Local expression of cytokines in idiopathic inflammatory myopathies

H. Lepidi, V. Frances, D. Figarella-Branger, C. Bartoli, A. Machado-Baeta & J-F. Pellissier (1998) Neuropathology and Applied Biology , 24, 73–79
Local expression of cytokines in idiopathic inflammatory myopathies
The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM), are regarded as autoimmune diseases. They are characterized by chronic lymphocytic and macrophagic infiltration in muscle tissue. Of particular importance in understanding the immune response to IIM is the specific pattern of locally produced cytokines. Frozen muscle tissues from IIM (5 DM, 3 PM, and 1 IBM) were used to investigate the cytokine responses. The RT-PCR technique was instrumental to determine the pattern of expression of pro-inflammatory (IL-1β, IL-6, TNF-α), Th1 (IFN-γ IL-2), and Th2 (IL-4) cytokines. Immunohistochemistry was also used to localize morphologically IFN-γ and IL-4. Our results show that pro-inflammatory cytokines and Th1 cytokines are mainly expressed in IIM. The accumulation of mononuclear inflammatory cells and the inflammatory syndrome in IIM are probably related in part to the production of pro-inflammatory cytokines. Moreover, the pattern of local cytokine expression is consistent with a Th1 immune response related to autoimmune diseases.     

Distal inflammatory myopathy: unusual presentation of polymyositis or new entity?

New classification of idiopathic inflammatory myopathy (IIM) defined three major entities, polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (s-IBM). We report the clinical, electrophysiological and pathological characteristics of three patients with a rare form of IIM not fulfilling the diagnostic criteria for any of these three major entities. The three patients presented with a subacute, distal asymmetrical weakness in upper limbs. Muscle biopsy showed an active myositis, with necrosis and regeneration, T cell infiltrates with invasion of non-necrotic fibers, without rimmed vacuoles, and diffuse major histocompatibility complex-I (MHC-I) immunostaining in muscle fibers. All patients responded to immunosuppressive agents. Seven others cases were identified in the literature. It is important to recognize this atypical presentation as it seems to respond to immunosuppressive agents.     

Increase in transglutaminase 2 in idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IMs), including dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (s-IBM), are characterized by inflammatory cell infiltration in muscle tissue and muscle fiber destruction, which leads to muscle weakness. Although the cause of IMs is unclear, an autoimmune pathogenesis may be involved in initiating the muscle inflammation. Recently, we have found an aberrant expression of transglutaminase 2 (TGase 2) in s-IBM, which is closely associated with insoluble inclusion body formation. TGase 2 is a cross-linking enzyme that generates a conformational change of molecules via a covalent isopeptide bond. The increase in the level of TGase 2 expression and the inappropriate presentation of substrates/cross-linked aggregates to the immune system may contribute to the autoimmune aspects of IMs. We investigated whether or not an increase in TGase 2 expression is a common factor in muscle inflammation. Duchenne muscular dystrophy (DMD) and normal tissues were employed as controls. Using immunocytochemistry and quantitative RT-PCR, the level of TGase 2 expression was found to be specifically increased in PM and DM, but not in DMD and normal controls. Therefore, the targeting of TGase inhibition in IMs will be a challenging therapeutic approach that should be investigated in the near future.     

Chemokine-like factor expression in the idiopathic inflammatory myopathies

Objectives –  We evaluated the expression of chemokine-like factor (CKLF) in biopsied muscle fibers in inflammatory myopathies, non-inflammatory myopathies and neurologically diseased controls.
Materials and methods –  We studied the expression of CKLF in 15 polymyositis (PM), five dermatomyositis (DM), 15 non-inflammatory myopathies and nine neurologically diseased patients by immunohistochemistry.
Results –  Chemokine-like factor was mostly expressed in small diameter muscle fibers surrounded by infiltrated lymphocytes of inflammatory myopathies patients. Parts of them were also positive for the staining of the developmental form of myosin heavy chain, a maker of regenerating muscle fibers. Thrombin immunoreactivity was observed in endomysium in PM and perimysium in DM. In vitro differentiation study showed a constitutive expression of CKLF in myoblasts that was abolished in myotubes during differentiation process and was induced again by thrombin. Thrombin regulates CKLF expression through protease-activated receptor-1 in myotubes. Treatment of a protein kinase C inhibitor partially blocked CKLF expression in myoblasts, while it remarkably inhibited that in myotubes.
Conclusion –  Chemokine-like factor expression is differentially regulated in myoblasts and myotubes. Thrombin could be a strong regulator for its expression. As CKLF is immunohistochemically positive in regenerating muscle fibers, we postulate here that CKLF is a useful marker for regenerating muscle fibers in inflammatory myopathies.     

