ELISA, protein immunoprecipitation and line blot assays for anti-TIF1-gamma autoantibody detection in cancer-associated dermatomyositis.

OBJECTIVES: Anti-TIF1-gamma autoantibodies can be detected with immunoprecipitation (IP), line blot (LB) and ELISA. We compared assay performance in patients with DM and the potential of these assays to detect anti-TIF1-gamma positive cancer-associated DM (CADM). METHODS: We included sera from 131 patients with DM followed at Karolinska University Hospital, Stockholm, Sweden and 82 healthy controls. Serum samples taken at DM diagnosis were tested for anti-TIF1-gamma autoantibodies with IP, two ELISAs (in-house and commercial) and LB. Cancer diagnosis and dates were obtained from the Swedish national cancer register. CADM was defined as a malignancy that developed within 3 years of DM diagnosis. RESULTS: Anti-TIF1-gamma autoantibodies were detected in 19/101 (18.8%), 15/113 (13.2%), 34/131 (26%) and 45/131 (34.4%) of the patients with IP, LB, in-house and commercial ELISA, respectively. The anti-TIF1-gamma results from the in-house ELISA were confirmed with IP in 93 of 101 (92%) cases, κ = 0.76, with a commercial ELISA in 110 of 131 (84%) cases, κ = 0.63, and with LB in 101 of 113 (89.3%) cases, κ = 0.67. Anti-TIF1-gamma results with IP were confirmed with LB in 85 of 92 (92.4%) cases, κ = 0.73. For detecting CADM, the anti-TIF1-gamma in-house ELISA had a sensitivity of 58% and specificity of 86%, the commercial ELISA had a sensitivity of 63% and specificity of 82%, IP had a sensitivity of 52% and specificity of 92%, LB had a sensitivity of 40% and specificity of 96%. CONCLUSION: The two anti-TIF1-gamma ELISA assays had advantages both for autoantibody detection and to identify anti-TIF1-gamma-positive CADM.

Association of Anti-Transcription Intermediary Factor 1γ Antibodies With Paraneoplastic Rheumatic Syndromes Other Than Dermatomyositis.

OBJECTIVE: An association between cancer and dermatomyositis (DM) is well recognized. The high frequency of malignancies detected close to DM diagnosis suggest that DM can be a paraneoplastic syndrome. Recently, anti-transcription intermediary factor 1γ (anti-TIF1γ) has been discovered to be associated with cancer and with DM. A meta-analysis reported the pooled sensitivity of anti-p155 for diagnosing cancer-associated DM to be 78% and the specificity to be 89%. Thus, anti-TIF1γ has shown promising results as a marker for cancer-associated DM. However, none of the studies evaluated the association of anti-TIF1γ with cancer with or without rheumatic diseases other than DM. To clarify the specificity of anti-TIF1γ antibodies as a biomarker for cancer-associated DM, we analyzed the frequency of anti-TIF1γ antibodies in other cancer-associated rheumatic syndromes, as well as in cancer patients and healthy controls. METHODS: Sera from patients with paraneoplastic rheumatic syndrome (n = 91), patients with solid cancer (n = 95), and healthy controls (n = 80) were analyzed for the frequency of anti-TIF1γ IgG by enzyme-linked immunosorbent assay using a commercially available recombinant TIF1γ protein as coating antigen. The cutoff value was calculated by adding 2 SD to the mean optical density value of 80 healthy controls. RESULTS: The rate of anti-TIF1γ IgG positivity was 3.3% (n = 3) in patients with paraneoplastic rheumatic syndrome, 3.1% (n = 3) in cancer patients, and 1.3% (n = 1) in healthy controls. There were no significant differences in positivity between the groups (P < 0.05). CONCLUSION: Anti-TIF1γ antibodies are rarely present in patients with solid cancers or paraneoplastic rheumatic syndromes. This finding strengthens the approach to using anti-TIF1γ IgG as a marker for cancer-associated DM.

CD4+ and CD8+ CD28(null) T Cells Are Cytotoxic to Autologous Muscle Cells in Patients With Polymyositis.

