Major histocompatibility complex and inflammatory cell subtype expression in inflammatory myopathies and muscular dystrophies
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.125264
Source of Support: None, Conflict of Interest: None
Background: In inflammatory myopathies muscle biopsy is a crucial diagnostic test. Misinterpretation between inflammatory myopathies and muscular dystrophies with inflammation is known. Materials and Methods: Thirty-one patients clinically and pathologically diagnosed to have polymyositis and dermatomyositis and 16 patients of muscular dystrophy with inflammation were studied for MHC-I, MHC-II, CD4 and CD8 expression in skeletal muscle tissue. Results: MHC-I upregulation was noted in all samples of PM and DM. Interstitial and perivascular inflammation in PM were predominantly CD8+ cells, in dermatomyositis, interstitial and perimysial perivascular inflammatory cells were CD4+ T cells and CD8+ T cells were seen around endomysial vessels. Interestingly MHC-I upregulation was seen in all 16 cases of muscular dystrophy with presence of inflammation. Conclusion: The pattern of MHC-I and II expression appeared to be similar in both inflammatory myopathies as well as in muscular dystrophies with inflammation and hence differentiating them on MHC - I expression was difficult.
Keywords: Dermatomyositis, inflammatory myopathies, MHC I and MHC II, muscular dystrophy, polymyositie
Muscle biopsy is the gold standard for the diagnosis of inflammatory myopathies (IM) and various muscular dystrophies. Early detection and confirmation of diagnosis of IM is important as they are potential treatable. The characteristic histological features can, however, be absent in IMs and is due to the disease process not being active and patchy nature of the disease process and sampling error.  Histologically, inflammation can also be a part of the disease process in muscular dystrophy, to such an extent that they can be misdiagnosed as inflammatory myopathies.  The diagnostic utility of major histocompatibility complex (MHC) has been studied in IMs with conflicting results. Earlier studies have demonstrated expression of MHC in inflammatory as well as X-linked and other muscular dystrophies. ,, In contrast, recent studies have strongly emphasized the high sensitivity and specificity of MHC I detection in distinguishing IMs from muscular dystrophies in the presence of inflammation in biopsy. , In the present study an attempt was made to ascertain the utility of MHC-I and II as well as MHC-CD8 complex in differentiating IMs from muscular dystrophies.
Thirty-one patients with clinical and histopathological evidence of inflammatory myopathy [polymyositis (PM) and dermatomyositis (DM)], and evaluated between December 2007 and January 2010, were identified from the archived muscle biopsy records maintained by the Department of Neuropathology. Clinical diagnosis of IMs was made according to the criteria proposed by Dalakas and Hohlfeld.  These muscle biopsies were compared with muscle biopsies from 16 patients with clinically and histologically confirmed muscular dystrophies with evidence of significant inflammation on biopsy. Serum creatine kinase (CK) was done in all patients. Findings of electromyography and nerve conduction study wherever available were recorded. Evaluation for autoimmune disorders and underlying malignancy were done wherever feasible. Muscle biopsies of patients clinically suspected to have myopathy, which were normal histologically and histochemically were used as controls. Six patients received immuno-therapy: steroids (n = 6), methotrexate (n = 1), intravenous immunoglobulin (n = 1) and mycophenolate mofetil (n = 1). In these patients, muscle biopsy was carried out after stopping immuno-therapy for a minimum of 4 weeks. The final diagnosis was dermatomyositis (n = 3) and muscular dystrophy with inflammation (n = 3) based on clinical examination and immunohistochemical studies. The study was approved by the Institutional Ethics Committee.