Expression of matrix metalloproteinases in the muscle of patients with inflammatory myopathies

OBJECTIVE: To investigate the role of matrix metalloproteinases (MMPs) in the pathogenesis of inflammatory myopathies and the amyloid formation in sporadic inclusion body myositis (s-IBM). BACKGROUND: MMPs comprise a family of calcium-dependent zinc endoproteinases induced by cytokines and secreted by inflammatory cells. They enhance T-cell migration or adhesion and degrade components of the extracellular matrix proteins. Some MMPs also have been implicated in the formation of beta-amyloid. METHODS: We examined the expression of MMPs with single and double immunocytochemistry using antibodies against MMP-2, MMP-3, MMP-7, MMP-9, major histocompatibility complex (MHC) class I, CD8+ cells, macrophage, and beta-amyloid precursor protein (beta-APP) on serial muscle biopsy sections from patients with s-IBM, polymyositis (PM), dermatomyositis (DM), and disease control specimens. The enzyme activity of MMPs was measured by gelatin substrate zymography. RESULTS: Only the gelatinases, MMP-9 and MMP-2, were expressed in the muscle. In s-IBM and PM, but not the control specimens, MMP-9 and MMP-2 immunostained the non-necrotic and MHC class-I-expressing muscle fibers, and MMP-9, but not MMP-2, immunostained the autoinvasive CD8+ cytotoxic T cells. Zymography in muscle homogenates confirmed the increased MMP-2 and MMP-9 enzymatic activity. MMP-2, but not MMP-9, immunostained the rimmed vacuoles in s-IBM and colocalized with beta-APP, suggesting a possible involvement with the amyloid deposits. CONCLUSIONS: Because collagen IV is prominent on the muscle membrane, the overexpression of matrix metalloproteinases (MMPs) 2 and 9 on the non-necrotic muscle fibers in polymyositis (PM) and sporadic inclusion body myositis (s-IBM) may facilitate lymphocyte adhesion and enhance T-cell-mediated cytotoxicity by degrading extracellular matrix proteins. The findings may have practical implications in considering therapeutic trials with MMP inhibitors in patients with PM and s-IBM.     

Diaphragmatic dysfunction in patients with idiopathic inflammatory myopathies

Polymyositis, dermatopolymyositis, and inclusion body myositis imply chronic inflammation of skeletal muscles. Pulmonary complications include aspiration pneumonia, interstitial pneumonitis, or respiratory muscle myositis. This study aims at better describing their impact on respiratory muscle. Twenty-three consecutive patients (12 PM, 5 DM, 6 IBM) were studied (static inspiratory and expiratory pressures; diaphragm function in terms of the mouth and transdiaphragmatic pressure responses to bilateral phrenic stimulation). Pulmonary parenchymatous abnormalities were mild (6 cases) or absent. The mouth pressure produced by phrenic stimulation was 6.83+/-3.01 cm H2O, with 18 patients (78%) diagnosed with diaphragm weakness (<10 cm H2O) and lower values in DM (4.35+/-1.48 cm H2O) than in IBM and in PM (P<0.05). Diaphragm weakness is frequent and probably overlooked in inflammatory myopathies. Further studies are needed to delineate the clinical relevance of these results.     

