular infiltrates, IL-1α, IL-1ß, IL-1Ra and major histocompatibility complex-class I by immunohistochemistry. Serum levels of IL-1Ra and granulocyte colony-stimulating factor (G-CSF) were measured by ELISA. T cell phenotype and functional assays were investigated by multicolour flow cytometry. RESULTS: Seven patients had clinical response according to IMACS, four of them also showed improved FI. Responders had higher baseline extramuscular score compared with non-responders. In muscle biopsies, baseline CD163 macrophages and IL-1α expression were inversely correlated with muscle performance after 6 months treatment; all responders had IL-1Ra expression in the post-treatment biopsies but only 3/8 non-responders. In serum, IL-1Ra levels were increased and G-CSF was decreased after 6 months treatment, but their levels and changes were not related to clinical response. For T cells, an inverse correlation between baseline frequency of CD4 activated/memory T cells and decreased creatine kinase levels was observed. Five of six patients demonstrated less IL-17A and more IFN-γ secreting CD4 T cells after 6 months treatment. Moreover, anakinra reduced IL-17A secretion in vitro. CONCLUSIONS: Patients with myositis may respond to anakinra. Extramuscular score, muscle CD163 macrophages and IL-1α expression, blood CD4 activated/memory T cells might associate with anakinra treatment response. Blocking the IL-1 receptor disfavoured Th17 cell differentiation both in vivo and in vitro.

Low serum levels of vitamin D in idiopathic inflammatory myopathies.

OBJECTIVES: To evaluate serum levels of 25(OH) vitamin D in patients with idiopathic inflammatory myopathies (IIM) (polymyositis (PM), dermatomyosistis (DM), inclusion body myositis (IBM) and juvenile DM (JDM)) and to compare these with healthy controls. METHODS: Serum samples from 149 patients with IIM and 290 healthy controls matched for gender and the month of blood sampling were analysed for 25(OH) vitamin D. ORs for vitamin D classes with 95% CI were calculated using a matched (conditional) logistic regression model. Groups were compared by the Kruskal-Wallis test and p values <0.05 were considered significant. RESULTS: Patients with IIM had significantly lower serum levels of 25(OH) vitamin D than healthy controls (median 39 (10-168) nmol/l vs 68 (19-197) nmol/l; p=0.0001). There was no significant difference in vitamin D levels between the myositis subgroups. When vitamin D levels were subclassified into deficient (<50 nmol/l), insufficient (50-74 nmol/l) and normal (≥75 nmol/l), most of the patients with PM (68%), DM (65%) and IBM (53%) had deficient levels compared with only 60 (21%) healthy individuals. In patients with IIM the OR for deficient versus normal was 17.7 (95% CI 8.1 to 38.6) and the OR for insufficient versus normal was 2.4 (95% CI 1.2 to 4.7). CONCLUSIONS: Low serum levels of vitamin D were found in most patients with IIM and may confer a risk factor for developing adult myositis, similar to some other autoimmune diseases.

Sera from anti-Jo-1-positive patients with polymyositis and interstitial lung disease induce expression of intercellular adhesion molecule 1 in human lung endothelial cells.

OBJECTIVE: To investigate whether sera or purified IgG from patients with polymyositis (PM) and patients with dermatomyositis (DM), with or without interstitial lung disease (ILD), can activate endothelial cells (ECs). METHODS: Patients' sera were selected based on the presence or absence of anti-Jo-1, anti-SSA, or anti-U1 small nuclear RNP autoantibodies. The presence of autoantibodies was determined by line blot assays. Cultured human microvascular ECs derived from lung tissue (HMVEC-L) were incubated with sera or purified IgG from 22 patients with PM, 7 patients with DM, and 10 healthy individuals as controls. Assessment of intercellular adhesion molecule 1 (ICAM-1) expression was conducted by immunofluorescence (n=22) and by cell-based enzyme-linked immunosorbent assay (ELISA) (n=20). Serum levels of soluble ICAM-1 (sICAM-1) were determined by ELISA. RESULTS: Sera from PM patients with ILD who were positive for anti-Jo-1 autoantibodies had a significantly stronger effect on the expression of ICAM-1 by HMVEC-L in comparison with sera from healthy controls and patients with other autoantibodies. Purified IgG did not induce ICAM-1 expression. Higher serum levels of sICAM-1 were found in patients with myositis compared with healthy controls. CONCLUSION: EC activation with ICAM-1 expression could contribute to the multiorgan involvement, including the development of myositis and ILD, in patients carrying anti-Jo-1 autoantibodies. The EC-activating factors are not the autoantibodies themselves, but might be systemic factors associated with these autoantibodies.

Use of a commercial line blot assay as a screening test for autoantibodies in inflammatory myopathies.

