Expression of myogenic regulatory factors and myo-endothelial remodeling in sporadic inclusion body myositis

Muscle repair relies on coordinated activation and differentiation of satellite cells, a process that is unable to counterbalance progressive degeneration in sporadic inclusion body myositis (s-IBM). To explore features of myo regeneration, the expression of myogenic regulatory factors Pax7, MyoD and Myogenin and markers of regenerating fibers was analyzed by immunohistochemistry in s-IBM muscle compared with polymyositis, dermatomyositis, muscular dystrophy and age-matched controls. In addition, the capillary density and number of interstitial CD34⁺ hematopoietic progenitor cells was determined by double-immunoflourescence staining. Satellite cells and regenerating fibers were significantly increased in s-IBM similar to other inflammatory myopathies and correlated with the intensity of inflammation (R > 0.428). Expression of MyoD, visualizing activated satellite cells and proliferating myoblasts, was lower in s-IBM compared to polymyosits. In contrast, Myogenin a marker of myogenic cell differentiation was strongly up-regulated in s-IBM muscle. The microvascular architecture in s-IBM was distorted, although the capillary density was normal. Notably, CD34⁺ hematopoietic cells were significantly increased in the interstitial compartment. Our findings indicate profound myo-endothelial remodeling of s-IBM muscle concomitant to inflammation. An altered expression of myogenic regulatory factors involved in satellite cell activation and differentiation, however, might reflect perturbations of muscle repair in s-IBM.     

PAX3/7 expression coincides with MyoD during chronic skeletal muscle overload

Paired box (Pax) proteins 3 and 7 are key determinants for embryonic skeletal muscle development by initiating myogenic regulatory factor (MRF) gene expression. We show that Pax3 and 7 participate in adult skeletal muscle plasticity during the initial responses to chronic overload (< or =7 days) and appear to coordinate MyoD expression, a member of the MRF family of genes. Pax3 and 7 mRNA were higher than control within 12 h after initiation of overload, preceded the increase in MyoD mRNA on day 1, and peaked on day 2. On days 3 and 7, Pax7 mRNA remained higher than control, suggesting that satellite cell self-renewal was occurring. Pax3 and 7 and MyoD protein levels were higher than control on days 2 and 3. These data indicate that Pax3 and 7 coordinate the recapitulation of developmental-like regulatory mechanisms in response to growth-inducing stimuli in adult skeletal muscle, presumably through activation of satellite cells.     

Pattern of myogenesis and vascular repair in early and advanced lesions of juvenile dermatomyositis

Regenerative processes that counteract perifascicular muscle atrophy and capillary loss in juvenile dermatomyositis (JDM) are not well characterized. We aimed to analyze the pattern of myo-regeneration in relation to vascular damage and repair in muscle specimens from JDM patients. Myogenic regulatory factors that are sequentially expressed during myogenesis were studied by immunohistochemistry. Capillary density, numbers of CD34⁺ endothelial progenitor cells within the endomysium and molecules implicated in angiogenesis were evaluated by double-immunofluorescence techniques. Myogenic regulatory factors were significantly up-regulated in JDM muscle exhibiting a different pattern in early and advanced lesions. In early lesions Pax7⁺ satellite cells and both MyoD⁺ and Myogenin⁺ myogenic cells were moderately increased. In lesions with advanced perifascicular atrophy Pax7⁺ satellite cells were numerous, but absence of MyoD⁺ in the context of increased Myogenin⁺ expression suggested a dysregulation of the myogenic regenerative pathway. The overall capillary density in JDM was decreased, but regions of capillary loss in advanced lesions alternated with focal increase of hyperplastic endothelial cells in early lesions. Up-regulation of endoglin in hyperplastic endothelial cells in conjunction with overexpression of TGF-β1 and VEGF suggested activation of neovascularization. Conversely, CD34⁺ endothelial progenitor cells were not increased arguing against relevant contribution to vascular repair. Our results demonstrate substantial induction of myogenesis in JDM. While the early phase of myogenesis appears to be associated with endothelial cell activation, an altered expression of MRFs in perifascicular regions with capillary depletion suggests an impairment of myogenic differentiation that may contribute to perifascicular muscle fiber atrophy in JDM.     

