Provision of an explanation for the inefficacy of immunotherapy in sporadic inclusion body myositis: quantitative assessment of inflammation and ß-amyloid in the muscle.

OBJECTIVE: In sporadic inclusion body myositis (IBM), inflammation and accumulation of ß-amyloid-associated molecules cause muscle fiber damage. We undertook this study to determine why intravenous immunoglobulin (IVIG) and prednisone are not effective in sporadic IBM despite their effectiveness in other inflammatory myopathies. METHODS: Relevant inflammatory and degeneration- associated markers were assessed by quantitative polymerase chain reaction and immunohistochemistry in repeated muscle biopsy specimens from patients with sporadic IBM treated in a controlled study with IVIG and prednisone (n = 5) or with prednisone alone (n = 5). Functional effects were assessed in a muscle cell culture model. RESULTS: In muscle biopsy specimens, messenger RNA (mRNA) expression of the proinflammatory chemokines CXCL9, CCL3, and CCL4 and of the cytokines interferon-γ (IFNγ), transforming growth factor ß, interleukin-10 (IL-10), and IL-1ß was significantly reduced after treatment in both groups. No consistent changes were observed for tumor necrosis factor α, IL-6, inducible costimulator (ICOS), its ligand ICOSL, and perforin. Messenger RNA expression of the degeneration-associated molecule ubiquitin and the heat-shock protein αB-crystallin was also reduced, but no changes were noted for amyloid precursor protein (APP) or desmin. By immunohistochemistry, a significant down-modulation of chemokines was observed, but not of inducible nitric oxide (NO) synthase, nitrotyrosine, IL-1ß, APP, and ubiquitin; ß-amyloid was reduced in 6 of 10 patients. Pronounced staining of IgG was observed in the muscle after treatment with IVIG, indicating penetration of infused IgG into the muscle and a possible local effect. In muscle cells exposed to IFNγ plus IL-1ß, IgG and/or prednisone down-regulated mRNA expression of IL-1ß 2.5-fold. Accumulation of ß-amyloid, overexpression of αB-crystallin, and cell death were prevented. In contrast, NO-associated cell stress remained unchanged. CONCLUSION: IVIG and prednisone reduce some inflammatory and degenerative molecules in muscle of patients with sporadic IBM and in vitro, but do not sufficiently suppress myotoxic and cell stress mediators such as NO. The data provide an explanation for the resistance of sporadic IBM to immunotherapy and identify markers that may help to design novel treatment strategies.

Upregulation of chemokines and their receptors in Duchenne muscular dystrophy: potential for attenuation of myofiber necrosis.

INTRODUCTION: In Duchenne muscular dystrophy (DMD), the infiltration of skeletal muscle by immune cells aggravates disease, yet the precise mechanisms behind these inflammatory responses remain poorly understood. Chemotactic cytokines, or chemokines, are considered essential recruiters of inflammatory cells to the tissues. METHODS: We assayed chemokine and chemokine receptor expression in DMD muscle biopsies (n = 9, average age 7 years) using immunohistochemistry, immunofluorescence, and in situ hybridization. RESULTS: CXCL1, CXCL2, CXCL3, CXCL8, and CXCL11, absent from normal muscle fibers, were induced in DMD myofibers. CXCL11, CXCL12, and the ligand-receptor couple CCL2-CCR2 were upregulated on the blood vessel endothelium of DMD patients. CD68(+) macrophages expressed high levels of CXCL8, CCL2, and CCL5. CONCLUSIONS: Our data suggest a possible beneficial role for CXCR1/2/4 ligands in managing muscle fiber damage control and tissue regeneration. Upregulation of endothelial chemokine receptors and CXCL8, CCL2, and CCL5 expression by cytotoxic macrophages may regulate myofiber necrosis.

The tumor necrosis factor superfamily of cytokines in the inflammatory myopathies: potential targets for therapy.