OBJECTIVE: Inflammatory T cell infiltrates in the skeletal muscle tissue of patients with polymyositis are dominated by CD28-negative effector (CD28(null) ) T cells of both the CD4 and CD8 lineage. These cells are potentially cytotoxic, and the aim of the present study was to develop a fully autologous cell culture system in which to investigate the functional contribution of such CD28(null) T cells to myotoxicity. METHODS: In vitro cocultures of autologous skeletal muscle cells and T cell subsets obtained from 5 polymyositis patients were performed. Myotoxicity of T cells was quantified by calcein release and flow cytometric analyses. T cell degranulation was blocked with concanamycin A. Specific blocking of perforin, cytokines, and HLA was performed using antibodies. RESULTS: Both CD4+CD28(null) and CD8+CD28(null) T cells induced more muscle cell death than did their CD28+ counterparts. Differentiated muscle cells (myotubes) were more sensitive to T cell-mediated cell death than were their precursors (myoblasts). Both CD8+ and CD4+ CD28(null) T cells displayed perforin polarization toward muscle cells and secreted higher levels of granzyme B and interferon-γ (IFNγ) in coculture than did CD28+ T cells. The myotoxic effects of CD28(null) T cells were reduced upon the blocking of perforin, cytokines, and HLA. Addition of IFNγ or tumor necrosis factor did not induce skeletal muscle cell death in the absence of T cells; however, it did up-regulate HLA expression on muscle cells. CONCLUSION: Myotoxicity of CD4+ and CD8+ CD28(null) T cells is mediated by directed perforin-dependent killing and can be further influenced by IFNγ-induced HLA expression on muscle cells. The data suggest that CD28(null) T cells are key effector cells that contribute to the muscle cell damage in polymyositis.

Cardiomyopathy in murine models of systemic sclerosis.

OBJECTIVE: Cardiomyopathy has emerged as a leading cause of death in patients with systemic sclerosis (SSc). However, the pathogenesis of SSc-related cardiomyopathy is poorly understood, and new therapies as well as platforms for testing are needed. The aim of this study was to characterize the histopathologic features of cardiomyopathy in patients with SSc and in common mouse models of SSc. METHODS: The histopathologic features of myocardial tissue specimens obtained at autopsy from 5 subjects with SSc and 5 control subjects matched for sex, age, and cardiovascular risk factors were evaluated and compared with those of myocardial tissue specimens obtained from 3 common mouse models of SSc with systemic manifestations: Fra-2-transgenic mice, mice with sclerodermatous chronic graft-versus-host disease (GVHD), and TSK-1 mice. RESULTS: Myocardial tissue from autopsy subjects with SSc and no clinically manifest cardiac involvement showed endothelial cell apoptosis with reduced capillary density, perivascular inflammation, myofibroblast differentiation, and accumulation of collagen. Only selected features of SSc-related cardiomyopathy were observed in the mice with chronic GVHD and TSK-1 mice. However, the myocardial tissue of Fra-2-transgenic mice mimicked all features of SSc-related cardiomyopathy and also demonstrated comparable vascular, inflammatory, and fibrotic manifestations. Of note, the expression of Fra-2 was also increased in the myocardium of autopsy subjects with SSc. CONCLUSION: We demonstrate that all typical manifestations of SSc-related cardiomyopathy are mimicked in Fra-2-transgenic mice. Moreover, overexpression of Fra-2 in the myocardium of autopsy subjects with SSc may suggest similar underlying pathogenic mechanisms. Thus, Fra-2-transgenic mice might be a suitable preclinical model with which to study the mechanisms of and therapeutic approaches to myocardial involvement in SSc.

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.

Immune mechanisms in polymyositis and dermatomyositis and potential targets for therapy.

PM and DM are characterized clinically by weakness and low endurance of skeletal muscle. Other organs are frequently involved, suggesting that idiopathic inflammatory myopathies (IIMs) are systemic inflammatory diseases. Involvement of immune mechanisms in IIMs is supported by the presence of T cells, macrophages and dendritic cells in muscle tissue, by the presence of autoantibodies and by HLA-DR being a strong genetic risk factor. T cells may have direct and indirect toxic effects on muscle fibres, causing muscle fibre necrosis and muscle weakness, but the target of the immune reaction is not known. A newly identified T cell subset, CD28(null) T cells, may have cytotoxic effects in the CD4(+) and CD8(+) T cell phenotype. These cells are apoptosis resistant and may contribute to treatment resistance. Several myositis-specific autoantibodies have been identified, but they are all directed against ubiquitously expressed autoantigens and the specificity of the T cell reactivity is not known. These autoantibodies are associated with distinct clinical phenotypes and some with distinct molecular pathways; e.g. sera from patients with anti-Jo-1 autoantibodies may activate the type I IFN system and these sera also contain high levels of B cell activating factor compared with other IIM subsets. The characterization of patients into subgroups based on autoantibody profiles seems to be a promising way to learn more about the specificities of the immune reactions. Careful phenotyping of infiltrating immune cells in muscle tissue before and after specific therapies and relating the molecular findings to clinical outcome measures may be another way to improve knowledge on specific immune mechanism in IIMs. Such information will be important for the development of new therapies.