Lack of muscle stem cell proliferation and myocellular hypertrophy in sIBM patients following blood-flow restricted resistance training

Sporadic inclusion body myositis (sIBM) is characterised by skeletal muscle inflammation, progressive muscle loss and weakness, which is largely refractory to immunosuppressive treatment. Low-load blood-flow restricted (BFR) training has been shown to evoke gains in myofibre cross sectional area (mCSA) in healthy adults. This could partially be due to the activation and integration of muscle satellite cells (SC) resulting in myonuclei addition. Consequently, this study investigated the effect of 12-weeks lower limb low-load BFR resistance training in sIBM patients on SC and myonuclei content, myofibre size and capillarization. Muscle biopsies from sIBM patients randomised to 12-weeks of low-load BFR resistance training (n = 11) or non-exercising controls (CON) (n = 9) were analysed for SC and myonuclei content, myofibre size and capillarization using three-colour immunofluorescence microscopy and computerised quantification procedures. No between-group differences (time-by-group interactions) or within-groups changes were observed for resident SCs (Pax7⁺/Six1⁺), proliferating SCs (Pax7⁺/ Ki67⁺), myonuclei (Six1⁺), type 1 mCSA or capillary number (CD31⁺). However, a time-by-group interaction for type 2 mCSA was observed (p = 0.04). Satellite cell content, myonuclei number, mCSA and capillary density remained unaffected following 12-weeks low-load BFR resistance training, indicating limited myogenic capacity and satellite cell plasticity in long-term sIBM patients.     

Coculture with autologous myotubes of cytotoxic T cells isolated from muscle in inflammatory myopathies.

T-cell lines were expanded from muscle of 10 patients with polymyositis, 5 with inclusion body myositis, 5 with dermatomyositis, and 5 with other muscle diseases. All cell lines uniformly expressed T-cell antigens, but not natural killer cell or B-cell antigens. The proportion of helper (CD4+) and cytotoxic (CD8+) T cells in the expanded lines was variable and showed no correlation with the diagnosis. Sixteen cell lines (6 polymyositis, 4 inclusion body myositis, 5 dermatomyositis, 1 other muscle disease) consisted predominantly of CD8+ T cells. None of these lines displayed natural killer-like cytotoxicity but all were capable of lectin-dependent cytotoxicity. Three of 6 polymyositis, 1 of 4 inclusion body myositis, and 1 of 5 dermatomyositis lines showed low but statistically significant cytotoxicity against autologous myotubes (6 to 27% specific 51Cr release; effector-target ratio, 20:1). The results demonstrate that functionally competent cytotoxic T cells can be expanded from muscle affected by inflammatory myopathies and are consistent with the hypothesis that some cytotoxic T cells recognize an autoantigen on myotubes. Further studies of this experimental system may define the molecular mechanism of T cell-mediated muscle fiber injury and may help to identify the relevant antigens.     

Invasive fibroblasts: Fundamental difference between sporadic inclusion body myositis and polymyositis

Introduction: Sporadic inclusion body myositis (sIBM) is a progressive disease that leads to extensive muscle weakness. The aim of this study was to determine whether the number and distribution of fibroblasts differ in sIBM when compared with polymyositis. Methods: Immunofluorescence double labeling was performed on 35 biopsies with antibodies directed against perlecan and CD90, procollagen I, CD34, and CD105. In addition, nonserial ultrathin sections were studied from 3 biopsies of each condition. Results: Fibroblasts expressing CD90 and CD34 accumulated in the endomysial compartment in polymyositis and sIBM. In addition, cells expressing CD90 were found beneath the basal lamina in both conditions. At the ultrastructural level in polymyositis, fibroblasts invaded the myofiber, with focal destruction of the basement membrane. In sIBM, by contrast, invasive fibroblasts were ensheathed by the intact myofiber basement membrane. Conclusions: The impact of intruding fibroblasts on satellite cells remains to be established. Muscle Nerve 49 : 175–180, 2014     