Muscle tissue was obtained by open method from a moderately weak muscle. Biceps or vastus lateralis was chosen after obtaining written informed consent. Fresh frozen sections were subjected to routine haematoxylin-Eosin (HE), modified Gomori trichrome (MGT) and a battery of enzyme stains including nicotinamide adenine dinucleotide- tetrazolium reductase (NADH-Tr), succinic dehydrogenase (SDH), adenosine triphosphatase (ATPase) pH 9.5, 4.6 and 4.3. Immunostaining to monoclonal antibodies against dystrophin (Rod domain, C-and N-terminal), sarcoglycans (α, β, γ, δ), β-dystroglycan, merosin, and dysferlin (Nova Castra laboratories) for classifying muscular dystrophies, CD4+ Tcell (4B12 Bio Genex) and CD8+ T cell (1A5 Bio Genex) for inflammatory cells and MHC-class I (HLA ABC) W6/32 (DAKO), and MHC-class II (HLA DR) LN3 (Bio Genex) were used as primary antibodies, with HRP- tagged LSAB+ (labeled streptavidin biotin) as secondary antibody.
Expression of MHC-class I and MHC-class II were assessed based on the labeling along the membrane, while the inflammatory infiltrates labeled for CD4+ T cells and CD8+ T cells were assessed in the interstitial tissue (endomysium and perimysium) and in the perivascular region (endo and perimysial vessels). The sarcolemmal labeling with MHC classes I and II antigens were calculated as percentage of muscle fibers estimated on two fields at ×10 magnification. This was based on an earlier publication and graded as: absent: -; scattered: +; <25%: +; 25-50%: ++; 50-75%: +++. 
Dermatomyositis (Group I)
There were 14 (8 (53.3%) males) in this group. The mean age at presentation was 26.7 ± 19.9 years (range: 5-62 years) and median duration of symptoms was 2 months (range: 15 days to 1.5 years). All had subacute onset, progressive, predominantly proximal limb weakness. All except 2 patients had the cutaneous rash characteristic of DM. Thirteen patients had neck flexor weakness and 3 had bulbar muscle involvement. Muscle pain was reported by 10 (71.4%) patients. One patient had respiratory distress. Preceding fever was present in 5 and 3 had arthralgia and 1 had arthritis. Oral ulcers, alopecia, and weight loss was present in one patient each [Table 1]. Seven patients did not satisfy the criteria for definite dermatomyositis (absence of characteristic rash in two, no CK elevation in two, absence of perifascicular atrophy in three). Mean serum CK level was 6678.6 ± 6187.96 IU/l (range: 118-22 526).
Polymyositis (Group II)
Of the 14 patients in this group, ten were females and seven males, with a mean age at presentation of 36.56 ± 13.7 years (range: 15-61 years). Mean age of onset was 35.7 ± 13.8 years and the mean duration of symptoms was 4 months (mean: 1.59 ± 3.26 years). All patients had moderate to severe weakness of proximal upper and lower limbs. Neck muscle weakness was present in 11, dysphagia in eight and nasal regurgitation in three patients. Two patients had relapsing-remitting symptoms. Muscle pain was present in eight patients. One patient was diagnosed to have carcinoma cervix [Table 1]. Five patients did not satisfy the definite criteria for polymyositis (age less than 18 years in one, absence of CK elevation in four); hence they were classified as probable polymyositis. Mean CK level was 4751.3 ± 4707.5 IU/l (range: 125-16 886 IU/l). ANA was positive in 6 of 13, ANCA in 2 of 4, and dsDNA in one of two. Electromyography showed myopathic pattern in 15 and mixed pattern in two patients.
Muscular dystrophies with inflammation (Group III)
Sixteen patients diagnosed as muscular dystrophies with significant inflammation were included -Duchenne muscular dystrophy (n = 2), dysferlinopathy (n = 1), calpainopathy (n = 1), limb girdle muscular dystrophy unclassified (n = 9), and facioscapulohumeral muscular dystrophy (n = 3). In addition to significant endomysial and perimysial inflammation seen in these patients, inflammation around non-necrotic fibers was seen in all patients with dystrophinopathy and in one patient with Fascio-scapulo-humeral muscular dystrophy. One patient in this group was diagnosed to have inflammatory myopathy on the basis of inflammation in muscle biopsy done at another center. He did not improve with immunomodulation with steroids and methotrexate. Immunohistochemistry confirmed the absence of dysferlin in the repeat biopsy done at our center. Similarly, another patient presented with a 2-year history of bifacial weakness and one year history of proximal limb weakness (upper limbs more involved than lower limbs). She was also diagnosed to have inflammatory myopathy based on biopsy findings, and was treated with steroids, mycophenolate mofetil and intravenous immunoglobulin, without benefit. Clinically, patient had typical features of facioscapulohumeral muscular dystrophy.