T cells in myositis.

T cells of both the CD4 and CD8 lineage are commonly found in affected tissues of patients with idiopathic inflammatory myopathies, but understanding the contribution of these cells to immunopathogenesis remains challenging. Given recent advances in identifying more myositis-associated autoantibodies and their putative targets, we suggest that studies on autoreactive T cells targeting those autoantigens are one way forward. Another (so far, more frequently used) approach comes from studies on effector T cells in the context of myositis. This review summarizes recent advances and current hypotheses in both of these contexts.

Activation of canonical Wnt signalling is required for TGF-ß-mediated fibrosis.

The transforming growth factor-ß (TGF-ß) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-ß and the canonical Wnt pathway. TGF-ß stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-ß receptor type I signalling and also prevents fibrosis in other TGF-ß-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-ß-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases.

Prevalence of paraneoplastic rheumatic syndromes and their antibody profile among patients with solid tumours.

The aims of this study were to assess the prevalence of paraneoplastic rheumatic syndromes in a cohort of patients with newly diagnosed solid tumours and to describe their autoimmune profile, comparing it to the controls. Screening questionnaires (3,770) were distributed, and during a three-step study, 94 patients were confirmed to have both paraneoplastic syndrome and oncology diagnoses. Three control groups-patients with undifferentiated arthritis, Raynaud's phenomenon for non-malignant causes and solid tumours only-were designed to compare with the paraneoplastic cases and their immunology profile. The prevalence of paraneoplastic rheumatic syndromes was 2.65% (95% CI 0.21-3.20). The group of patients with arthritis and the group of patients with Raynaud's syndrome were found to prevail among other clinical presentations of paraneoplastic rheumatic syndromes. Both paraneoplastic syndromes were linked to malignancies of the urogenital system. Antinuclear antibodies were found to be similarly frequent in the paraneoplastic arthritis, paraneoplastic Raynaud's phenomenon and the solid tumour groups. No differences were observed when comparing paraneoplastic arthritis and undifferentiated arthritis, except that the patients with paraneoplastic arthritis were older. Comparing paraneoplastic Raynaud's to Raynaud's phenomenon, male preponderance in the paraneoplastic Raynaud's phenomenon group was observed, and the patients were obviously older. Paraneoplastic rheumatic syndromes are rare and more often occur in older patients. Among them, paraneoplastic arthritis and Raynaud's syndrome were the most frequent. The immunology profile does not help in discriminating between arthritis and paraneoplastic arthritis patients and is of limited use in Raynaud's differential diagnosis.

The transcription factor Fra-2 regulates the production of extracellular matrix in systemic sclerosis.

OBJECTIVE: Fra-2 belongs to the activator protein 1 family of transcription factors. Mice transgenic for Fra-2 develop a systemic fibrotic disease with vascular manifestations similar to those of systemic sclerosis (SSc). The aim of the present study was to investigate whether Fra-2 plays a role in the pathogenesis of SSc and to identify the molecular mechanisms by which Fra-2 induces fibrosis. METHODS: Dermal thickness and the number of myofibroblasts were determined in skin sections from Fra-2-transgenic and wild-type mice. The expression of Fra-2 in SSc patients and in animal models of SSc was analyzed by real-time polymerase chain reaction and immunohistochemistry. Fra-2, transforming growth factor beta (TGFbeta), and ERK signaling in SSc fibroblasts were inhibited using small interfering RNA, neutralizing antibodies, and small-molecule inhibitors. RESULTS: Fra-2-transgenic mice developed a skin fibrosis with increases in dermal thickness and increased myofibroblast differentiation starting at age 12 weeks. The expression of Fra-2 was up-regulated in SSc patients and in different mouse models of SSc. Stimulation with TGFbeta and platelet-derived growth factor (PDGF) significantly increased the expression of Fra-2 in SSc fibroblasts and induced DNA binding of Fra-2 in an ERK-dependent manner. Knockdown of Fra-2 potently reduced the stimulatory effects of TGFbeta and PDGF and decreased the release of collagen from SSc fibroblasts. CONCLUSION: We demonstrate that Fra-2 is overexpressed in SSc and acts as a novel downstream mediator of the profibrotic effects of TGFbeta and PDGF. Since transgenic overexpression of Fra-2 causes not only fibrosis but also vascular disease, Fra-2 might be an interesting novel candidate for molecular-targeted therapies for SSc.