Inflammatory myopathies: evaluation and management

The inflammatory myopathies, including dermatomyositis, inclusion body myositis, and polymyositis, are poorly understood autoimmune diseases affecting skeletal muscle. Dermatomyositis is a disease mainly of skin and muscle, but may affect lung and other tissues. Proximal or generalized weakness or skin rash are the typical presenting features. Inclusion body myositis has a specific clinical pattern of weakness that generally distinguishes it from other inflammatory myopathies, with prominent involvement of wrist and finger flexors, and quadriceps. Polymyositis generally presents with proximal or generalized weakness. Typical dermatomyositis muscle pathology is quite distinct, with perivascular inflammatory cells that include plasmacytoid dendritic cells, and abnormal capillaries and perimysial perifascicular myofibers. Both inclusion body myositis and polymyositis usually have infiltration into muscle of large numbers of inflammatory cells, typically surrounding and displacing, and sometimes invading, myofibers. Inclusion body myositis is refractory to corticosteroids and to several immunomodulating therapies that have been used. Dermatomyositis and polymyositis are treated with corticosteroids and a variety of agents. Osteoporosis and opportunistic infections pose a significant risk during treatment of patients. This review discusses the clinical manifestations, pathology, and treatment approaches for the inflammatory myopathies.     

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.     

Inclusion body myositis: Clinical and pathological boundaries

Inclusion body myositis, polymyositis, and dermatomyositis are three distinct categories of inflammatory myopathy. Some authorities commented on the selective early weakness of the volar forearm muscles, quadriceps, and ankle dorsiflexors in inclusion body myositis. The most important feature distinguishing inclusion body myositis from the other two inflammatory myopathies is the lack of responsiveness to immunosuppressive treatment. Although most patients with inclusion body myosities have characteristic muscle biopsy findings, some cannot be distinguished histologically early from polymyositis. Presdicting responsiveness to mmunosuppressive medications, independent of muscle histology, would be valuable to clinicians. We retrospectively reviewed the pattern of weakness and other clinical features of 46 patients newly diagnosed with either inclusion body myositis, polymyositis, or dermatomyosities. Asymmetrical muscle weakness with prominent wrist flexor, finger flexor, and knee extensor involvement was specific for inclusion body myositis and unresponsive polymyositis. Male sex, lower creatine kinase levels, slower rate of progression, and peripheral neuropathy were also more common in inclusion body myositis and unresponsive polymyositis than in responsive polymyositis and dermatomyositis patients. Repeat muscle biopsy in 2 patients in teh unresponsive polymyositis group demonstrated histological features of inclusion body myositis. We suspect that patients with clinical features of inclusion body myositis but lacking histological confirmation may nonetheless have inclusion body myositis. Our study supports the recently proposed criteria for defintie and possible inclusion body myositis.     

Monoclonal antibody analysis of mononuclear cells in myopathies. I: Quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by T cells

In 76 muscle specimens (normal controls, 9; Duchenne dystrophy, 11; scleroderma, 11; dermatomyositis, 13; polymyositis, 15; inclusion body myositis, 17), mononuclear cells were analyzed at perivascular, perimysial, and endomysial sites of accumulation. Monoclonal antibodies reactive for B cells, T cells, T cell subsets, killer (K) or natural killer (NK) cells, and the Ia antigen were used for cell typing. Macrophages were identified by the acid phosphatase reaction. Few extravascular mononuclear cells occurred in normal muscle. In all inflammatory myopathies, a mixed exudate of T cells, B cells, and macrophages was present. Mature K/NK cells were rare in all diseases. In dermatomyositis, polymyositis, and inclusion body myositis, there was a positive gradient for T cells, T8+ cells, and activated T cells and a negative gradient for B cells and T4+ cells between perivascular and endomysial sites. In scleroderma the predominant perimysial exudate consisted mostly of T cells and macrophages. The percentage of B cells at all sites, and the T4+/T cell ratio in the endomysium, were significantly higher in dermatomyositis than in the other diseases. In polymyositis and inclusion body myositis, the endomysial exudate contained a large number of T cells, T8+ cells, and activated T cells but only sparse B cells. T cells accompanied by macrophages focally surrounded and invaded nonnecrotic fibers in polymyositis and inclusion body myositis. Rare fibers in Duchenne dystrophy and a very few fibers in dermatomyositis and scleroderma were similarly affected. We infer that (1) T-B, T-T, and T-macrophage cooperativities are likely to exist in muscle in different myopathies; (2) T cell-mediated fiber injury plays a role in polymyositis and inclusion body myositis; (3) T cell-mediated fiber injury can also occur in inherited diseases, such as Duchenne dystrophy; and (4) a local humoral response may occur in muscle in dermatomyositis and possibly in polymyositis and inclusion body myositis.     