Inflammatory myopathies (Group I and II)
In patients diagnosed to have DM and PM (idiopathic and secondary), immunohistochemical staining with MHC-I showed uniform labeling along the membrane of all muscle fibers [Figure 1]b, g, and . This suggested upregulation of MHC-I in all fibers irrespective of the presence of inflammatory cells or necrosis. In cases diagnosed as DM, the atrophic fibers in the perifascicular region did not show upregulation of MHC-I [Figure 1]a and b. Some of the inflammatory cells in the endo- and perimysium also labeled MHC-I. Upqregulation of MHC-II was seen along the membrane only in those fibers undergoing myophagocytosis. None of the other muscle fibers including the perifascicular atrophic fibers labeled MHC-II [Figure 1]c, h, and m.
In PM the inflammatory cells were seen singly or in clusters in the interstitium as well as in the perivascular region and consisted predominantly of CD8+ cells, with occasional CD4+ cells [Figure 1]f, i-k n, and o. Interestingly, fibers with myophagocytosis, necrotic fibers and non-necrotic fibers showed predominantly CD4+ T cells within and around the fibers with occasional CD8+ T cells. In contrast, in DM, the inflammatory cells in the interstitial region consisted mainly of CD4+ T cells with a few CD8+ Tcells [Figure 1]d and e. Clusters of CD4+ T cells were predominantly noted around the perimysial vessels, while CD8+ T cells were seen around the endomysial vessels.
Muscular dystrophies with inflammation
Sixteen patients, clinically and histologically confirmed as muscular dystrophies [Figure 1]p revealed uniform labeling of MHC-I along the membrane in all muscle fibers, including non-necrotic fibers, in all the biopsies studied [Figure 1]q. MHC II positive labeling was seen in a few fibers in one of the cases of dystrophinopathy and a few inflammatory cells around the endomysial vessels in one patient with calpainopathy [Figure 1]r. The inflammatory cells were CD4+ T cells in the interstitium and perivascular region with an occasional CD8+ T cell [Figure 1]s and t. The fibers with myophagocytosis showed predominantly CD4+ T cells in all, except one patient with calpainopathy, where cells within the fibers undergoing myophagocytosis were not labeled. Non necrotic fibers were surrounded by CD4+ T cells. The histopathological features and expression pattern of MHC-I and II are represented in [Table 2] and [Table 3].