Expression and distribution of the nitric oxide synthases in idiopathic inflammatory myopathies

Different immune effector mechanisms have been characterised in the idiopathic inflammatory myopathies (IIM): in polymyositis (PM) and sporadic inclusion body myositis (sIBM), T-cell-mediated cytotoxicity targets nonnecrotic muscle fibres, whereas in dermatomyositis (DM) the complement-mediated immune response is directed against the microvasculature. As nitric oxide (NO) has an important function in cell signalling and in the cytotoxicity displayed by activated macrophages, it is potentially involved in the immunopathogenesis of IIM. Using immunohistochemical, in situ hybridisation and Western blotting techniques, we visualised the three isoforms of NO synthase (NOS) in muscle tissues from normal controls and from patients diagnosed with IIM. The levels of both constitutive isoforms of NOS (endothelial, i.e., eNOS, and neuronal, i.e., nNOS) were unchanged in IIM as compared with normal muscle. Both protein and mRNA of the inducible form (iNOS) were detected in half of the control biopsies. Constant and increased iNOS protein expression was found in endomysial infiltrates of PM and sIBM, whereas perimysial inflammatory cells in DM were largely negative. We developed a quantitative Western blotting protocol which confirmed the constitutive nature of nNOS and eNOS and the significant induction of iNOS in PM. Our results appoint iNOS with a dual function: a limited and transient role in normal muscle physiology and an active cytotoxic role in PM and sIBM.     

Expression of myositis specific autoantigens during post-natal myogenesis

Idiopathic inflammatory myopathies such as polymyositis (PM) and dermatomyositis (DM) are a group of rare autoimmune diseases, characterized by an inflammatory infiltrate within the skeletal muscle and high titer of circulating autoantibodies in the patient's serum. The etiopathogenesis of these diseases is not known and the relationship between the specific muscle involvement and the ubiquitary presence of the targeted antigens is still unclear. The enhanced expression of myositis specific autoantigens in regenerating muscle fibers from biopsies of PM and DM patients compared to normal muscle has been recently demonstrated. In order to understand whether candidate autoantigens in myositis are expressed during post-natal myogenesis, we performed immunolocalization studies of myositis specific autoantigens in skeletal muscle from newborn and adult rats. Our observations indicate the presence of myositis specific autoantigens during post-natal myogenesis, with possible implications for the induction and/or amplification of the immune-inflammatory response, in patients affected with autoimmune myositis.     

Beta-chemokine receptor expression in idiopathic inflammatory myopathies

Beta-chemokines attract and activate T cells and monocytes and have a key role in chronic inflammation. Certain beta-chemokines, such as monocyte chemoattractant protein-1 (MCP-1), have been reported to be upregulated in the idiopathic inflammatory myopathies (IIM). We studied the distribution of beta-chemokine receptors in polymyositis (PM), sporadic inclusion-body myositis (sIBM), dermatomyositis (DM), and control samples. CCR1-5 were localized to blood vessels in all samples. In addition, increased endothelial expression of CCR2A was observed in IIM. Subsets of inflammatory cells, identified as macrophages and T cells, in all three types of IIM expressed CCR2A, CCR2B, CCR3, CCR4, and CCR5. In contrast to an earlier report, we found CCR2B to be the most prominent MCP-1 receptor on inflammatory cells in IIM, especially in PM and sIBM. Strong CCR4 expression was present on myonuclei of regenerating muscle fibers. The prominence of the CCR2 receptors further underlines the importance of the interaction with their ligand MCP-1 in the immunopathogenesis of IIM and puts CCR2B forward as a potential target for future therapeutic intervention.     

Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies

The inflammatory myopathies include dermatomyositis (DM), polymyositis (PM), and sporadic inclusion-body myositis (s-IBM). In DM, the main immune effector response appears to be humoral and directed against the microvasculature, whereas in both PM and s-IBM, cytotoxic CD8+ T cells and macrophages invade and eventually destroy nonnecrotic muscle fibers expressing major histocompatibility complex class I. The need for more specific and safer therapies in inflammatory myopathies has prompted researchers to better decipher the molecular events associated with inflammation and muscle fiber loss in these diseases. The complex specific migration of leukocyte subsets to target tissues requires a coordinated series of events, namely activation of leukocytes, adhesion to the vascular endothelium, and migration. Cell adhesion molecules (CAM) and chemokines play a major role in this multistep process. In addition, cytokines by stimulating CAM expression and orchestrating T-cell differentiation also influence the immune response. This review focuses on recent advances in defining the molecular events involved in leukocyte trafficking in inflammatory myopathies. Specific topics include a concise summary of clinical features, pathological findings and immunopathology observed in inflammatory myopathies, background information about cytokines, chemokines and cell adhesion molecules, and the expression of these molecules in inflammatory myopathies.     