Differential expression of chemokines in inflammatory myopathies

BACKGROUND: Chemokines represent a family of small-molecular-weight cytokines that recruit and activate inflammatory cells in response to inflammation. Invasion of cytotoxic memory T cells and macrophages in nonnecrotic muscle fibers characterizes polymyositis and sporadic inclusion body myositis. Dermatomyositis is a complement-mediated endotheliopathy. Elucidation of the mechanisms guiding lymphocyte diapedesis and trafficking could lead to selective therapeutic interventions. METHODS: Immunoblots and multistep immunofluorescence studies with non-cross-reactive antibodies recognizing interleukin-8, monocyte chemoattractant protein-1 (MCP-1), MCP-3, TARC (thymus and activation regulated cytokine), and RANTES (regulated upon activation, normal T-cell expressed and secreted), using appropriate positive and negative controls. In situ hybridization was used to localize MCP-1 mRNA. RESULTS: MCP-1 protein was strongly expressed on T cells and a subset of macrophages actively invading a proportion of the nonnecrotic muscle fibers in polymyositis and inclusion body myositis alike. Capillaries and arterioles in the vicinity of endomysial inflammatory foci were immunoreactive for MCP-1, with faint or no expression in unaffected parts of the tissue. By contrast, widespread and strong endothelial MCP-1 expression occurred on perifascicular and perimysial endothelia in dermatomyositis, also at sites remote from inflammatory infiltrates. In some control specimens, a subset of capillaries also expressed MCP-1, possibly reflecting a role of this chemokine in normal immune surveillance. MCP-1 mRNA was detected in scattered macrophages in each inflammatory myopathy. All other chemokines were absent. CONCLUSION: Chemokines are differentially expressed in the symptomatic stage of inflammatory myopathies. MCP-1 plays a major role in the myocytotoxicity in polymyositis and inclusion body myositis. MCP-1 may be induced by membranolytic attack complex binding to endothelial cells in dermatomyositis.     

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.     

Expression of immunoreactive major histocompatibility complex products in human skeletal muscles

Immunoreactive class 1 and class 2 major histocompatibility complex gene products (MHCP) and beta 2 microglobulin (beta 2 MG) were demonstrated by microscopic immunocytochemistry in cryostat sections of skeletal muscle biopsies of 67 patients with various neuromuscular diseases. Diagnoses included normal muscle, chronic partial denervation, Duchenne dystrophy, polymyositis, dermatomyositis, inclusion body myositis, and miscellaneous neuromuscular diseases. Normal mature muscle fibers did not express MHCP, but blood vessels showed both class 1 and 2 MHCP and beta 2 MG. Regenerating muscle fibers showed consistent sarcolemmal class 1 MHCP expression irrespective of the disease. In polymyositis, the majority of extrafusal muscle fibers of most patients showed strong sarcolemmal class 1 MHCP expression. In dermatomyositis, muscle fibers situated either in perifascicular or in randomly clustered distribution revealed strong class 1 MHCP reactivity. In inclusion body myositis, scattered small clusters of muscle fibers were positive for class 1 MHCP. In polymyositis and inclusion body myositis, particularly strong class 1 MHCP expression was invariably seen in nonnecrotic muscle fibers partially invaded by lymphocytes whose cytotoxic effects are believed to be class 1 MHCP restricted. Factors or agents that trigger class 1 MHCP expression are presumed also to sensitize lymphocytes to muscle fibers in these diseases, but their identity remains obscure at this time. In dermatomyositis, the expression of MHCP in perifascicular muscle fibers and in areas of capillary loss may represent the triggering of MHCP expression by a nonspecific cellular stress reaction, in this case probably low-grade ischemia.     