Relationship between serum levels of TGF-beta1 and clinical parameters in patients with rheumatoid arthritis and Sjögren's syndrome secondary to rheumatoid arthritis.

TGF-beta1 is a pleiotropic cytokine, which prevents inappropriate autoimmune responses and balances the requirements of proper immune cell levels during pathologic states that trigger the immune response. We assessed the serum levels of TGF-beta1 and determined the relationship between TGF-beta1 and clinical parameters in patients with rheumatoid arthritis (RA) and Sjögren's syndrome (SS) secondary to RA (SS + RA). Comparison of the serum levels of TGF-beta1 in patients with RA, SS + RA and NHD differed significantly (51.7 +/- 12.4 ng/ml (RA); 33.0 +/- 3.1 ng/ml (SS + RA) and versus 31.6 +/- 2.0 ng/ml (NHD)). We further found correlations between TGF-beta1 levels and radiologically defined joint damage determined by the Steinbrocker scoring system, symptoms and signs of SS. We conclude that serum levels of TGF-beta1 may reflect ongoing autoimmune inflammation and correlate with joint damage in RA.

Treatment with imatinib prevents fibrosis in different preclinical models of systemic sclerosis and induces regression of established fibrosis.

OBJECTIVE: Imatinib is a small-molecule tyrosine kinase inhibitor capable of selective, dual inhibition of the transforming growth factor beta and platelet-derived growth factor (PDGF) pathways. Imatinib has previously been shown to prevent the development of inflammation-driven experimental fibrosis when treatment was initiated before administration of the profibrotic stimulus. The aim of this study was to confirm the efficacy of imatinib in a murine model of systemic sclerosis (SSc) that is less driven by inflammation and to investigate whether imatinib is also effective for the treatment of established fibrosis. METHODS: The tight skin 1 (TSK-1) mouse model of SSc was used to evaluate the antifibrotic effects of imatinib in a genetic model of the later stages of SSc. In addition, the efficacy of imatinib for the treatment of preestablished fibrosis was analyzed in a modified model of bleomycin-induced dermal fibrosis in which the application of bleomycin was prolonged and the onset of treatment was late. RESULTS: Treatment with imatinib reduced dermal and hypodermal thickening in TSK-1 mice and prevented the differentiation of resting fibroblasts into myofibroblasts. In the model of preestablished dermal fibrosis, imatinib not only stopped further progression of fibrosis but also induced regression of preexisting dermal fibrosis, with a reduction in dermal thickness below pretreatment levels. CONCLUSION: These results indicate that combined inhibition of the tyrosine kinase c-Abl and PDGF receptor might be effective in the later, less inflammatory stages of SSc and for the treatment of established fibrosis. Thus, imatinib might be an interesting candidate for clinical trials in patients with longstanding disease and preexisting tissue fibrosis.

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo.

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.

Src kinases in systemic sclerosis: central roles in fibroblast activation and in skin fibrosis.

OBJECTIVE: Src kinases are nonreceptor tyrosine kinases, which have been implicated in cytoskeletal organization and cell mobility. This study was undertaken to evaluate the potential of Src kinases as novel targets of antifibrotic therapies. METHODS: Fibroblast cultures were obtained from 10 patients with systemic sclerosis (SSc) and 5 healthy subjects. Src signaling was inhibited using small-molecule inhibitors and overexpression of a dominant-negative mutant of Src and of the endogenous inhibitor Csk. The expression of extracellular matrix proteins was analyzed by real-time polymerase chain reaction and by SirCol collagen assay. Toxic effects were excluded by MTT assay and staining for annexin V and propidium iodide. The mouse model of bleomycin-induced dermal fibrosis was used to assess the role of Src kinases in dermal fibrosis in vivo. RESULTS: Stimulation with transforming growth factor beta and platelet-derived growth factor activated Src signaling in dermal fibroblasts from patients with SSc and healthy donors. Incubation with the Src kinase inhibitors or overexpressed mutant Src or Csk reduced the synthesis of messenger RNA for COL1A1, COL1A2, and fibronectin 1. A dose-dependent reduction in collagen release was also observed at the protein level. No inhibitory effects on proliferation and no increase in the number of apoptotic or necrotic fibroblasts were observed. Consistent with the in vitro data, inhibition of Src kinases prevented experimental dermal fibrosis. Dermal thickness, the amount of collagen protein, and the number of myofibroblasts were reduced in a dose-dependent manner. CONCLUSION: These findings indicate that Src kinases play important roles in the activation of fibroblasts and in the development of experimental fibrosis. Thus, Src kinases might be interesting targets for novel antifibrotic therapies in SSc.