Clinical and serological characteristics of 125 Dutch myositis patients

The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of systemic diseases that include the familiar disease entities of dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM). A subset of patients has unique autoantibodies which are specific for IIM (myositis specific autoantibodies; MSAs). We studied the clinical and serological characteristics of IIM in 125 Dutch patients. Sera were analysed by immunoblotting, enzyme-linked immunosorbent assay, and immunoprecipitation. The most frequently encountered MSA was the anti-Jo-1 autoantibody (20%), followed by anti-tRNAHis (6%), anti-Mi-2 (6%), and anti-SRP (4%). The presence of certain MSAs was clearly associated with specific clinical characteristics. Anti-Jo-1 and anti-tRNAHis were associated with the anti-synthetase syndrome, anti-SRP with PM with severe myalgia and arthralgia and a moderate response to immunosuppressive treatment. A novel finding was the presence of anti-Mi-2, not only in DM, but also in PM. MSAs were frequently present in DM/PM sera, but were hardly ever detected in the sera of IBM patients. The few IBM patients with MSAs demonstrated a significant response to immunosuppressive treatment. It can be concluded that MSAs define specific clinical syndromes within the spectrum of IIM and that they can assist in the differential diagnosis and treatment plan of these enigmatic disorders by virtually excluding IBM by their presence, and by potentially identifying a subgroup of steroid-responsive IBM patients.     

Tau aggregates are abnormally phosphorylated in inclusion body myositis and have an immunoelectrophoretic profile distinct from other tauopathies

Sporadic inclusion body myositis (s-IBM) is the most frequent progressive acquired inflammatory myopathy in people older than 50 years. Abnormal aggregates of 'Alzheimer's proteins', including tau proteins, have been previously demonstrated in s-IBM. In the present study, we have investigated by immunohistochemistry and immunoblotting analysis the presence of tau proteins in muscle biopsy samples from patients with s-IBM and other myopathies with rimmed vacuoles, using newly developed antibodies raised against tau protein epitopes found in Alzheimer's disease brain. Tau immunoreactivity was shown in rimmed vacuoles or inclusions, preferentially with antibodies directed against phosphorylated carboxy-terminal epitopes of tau proteins. Cytoplasmic reactivity was also demonstrated in atrophic, nonvacuolated fibres, as well as in non-necrotic fibres invaded by inflammatory cells. Abnormally phosphorylated tau aggregates were also found in other compartments of the muscle fibre in s-IBM and other myopathies. Tau immunoblotting showed an electrophorectic profile of a doublet within the range of 60–62 kDa isovariants, which was different from tauopathies of the central nervous system. Finally, the unique pattern of immunoreactivity of s-IBM samples towards anti-tau antibodies is a new clue to a possible distinct subclass of peripheral tauopathy, different from the tauopathies of the central nervous system.     

Evidence for heterogeneity of T cell expansion in polymyositis and inclusion body myositis

Vbeta usage of muscle-infiltrating T lymphocytes in polymyositis (PM) and sporadic inclusion body myositis (s-IBM) was correlated with clinical and histopathological features. Immunohistochemical analysis was combined with complementarity-determining region 3 (CDR3) length analysis in nine muscle biopsies of eight PM patients and six biopsies of five s-IBM patients. Vbeta usage was heterogeneous in seven patients. Four of these patients had definite PM with endomysial located T cell infiltrates, but T cells specifically surrounding and invading individual non-necrotic fibers were not found. In two s-IBM patients, Vbeta 2 usage was increased. In one of them, a repeat biopsy showed a heterogeneous Vbeta usage. We conclude that clonal expansion of muscle-infiltrating T cells could only be detected in part of the patients. Explanations may be that clonal expansion does not take place in all disease phases and that PM is a heterogeneous disease with respect to pathogenesis.     