AIMS: To evaluate the clinical utility of a commercial immunoblot assay for the detection of myositis-specific autoantibodies. METHODS: Serum samples from 153 myositis patients and 77 disease controls were investigated. The commercial Euroline assay with seven autoantigens (Mi-2, Ku, PM-Scl, Jo-1, Pl-7, Pl-12 and SSA/Ro-52) was used according to the manufacturer s instructions, and supplemented with an anti-SRP strip. In a separate experiment analyses were performed at different temperatures. Results were recorded with densitometry. RESULTS: Anti-Jo-1 was found in 18 myositis and one systemic sclerosis patient. Antibodies against Mi-2 were found in 5 myositis patients, and eleven myositis patients had antibodies against PM-Scl. Four myositis patients showed anti-Pl-7 reactivity, whereas no patients had antibodies against Pl-12. Anti-Ku antibodies were found in 4 myositis and 2 primary Sjögren's syndrome patients. Anti-SRP was found in 8 myositis patients as well as in two disease controls. Antibodies against SSA/Ro52 ranged between 23-62% in all groups except juvenile dermatomyositis patients. Most autoantibody reactivities were clearly positive, only 11% (14/127) were borderline positive. Higher assay temperature increased antibody reactivities. CONCLUSIONS: Except for anti-SSA/Ro-52 and anti-Ku the antibody reactivities were rather myositis-specific, supporting the use of this immunoblot assay. However, assay validation needs to be determined against other methods.

A possible mechanism for endogenous activation of the type I interferon system in myositis patients with anti-Jo-1 or anti-Ro 52/anti-Ro 60 autoantibodies.

OBJECTIVE: To investigate type I interferon (IFN) system activation and its correlation with autoantibodies and organ manifestations in polymyositis (PM), dermatomyositis (DM), and inclusion body myositis. METHODS: Sera from 30 patients and 16 healthy controls, or purified IgG, were combined with material released from necrotized cells to stimulate IFNalpha production by peripheral blood mononuclear cells (PBMCs) from healthy blood donors. Muscle biopsy specimens from 25 patients and 7 healthy controls were investigated for blood dendritic cell antigen 2 (BDCA-2)-positive plasmacytoid dendritic cells (PDCs) and IFNalpha/beta-inducible myxovirus resistance 1 (MX-1) protein. RESULTS: Sera from 13 patients who were positive for anti-Jo-1 or anti-Ro 52/anti-Ro 60 autoantibodies induced IFNalpha production in PBMCs when combined with necrotic cell material. In addition, IgG prepared from anti-Jo-1-positive PM sera induced IFNalpha with necrotic material, but not when the latter was treated with RNase. BDCA-2 expression in PDCs in muscle tissue was increased in PM patients with anti-Jo-1 autoantibodies, while MX-1 staining in capillaries was increased in DM patients, compared with healthy individuals. IFNalpha-inducing capacity correlated with interstitial lung disease, while MX-1 expression in the capillaries correlated with DM. CONCLUSION: Immune complexes containing anti-Jo-1 or anti-Ro 52/anti-Ro 60 autoantibodies and RNA may act as endogenous IFNalpha inducers that activate IFNalpha production in PDCs. These PDCs could be of importance for inducing myositis, whereas in DM patients without autoantibodies the presence of MX-1 protein in capillaries suggests another cellular IFNalpha source and induction mechanism. Consequently, the type I IFN system may be of importance in both PM and DM, but via different pathways.

Limited effects of high-dose intravenous immunoglobulin (IVIG) treatment on molecular expression in muscle tissue of patients with inflammatory myopathies.

OBJECTIVES: The study was conducted with the aim of achieving an improved understanding of the molecular mechanisms of high-dose intravenous immunoglobulin (IVIG) in inflammatory myopathies by investigating the effects on muscle function and immunological molecules in skeletal muscle of polymyositis (PM), dermatomyositis (DM) and inclusion body myositis (IBM) patients. METHODS: Thirteen treatment-resistant patients, 6 PM, 4 DM, 2 IBM and 1 juvenile DM, were treated with 2 g/kg of IVIG, three times at monthly intervals. Functional Index in Myositis and serum creatinine kinase (CK) levels were determined, and muscle biopsies were performed before treatment and after the third IVIG infusion. Immunological molecules were also studied in biopsies taken 24-48 h after the first infusion. RESULTS: Improved muscle function was observed in three patients (1 PM, 1 DM and 1 IBM) and CK levels decreased in five. T cells, macrophages, major histocompatibility complex (MHC) class I antigen on muscle fibres, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and membranolytic attack complex (MAC) deposits on capillaries were present to an equal degree in biopsies before and after IVIG treatment. No correlation between the clinical response and molecular changes was found. CONCLUSIONS: The clinical effects of high-dose IVIG on muscle function in patients with refractory inflammatory active myositis did not correspond to effects on any of the investigated molecules in our study. T cells, macrophages, phenotypical changes in muscle fibres and endothelial cell activation were still present after treatment. These observations question a role for IVIG as an immune-modulating therapy in patients with inflammatory myopathies.