In this study, an attempted was made to evaluate the expression pattern of MHC I and II in inflammatory myopathies and explore their role in distinguishing these disorders from muscular dystrophies with the presence of inflammation on muscle biopsy. The diagnosis of muscle disorders has undergone tremendous changes from routine histology on paraffin sections to enzyme histochemistry in 1960s to immunocytochemistry in 1990s,  yet, well defined diagnostic markers of inflammatory myopathies are lacking. Although, the presence of inflammation in muscle biopsy is considered diagnostic of inflammatory myopathies, errors do occur because histopathologicall changes in inflammatory myopathies may be patchy and hence prone to sampling error and intense inflammation may be present in muscular dystrophies, thus making the diagnosis on histology difficult.  Recently MHC-I antigen immunostaining has been used to differentiate inflammatory myopathies from muscular dystrophies with presence of inflammation. ,
The MHC antigens are integral membrane glycoproteins expressed consistently in all nucleated cells. In contrast, normal skeletal and cardiac muscle fibers express neither MHC I nor II. In this study, it was observed that MHC-I was upregulated along the membrane of all muscle fibers in patients with inflammatory myopathy, but interestingly the perifascicular atrophic fibers in dermatomyositis did not demonstrate upregulation of MHC-I. While the results of studies of MHC-I expression in inflammatory myopathies by McDouall et al.,  and Jain et al.,  is in concurrence with the findings of our study, the striking difference was the absence of MHC I expression in the perifascicular atrophic fibers in dermatomyositis. In polymyositis, some of the inflammatory cells in the endo and perimysium also labeled MHC-I. Internal staining of sarcoplasm was not noted in any of the muscle fibers including the necrotic and regenerating fibers and this has been reported earlier. ,
Previous studies have demonstrated MHC-I to be absent or expressed only in the muscle fibers surrounded by inflammatory cells. ,, These studies strongly emphasize the high sensitivity and specificity of MHC-I detection in distinguishing inflammatory myopathies from muscular dystrophies with the presence of inflammation. Interestingly, in the present study, immunostaining with MHC-I of muscle biopsies from patients with muscular dystrophies revealed uniform, intense labeling along all fibers including the non-necrotic fibers and also fibers without inflammation. The intensity and pattern of labeling was indistinguishable from that of inflammatory myopathies. Our findings were strikingly different from previous reports. ,, Thus, MHC I expression may be less sensitive method in assisting the neuropathologist to strongly differentiate inflammatory myopathies from muscular dystrophies with inflammation.
Curiously, the earliest studies of expression of MHC in normal and abnormal muscles have shown that there is positive membrane labeling of MHC-I in dystrophic muscle fibers, although of varying intensity. The intensity of staining was unrelated to the degree of pathological change in the muscle. ,, In a recent study of a cohort of 10 patients with dysferlinopathy, a non-uniform MHC-I labeling was demonstrated in seven patients.  However, in the present study there was no difference in the intensity of expression of MHC-I and II as well.
There are conflicting reports in literature with respect to the expression of MHC-II in diseased muscle, and hence the utility of the same in diagnosis. While in an earlier study the authors failed to demonstrate the expression of MHC-II in normal muscle as well as in inflammatory myopathies and muscular dystrophies , a more recent study showed a significant proportion of patients with inflammatory myopathies showing MHC-II expression particularly along the perifascicular atrophic fibers in dermatomyositis. , MHC-II labeling was not present in muscular dystrophies in any of these studies. In the current study, membrane labeling of MHC-II was present in some of the muscle fibers undergoing myophagocytosis in dermatomyositis and secondary polymyositis as well as in a few fibers in a patient with DMD. Our finding of lack of MHC II expression is in accordance with previous studies, , but contrasts with findings in other reports which have demonstrated that a significant proportion of muscle fibers express MHC II.  Jain et al., demonstrated a high specificity of MHC II expression in the diagnosis of inflammatory myopathies, as none of the muscular dystrophies in their cohort expressed MHC II.  However, the utility of MHC II in the diagnosis of inflammatory myopathy could not be demonstrated in the current study. The value of MHC I and II expression in the evaluation of inflammatory myopathies is controversial. The conflicting results in different studies described above could partly be attributed to differences in methodology. While some have used PHM4 antibody,  others, including the current study have used W6/32 antibody in the staining technique. ,,,, Another important methodological difference in these studies is the system of grading sarcolemmal positivity for MHC I/II expression. The 'percentage' of muscle fibers expressing MHC classes I and II antigens on two fields (at ×10 magnification) has been used to grade sarcolemmal expression for MHC I/II from absent to +++ (absent: '- '; scattered: '+'; <25%: '+'; 25-50%: '++'; 50-75%: '+++') in some studies.  An alternative method that has been more commonly employed is subjective and grades sarcolemmal expression based on the 'intensity' of staining from absent to +++ (absent: '-'; distinct but poorly stained/weak: '+'; distinct: '++'; marked: '+++'). , There are also variations in the grading of MHC II expression with respect to the 'percentage' of muscle fibers that show positivity.  Some studies have not clearly defined the technique used for determining/grading MHC expression.  A recent study has shown that by objectively quantifying sarcolemmal MHC expression, the positive and negative predictive values for the diagnosis of inflammatory myopathies are 100% and 94% respectively, when the percentage of MHC I positive fibers is greater than 50%.  While this has been demonstrated in occasional patients with dysferlinopathy and inflammatory changes in biopsy,  a formal comparison of the quantitative expression of MHC in inflammatory myopathies and muscular dystrophies with inflammation remains to be established.