Assessment of satellite cell number and activity status in human skeletal muscle biopsies

The primary aim of our study was to validate the assessment of myonuclear and satellite cell number in biopsies from human skeletal muscle. We found that 25 type I and 25 type II fibers are sufficient to estimate the mean number of myonuclei per fiber. In contrast, the assessment of satellite cells improved when more fibers were included. Second, we report that small differences in counting satellite cells using CD56 and Pax7 antibodies can be attributed to the different staining profiles. Third, we provide support for the use of Ki67 in evaluating the proportion of active satellite cells. We observed very few (up to 1.3%) active satellite cells in healthy adult skeletal muscle at rest, but they increased significantly (up to 7‐fold) following muscle activity. This study provides valuable tools to assess the behavior of satellite cells, both in pathological conditions and in response to physiological stimuli. Muscle Nerve 40: 455–465, 2009     

Monoclonal antibody analysis of mononuclear cells in myopathies. III: Immunoelectron microscopy aspects of cell-mediated muscle fiber injury

We have previously obtained light microscopical immunocytochemical evidence for cell-mediated muscle fiber injury and destruction in polymyositis and inclusion body myositis. To evaluate further interactions of the different cell phenotypes with each other and with the muscle fibers, the T8, T4, and Leu 7 markers in 7 cases of polymyositis and in 9 cases of inclusion body myositis were localized by immunoelectron microscopy. In the early stages of the cell-mediated process, T8+ cells and macrophages are apposed against, and/or send spikelike processes into, nonnecrotic muscle fibers. Leu-7+ cells penetrate fibers infrequently, and T4+ cells do not penetrate muscle fibers. Subsequently, an increasing number of T8+ cells and macrophages traverse the basal lamina; focally replace, displace, or compress the fiber; and spikes from these cells honeycomb the adjacent muscle fiber regions. The macrophages contain only few heterophagic vacuoles and therefore act in a cytotoxic rather than a phagocytic capacity. The integrity of the muscle fiber surface membrane facing the invading cells is maintained, but the possibility also exists that the membrane is damaged and rapidly repaired, or that the damage cannot be detected by electron microscopy. Nearby fiber regions often show either degenerative or regenerative changes. Ultimately, segments of the entire muscle fiber are replaced by the invading cells.     

Toxoplasmic myositis as a presenting manifestation of idiopathic CD4 lymphocytopenia

Toxoplasma gondii encysts in skeletal muscle. Although only rarely found at muscle biopsy, this parasite has previously been regarded as a possible cause of polymyositis. We report a case of biopsy-proven toxoplasmic myositis in a non-HIV-infected patient that led to recognition of idiopathic CD4 lymphocytopenia (ICL), a rare condition typically associated with opportunistic infections. Interestingly, the CD25(+) subset that corresponds to the CD4(+) regulatory T cells controlling autoimmune processes was lacking. Steroid and antiprotozoal therapy led to recovery.     

Blood vessels expressing CD90 in human and rat brain tumors

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90⁺ cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90⁺ cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90⁺ cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α‐smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90⁺ vasculatures were surrounded by tumor‐associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90⁺ vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth.     

Muscle satellite cell and atypical myogenic progenitor response following exercise

Skeletal muscle satellite cells play an essential role in muscle regeneration and exercise adaptation. In recent years atypical myogenic progenitors (non-satellite-cell muscle stem cells) have been identified in skeletal muscle and have been hypothesized to play an important role in the process of muscle regeneration. It remains unknown, however, whether any populations other than satellite cells play a significant role in repair and adaptation following exercise-induced damage. We assessed the response of the satellite cell population and the CD45+:Sca-1+ cell population, previously shown to support muscle regeneration following cardiotoxin-induced injury, after acute eccentrically biased exercise in wild-type mice. We observed evidence of focal muscle damage and repair following the exercise protocol using electron microscopy, hematoxylin-eosin staining, and single-fiber analysis. In addition, we observed an approximately sixfold increase in the number of Myf5-expressing cells by 48 h, which remained elevated until at least 96 h following exercise. We did not, however, observe any significant expansion of the CD45+:Sca-1+ cell population or commitment of resident CD45+:Sca-1+ cells to the myogenic lineage. Furthermore, expression of Wnt gene family members, previously associated with myogenic specification of CD45+:Sca-1+ cells, did not differ following exercise. Therefore, we conclude that muscle satellite cells are the primary responders to exercise-induced stress and that the CD45+:Sca-1+ myogenic progenitors do not contribute to muscle repair/adaptation following exercise.