The idiopathic inflammatory myopathies (IM) represent a heterogeneous group of autoimmune diseases, of which dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (IBM) are the most common. The crucial role played by tumor necrosis factor alpha (TNFα) in the IM has long been recognized. However, so far, 18 other members of the TNF superfamily have been characterized, and many of these have not yet received the attention they deserve. In this paper, we summarize current findings for all TNF cytokines in IM, pinpointing what we know already and where current knowledge fails. For each TNF family member, possibilities for treating inflammatory diseases in general and the IM in particular are explored.

Role of cytokines and chemokines in idiopathic inflammatory myopathies.

PURPOSE OF REVIEW: Cytokines and chemokines are essential players in the initiation and progression of the idiopathic inflammatory myopathies (IIMs). This review focuses on the most recent data and the new insight they provide for the disease mechanisms of dermatomyositis, polymyositis and sporadic inclusion body myositis. RECENT FINDINGS: Interferon-alpha and beta are implicated in the innate immune responses underlying dermatomyositis, whereas interferon-gamma stands forward as a more general regulator of the IIMs, reflected by the induction of many interferon-gamma-inducible genes in patients. Interleukin-1beta and interleukin-18 are localized to the inflammatory cells present in IIM muscle, where they may focally induce further recruitment of immune cells. Lymphotoxins are implicated in the cytotoxic activities toward polymyositis and inclusion body myositis muscle fibers, and in the organization and antibody production by B-cells in dermatomyositis. The alpha-chemokines CXCL9, CXCL10 and the beta-chemokines CCL2, CCL3, CCL4, CCL19 and CCL21 are expressed in IIM muscle. The B-cell activator CXCL13 is particularly prominent in the larger perimysial infiltrates of dermatomyositis. SUMMARY: The cytokine-chemokine patterns described in recent studies provide further evidence for predominance of Th1-mediated reactions in the different IIMs, inflammation-induced degenerative phenomena in inclusion body myositis, and a possible role for lymphoneogenesis in the sustained inflammatory response in dermatomyositis.

Chemokines in idiopathic inflammatory myopathies.

The idiopathic inflammatory myopathies (IIM) represent a heterogeneous group of acquired muscle diseases. The three best-studied subgroups: dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (IBM), differ considerably both clinically and pathophysiologically. DM is a chiefly humoral endotheliopathy often associated with characteristic skin manifestations. In PM and IBM nonnecrotic muscle fibers are invaded by auto-aggressive cytotoxic T-cells and macrophages. IBM presents with additional structural abnormalities of myofibers, including rimmed vacuoles and depositions of ectopic proteins. Data accumulates implicating the chemokines in the immunopathogenesis of the different IIM. This review bundles current knowledge of the chemokine and chemokine receptor expression in the skeletal muscle of DM, PM and IBM patients. The IIM are characterized by a general increase of specific chemokines, while the chemokine distribution reflects the two different immune responses represented within these muscle diseases: (I) The endotheliopathy of DM is characterized by increased levels of CXCL12 and CCL2 in the affected blood vessels, (II) In the myocytotoxic immune response of PM and IBM, active invasion of nonnecrotic myofibers by inflammatory cells is associated with CXCL10 and CCL2 upregulation. The ever accumulating data illustrates the important role of the chemokine system in IIM, indicating the therapeutic potential of interfering agents. This raises hopes for future treatments for DM and PM with fewer side effects, and the possible establishment of a therapy suited for IBM, a myopathy which has proven unresponsive to all available immuno-modulating interventions up till now.

Distribution of glucocorticoid receptor alpha and beta subtypes in the idiopathic inflammatory myopathies.