Specific matrix metalloproteinase expression in focal myositis: an immunopathological study

OBJECTIVES: The aim of our study was to investigate immunoexpression of matrix metalloproteinases MMP2, MMP7 and MMP9 in muscle specimens from patients with focal myositis (FM) vs polymyositis (PM) and dermatomyositis. MATERIALS AND METHODS: We studied muscle biopsy samples from seven patients affected by FM; samples from five patients each with PM and dermatomyositis were studied as disease controls. RESULTS: MMP2 immunoreactivity was present in PM and dermatomyositis, MMP7 only in PM, and MMP9 in PM, dermatomyositis and FM. CONCLUSIONS: Our results confirm that increased MMP9 immunoreactivity in muscle fibres is a common feature of all inflammatory myopathies and suggest that MMP2 and MMP7 cannot be implicated in the inflammatory events of FM.     

Changes in CSF acetyl- and butyrylcholinesterase activity after long-term treatment with AChE inhibitors in Alzheimer's disease

Jain A, Sharma MC, Sarkar C, Bhatia R, Singh S, Gulati S, Handa R. Detection of the membrane attack complex as a diagnostic tool in dermatomyositis.
Acta Neurol Scand: 2011: 123: 122–129.
© 2010 John Wiley & Sons A/S. Background – Currently there is no reliable diagnostic marker to distinguish between the subgroups of idiopathic inflammatory myopathies (IIMs), i.e. dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM). Membrane attack complex (MAC) has been shown to be involved in the pathogenesis of dermatomyositis but its role as a diagnostic marker has not been evaluated. Aim – To assess the diagnostic utility of MAC deposition in distinguishing dermatomyositis from other neuromuscular disorders. Material and methods – Immunohistochemical detection of MAC deposition on endomysial microvessels was carried out on 127 muscle biopsies comprising of 21 cases of dermatomyositis, 42 other IIMs and 64 non‐IIM neuromuscular diseases. Results – MAC deposition showed a high sensitivity (80.9%) and specificity (85%) to differentiate DM from other IIMs. Its specificity was higher (98.4%) in discriminating DM from non‐IIM muscular diseases and IIM from non‐IIMs. Conclusion – MAC deposition can serve as a reliable marker to distinguish DM from other IIMs (i.e. PM and IBM) as well as from non‐IIM diseases. It can also serve as a useful adjunct in diagnosis of IIMs when there is diagnostic dilemma with their morphologic similarities. These results provide further credence to the long‐standing view that MAC‐mediated capillary destruction is involved in the immunopathogenesis of DM.     

Alpha-chemokine receptors CXCR1–3 and their ligands in idiopathic inflammatory myopathies

The idiopathic inflammatory myopathies (IIM) are a heterogeneous group of neuromuscular disorders subdivided into polymyositis (PM), sporadic inclusion body myositis (sIBM) and dermatomyositis (DM). Chemokines play an essential role in sustained inflammation associated with IIM. We studied the distribution of the -chemokine receptors CXCR1, 2, 3 and their ligands interferon- (IFN-)-inducible T cell chemoattractant (I-TAC), IFN--inducible protein of 10 kDa (IP-10), monokine induced by IFN- (MIG) and growth-related oncogene (GRO) in IIM using immunohistochemistry, immunofluorescence and Western blotting. Abundant expression of IP-10 was observed on macrophages and T cells surrounding and invading non-necrotic muscle fibers in PM and sIBM and in T cells in perimysial infiltrates of DM. IP-10 was also localized to blood vessel endothelial cells in all inflammatory and normal muscle tissues. The distribution of other -chemokines was variable: Only low levels of MIG and I-TAC were detected; GRO was localized to the endomysial infiltrates of some PM and sIBM samples, but not in DM. Muscle tissues were invariably CXCR1 negative, while a subset of inflammatory cells in all IIM were CXCR2 positive. Strong CXCR3 expression was observed on the majority of T cells in each IIM. We describe the differential repertoire of -chemokines in IIM, and offer additional proof of the predominance of Th1-driven reactions in the immunopathogenesis of all three diagnostic subgroups. We suggest the Th1-mediated immunity in general, and the CXCR3/IP-10 interaction in particular, as potential targets for novel therapeutic intervention in IIM.