Immunophenotyping of inflammatory cell infiltrate is known to assist in the diagnosis as well as in understanding the possible immunopathogenesis. In dermatomyositis, the inflammatory infiltrate is reported to consist predominantly of CD4 cells and B lymphocytes and is consistent with a humoral mediated process while in polymyositis, CD8-positive cells invade muscle fibers expressing MHC I antigen, suggesting a cell-mediated process.  In our study, the pattern of inflammatory infiltrate in dermatomyositis and polymyositis was similar.
Interestingly the salient finding in our study was the presence of uniform expression of MHC I in sarcolemma in both inflammatory myopathies as well as muscular dystrophies. The major role of MHC is presentation of antigens to the immune system and hence, they have an important role in provoking autoimmune diseases.  Normal muscle does not express MHC I; this phenomenon is possibly a defense mechanism against autoimmune attack. It has been shown in a transgenic mouse model that over expression of MHC-I using a controllable muscle-specific promoter system causes clinical, biochemical, histological and immunological features akin to human myositis.  In humans, the appearance of MHC I on muscle fibers distant from the inflammatory infiltrate as shown in the current and previous studies concur with the fact that MHC plays a role in initiating and maintaining the ongoing muscle damage. The factors inciting expression of MHC I are varied. Every infection or trauma does not result in myositis. Only a prolonged upregulation of MHC I in certain individuals, perhaps with a predisposing genetic background or abnormal expression of sarcolemmal proteins, provokes a self-sustaining muscle inflammation.  In the current study, all patients were drug naοve as immunomodulation was discontinued for at least 4 weeks prior to muscle biopsy. Thus, it is unlikely that the findings on muscle biopsy were altered or masked by use of immunomodulatory agents.
In case of muscular dystrophies, the loss of linkage between extracellular matrix and actin cytoskeleton is reported to result in the loss of stability of muscle fiber membranes. It has been hypothesized that altered expression of different proteins in various forms of muscular dystrophies triggers inflammatory response and is involved in muscle degeneration. Inflammation has been found to be an important pathophysiological event in muscular dystrophies. There are numerous evidences for immunological involvement in pathological expression of various muscular dystrophies such as onset triggered by viral respiratory infections, presence of inflammation in muscle biopsy, upregulation of MHC I, and response to steroid therapy although transient. , The caveat being SJL/L mice deficient for dysferlin with MHC I knock-out gene did not differ in the extent of myopathy.  A strong persistent expression of MHC I in muscle fibers of patients with chronic inactive polymyositis and dermatomyositis has also been demonstrated.  Hence, the role of MHC in induction of inflammation in muscular dystrophies as well as inflammatory myopathies remains to be elucidated. Thus, in the present study, we were unable to distinguish inflammatory myopathies from muscular dystrophies with inflammation on the basis of MHC I expression.
The knowledge of pathophysiology of inflammatory myopathies has tremendously increased in the last half a century. In spite of this a number of uncertainties exist, particularly in the nature of factors inciting MHC I expression and definitive marker for establishing conclusive diagnosis. Expression of MHC-I on diseased muscle may make muscle a target for cytotoxic T cells and thus precede muscle fiber destruction in inflammatory myopathies and other dystrophies.
[Table 1], [Table 2], [Table 3]