In contrast with dermatomyositis and polymyositis, inclusion body myositis is unresponsive to glucocorticoid treatment. Glucocorticoid action is mediated through an active glucocorticoid receptor-alpha and negatively regulated by another glucocorticoid receptor isoform. In several autoimmune diseases glucocorticoid receptor-beta up-regulation is involved in glucocorticoid resistance. We studied glucocorticoid receptor distribution in normal and inflammatory myopathy muscle and investigated whether differences in glucocorticoid receptor-alpha and glucocorticoid receptor-beta protein expression are involved in the differential glucocorticoid sensitivity in inclusion body myositis versus polymyositis. Multistep immunofluorescence and Western blotting on fractionated cytoplasmic or nuclear muscle samples were used. Glucocorticoid receptor-alpha was the predominant receptor subtype in muscle and occurred abundantly in myonuclei of control and diseased muscle alike. Glucocorticoid receptor-beta was constitutively expressed on a subset of endothelial cells. No differences between dermatomyositis and the other idiopathic inflammatory myopathies were observed. Increased nuclear glucocorticoid receptor that has dissociated from heat shock protein 90 was found in glucocorticoid treated subjects. Glucocorticoid receptor-alpha and -beta isoform levels were unaltered in muscle tissues from control subjects that had received glucocorticoid treatment prior to biopsy. No differences in relative glucocorticoid receptor-alpha and glucocorticoid receptor-beta protein expression were seen in inclusion body myositis versus polymyositis specimens. Our study indicates that the different glucocorticoid sensitivity in the idiopathic inflammatory myopathies is not related to up- or down-regulation of a given glucocorticoid receptor isoform at the protein level.

Chemokine profile of different inflammatory myopathies reflects humoral versus cytotoxic immune responses.

The idiopathic inflammatory myopathies (IM) are subdivided into dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (IBM). These autoimmune muscle diseases represent different immunopathological entities. DM is a humoral endotheliopathy initiated by complement deposition in intramuscular blood vessels, and characterized by perimysial inflammation and muscle fiber atrophy in perifascicular regions. In PM and IBM, nonnecrotic muscle fibers are actively invaded by autoaggressive macrophages and cytotoxic T cells. Chemokines are key mediators of inflammatory disease as they regulate leukocyte recruitment to tissue target sites. We studied a large selection of alpha/beta-chemokines and receptors in normal controls and in the IM using immunohistochemistry, immunofluorescence, in situ hybridization, and Western blotting. We showed that the chemokine array of normal myocytes was limited, while the blood vessels in normal skeletal muscle tissue displayed a broad chemokine profile. The IM were characterized by a general increase of specific chemokines and chemokine receptors, while chemokine distribution reflected the two different immune responses represented within these muscle diseases. In DM, endothelial expression of CCL2 and CXCL12beta was highly increased. In PM and IBM, macrophages and cytotoxic T cells actively invading nonnecrotic muscle fibers expressed highest levels of CXCL10 and CCL2. Some chemokines were selectively expressed by different IM infiltrates: CCL4 was present only in the perimysial inflammatory foci of a subset of DM biopsies, while CXCL1, CXCL2, CXCL3, and CCL7-positive cells were exclusively detected in endomysial infiltrates of a number of PM and IBM samples. The chemokine receptor profile of the IM indicated the predominance of Th1-mediated immune responses in all three IM. Our studies identified three ligand-receptor pairs, namely CXCL10/CXCR3, CXCL12/CXCR4, and CCL2/CCR2, as potential targets for chemokine-based therapy in IM.

Potential therapeutic targets for idiopathic inflammatory myopathies.

The inflammatory myopathies essentially comprise three diseases with different immunopathologic features. Dermatomyositis (DM) is a complement-mediated microangiopathy. The immune response in polymyositis (PM) and sporadic inclusion body myositis (IBM) is a CD8+ T-cell-mediated cellular reaction against an unknown muscle fiber antigen. The multiple immune factors that guide inflammatory cell diapedesis and trafficking have been elucidated over the past two decades. Many of these molecules can now be targeted by monoclonal antibodies and other pharmacologic approaches. Randomized controlled trials are being started on a number of new agents to find out whether more specific immune interventions than the currently used glucocorticosteroids can treat DM and PM patients with fewer side effects, and may represent a first treatment modality for IBM, an entity unresponsive to all currently available pharmacological treatments.

The multifaceted character of lymphotoxin ß in inflammatory myopathies and muscular dystrophies.

Lymphotoxin beta (LTß) regulates some inflammatory mechanisms that could be operative in idiopathic inflammatory myopathies (IM). We studied LTß and LTßR in inflammatory myopathies, normal and disease controls with immunohistochemistry, Western blotting and in situ hybridisation. LTß occurs in myonuclei of normal controls, implying its role in normal muscle physiology. LTß is strongly upregulated in regenerating muscle fibres in all myopathies, but not in denervated myofibres. Normal-appearing myofibres in inflammatory myopathies and muscular dystrophies express LTß possibly reflecting early myofibre damage, representing a hitherto undescribed pathologic hallmark. Furthermore, we visualised LTß in several inflammatory cell types in inflammatory myopathies, suggesting its involvement in the different inflammatory mechanisms underlying inflammatory myopathy subgroups.

Heat shock protein families 70 and 90 in Duchenne muscular dystrophy and inflammatory myopathy: balancing muscle protection and destruction.

Heat shock proteins are important factors in skeletal muscle physiology and stress response. We examined the effects of chronic inflammation on the distribution of heat shock protein families 70 and 90 using immunofluorescence and Western blotting, in muscle biopsies from 33 idiopathic inflammatory myopathy patients [aged 26-66 (dermatomyositis), 17-78 (polymyositis) and 57-80 (sporadic inclusion body myositis) years], and seven Duchenne muscular dystrophy patients (aged 3-19 years). Our results reveal the multifaceted role played by chaperones in inflammatory muscle tissue. On the one hand, regenerating, atrophic and vacuolated muscle fibers displayed upregulation of both protein families. Higher levels of chaperones in challenged fibers point to the myocyte's attempt to restore and regenerate. On the other hand, heat shock proteins of the 90 family were strongly upregulated in macrophages and cytotoxic T-cells actively invading nonnecrotic muscle fibers of sporadic inclusion body myositis and polymyositis, probably conferring enhanced myocytotoxic capacity. Our data provide positive arguments for exploring heat shock protein 90-based therapy in inflammatory muscle disease.

Idiopathic inflammatory myopathies and the classical NF-kappaB complex: current insights and implications for therapy.

The idiopathic inflammatory myopathies (IIM) comprise a heterogeneous group of muscle diseases. The three best-studied subgroups are dermatomyositis (DM), polymyositis (PM) and sporadic inclusion body myositis (IBM). The latter represents a steroid-refractory condition. PM and IBM are characterized by a cell-mediated immune response directed against non-necrotic fibers expressing Major Histocompatibility Complex class I (MHC class I). IBM presents with additional degenerative features, including rimmed vacuoles and depositions of aberrant proteins. DM is a complement-mediated endotheliopathy often accompanied by characteristic skin manifestations. The ubiquitously expressed transcription factor NF-kappaB is considered essential for the development of auto-immunity. This review describes data gathered so far concerning the distribution of the classical heterodimer p65/p50 and its inhibitor I-kappaBalpha in IIM skeletal muscle. Data suggest that the NF-kappaB complex plays a role in the endotheliopathy characterizing DM and might be involved in myofiber regeneration, and appoint CD4+ and CD68+ mononuclear cells with a more prominent role than previously assumed. Fragmentary knowledge of the immunopathogenesis of IIM hampers the development of therapeutic strategies suited to all patient groups. Unravelling the precise involvement of NF-kappaB subunits in IIM immunopathogenesis can shed new light onto the etiology of these diseases and may offer a novel therapeutic target.

Distribution of the NF-kappaB complex in the inflammatory exudates characterizing the idiopathic inflammatory myopathies.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a ubiquitously expressed protein family that is considered crucial in autoimmunity. We describe NF-kappaB p50 and p65, and the inhibitor I-kappaB alpha in the inflammatory exudates characteristic for the different idiopathic inflammatory myopathies (IIM), that is, endomysial CD8(+) cytotoxic T cells invading non-necrotic fibers in polymyositis (PM) and sporadic inclusion body myositis (sIBM), and the perimysial/perivascular CD20(+) B cells and CD4(+) T cells in dermatomyositis (DM). We also analyzed other inflammatory cells in the vicinity of active inflammation sites. Strikingly, actively invading CD4(+) cells in PM and sIBM contained both p65 and p50, whereas I-kappaB alpha was absent. This could point to a high activation state in which these cells are capable of expressing a variety of inflammatory mediators, contributing to the degradation of non-necrotic fibers. Secondly, CD68(+) macrophages in the infiltrates in all three IIM subtypes showed strong nuclear p50 and I-kappaB alpha staining. This may point to a role for p50 in counteracting inflammation, a reaction that could be enhanced by an upregulation of I-kappaB alpha as we observed with immunofluorescence. These results shed further light on the immunopathology of PM, sIBM and DM. CD4(+) and CD68(+) mononuclear cells may play a more prominent role than previously assumed.

A dual role for HSP90 and HSP70 in the inflammatory myopathies: from muscle fiber protection to active invasion by macrophages.

Chaperones assist the metamorphosis of polypeptides to fully functional proteins. The family of heat shock proteins 70 (HSP70) bind at an early stage, while the HSP90 bind to proteins near their active conformation. We studied HSP90 and HSP70 in muscle biopsies from controls and patients diagnosed with the three main idiopathic inflammatory myopathies (IIM), namely dermatomyositis (DM), sporadic inclusion body myositis (IBM), and polymyositis (PM). Human skeletal muscle displayed constitutive levels of protein, but in IIM both HSP90 and HSP70 were upregulated. Regenerating muscle fibers of IIM and muscular dystrophy patients showed increased expression of HSP90 and HSP70, as did atrophic perifascicular muscle fibers of DM and vacuolated fibers of IBM. The infiltrates of IIM displayed weak homogeneous HSP70 staining. HSP90 were specifically upregulated in the CD68(+) cells, actively invading non-necrotic muscle fibers of IBM/PM and co-localized with inducible NO synthase. HSP90:HSP70 ratios were increased in IBM and PM tissues displaying high inflammation grade. Our results point to a multifaceted role for chaperones in muscle tissue: a general protective role for both HSP90 and HSP70 families in damaged muscle fibers, and a specific cytotoxic role for HSP90 in muscle fiber invasion typically associated with IBM and PM.

TNF alpha receptor genotype influences smoking-induced muscle-fibre-type shift and atrophy in mice.

Systemic manifestations of chronic obstructive pulmonary disease (COPD) include muscle wasting, and tumour necrosis factor alpha (TNFalpha) could represent a major inducer of these processes. We studied skeletal muscle histology in a murine model of cigarette smoke (CS)-induced COPD, comparing mice with different TNFalpha receptor genotypes. Muscles from hind limbs of wild type (WT), TNFalpha receptor 1 knockout (TNF alpha R1KO) and TNF alpha R2KO mice were prepared and weighed. The lower body weight, which was observed in CS-exposed WT and TNF alpha R1KO mice, was paralleled by reduced weights of gastrocnemius and biceps femoris muscle. The gastrocnemius muscle was evaluated for muscle fibre apoptosis and atrophy, and fibre-type distribution. CS-induced apoptosis was observed in all genotypes, while a significant reduction of cross-sectional areas of myofibres was present only in TNF alpha R2KO mice. A CS-induced fibre-type shift from the IIa to the IIb phenotype was observed in WT mice, an increase of muscle-fibre-type IIx was noticed in CS-exposed TNF alpha R2KO mice. Our data suggest that the skeletal muscle manifestations associated with this murine COPD model are under complex regulation by both TNFalpha receptors, but that TNF alpha R2 may be the most important determinant for the outcome of CS-induced myofibre apoptosis.