Immunopathological Diagnosis And Molecular Immunopathogenesis Of Idiopathic Inflammatory Myopathies

BackgroundThe idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of autoimmune diseases of skeletal muscle characterized by progressive muscle weakness associated with inflammatory cells infiltration within the muscle. Based on well-defined clinical and histopathological features, IIMs can be subdivided broadly into dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM), of which PM and DM are especially clinically important because they are potentially treatable. Hence accurate diagnosis is crucial for treatment and prognosis. Unlike DM, in which the characteristic rash or unique perifascular atrophy in muscle biopsies secures early recognition, the timely and accurate diagnosis of PM has always been a thorny issue within the neuromuscular and rheumatologic medical community.Although various other criteria have been proposed, to date, diagnosis of PM is still largely based on criteria established by Bohan and Peter in which presence of mononuclear cellular infiltrates in skeletal muscle tissue is essential for "definite PM". However, these infiltrates are not always present, thus casting doubt on the diagnosis. Furthermore, secondary inflammatory infiltrates frequently found in some types of muscular dystrophies, such as dysferlinopathy and facialscapulohumeral muscular dystrophy (FSHD), may cause pathological confusion with definite PM according to Bohan and Peter's criteria. In addition, the criteria also cannot distinguish PM from certain IBM cases in the absence of rimmed vacuoles.In 2003, new diagnostic criteria based on distinctive immunohistopathological features were proposed by Dalakas and Hohlfeld in which the importance of expression of MHC-I for the diagnosis of PM was emphasized. Particularly, the term CD8/MHC-I complex, which denotes CD8-positive lymphocytes invading non-necrotic fibers that express MHC-I antigen, was first introduced as a prerequisite of diagnosis for definite PM and as the best means of separating PM from other myopathies.Although several studies focused on the diagnostic value of MHC-I alone or in combination with MHC class II antigen for IIMs have been carried out, there is still much controversy and discussion regarding this issue. Does lack of MHC-I expression per se exclude the diagnosis of IIMs or can the presence of MHC-I establish the diagnosis? In addition, to our knowledge, there are limited data to date regarding the value of CD8/MHC-I complex for the diagnosis of PM.As opposed to previous studies that viewed the IIMs as a whole, the present study focused on the diagnostic value of MHC-I and CD8/MHC-I complex in PM. We first investigated the immunohistopathological features of MHC-I expression in PM, DM, IBM, dystrophies with secondary inflammation in muscle biopsies and steroid-responsive necrotizing myopathy and then assessed the diagnostic value of CD8/MHC-I complex for definite PM. MethodsThis is a retrospective study carried out between September 2004 and December 2008 at the Neuromuscular Institute of QiLu hospital affiliated to Shandong University. We selected 5 groups of patients. The first group comprised 20 cases of PM with foci of inflammation in muscle biopsies which fulfilled Bohan and Peter's criteria for definite PM. The second group consisted of 20 cases of DM, diagnosed by the criteria of Bohan and Peter, in which either characteristic skin rash or perifascicular atrophy in muscle biopsies was identified.5 cases of IBM patients fulfilled Griggs's criteria were selected in the third group. The forth group included 10 cases of dystrophies mimicking PM pathologically with foci of inflammation in muscle biopsies (inflammatory MD); among them were 6 cases of FSHD and 4 cases of dysferlinopathies. The fifth group included 10 patients with clinically suspected PM, but without serum autoantibodies and mononuclear cell infiltrates in muscle biopies. The diagnosis of steroid-responsive necrotizing myopathy was established in this group of patients based on their well responsiveness to adequately dosed and sustained steroids treatment. Known causes of necrotizing myopathy, including some types of dystrophies, exposure to myotoxic drugs or toxins and rhabdomyolysis were ruled out. We also selected 10 patients with clinical signs of muscle weakness but with no positive findings on biopsies as normal control, which was validated as other systemic disorder finally. Immunohistochemical detection of MHC-I was performed in all 5 groups of patients using a polymer detection system technique. Double immunofluorescent labeling for CD8 and MHC-I was used to detect the existence of CD8/MHC-I complex in muscle biopsies. ResultsExpression of MHC-I in the 5 groups of patientsMHC-I antigen was absent in normal muscle fibers and expressed only on endothelial cells of small vessels in normal biopsies.(1) PM:Non-necrotic fibers with MHC-I immununoreactivity can be found in 80%(16/20) of PM cases. Focal expression of MHC-I in scattered or small grouped non-necrotic fibers along sarcolemma was noted. There was a great discrepancy in the percentage of immunostained fibers from one patient to another in PM. The number of MHC-I immunoreactive fibers ranged from 10% to 60% in PM. The staining intensity of MHC-I in PM usually ranged from weakly positive to positive (1+to 2+).(2) DM:All biopsies (including the cases without inflammation) were positive for MHC-I (100% positivity). Intense sarcolemmal MHC-I immunoreactivity was found diffusely throughout the biopsies of DM patients, but was stronger in perifascicular areas if perifascicular atrophy occurred. Some non-necrotic fibers also showing sarcoplasmic expression of MHC-I were scattered and distributed in a mosaic pattern.(3) IBM:All muscle specimens of the 5 IBM patients were positive for MHC-I (100% positivity). Diffuse MHC-I expression pattern were found in 3 of 5 cases of patients, wherase the other 2 cases showen focal MHC-I expression in muscle biopsies. The number of MHC-I immunoreactive fibers ranged from 10% to 100% in this group of IBM patients.(4) Inflammatory MD:In the present study,60%(6/10) of the dystrophy cases, including 4 FSHD and 2 dysferlinopathies, showed weak sarcolemmal expression of MHC-I in a few (usually less than 5%) scattered non-necrotic fibers. No correlation between intensity of staining to inflammation was observed. (5) Steroid-responsive necrotizing myopathy:All muscle biopsy specimens of the 10 patients expressed MHC-I on capillaries and necrotic or regenerating fibers, but only minimal MHC-I staining of nonnecrotic muscle fibers was found in 5 of 10 (50%) patients. The number of MHC-I immunoreactive fibers ranged from 5% to 50% in steroid-responsive myopathy. Similar with PM, the staining pattern of MHC-I in this group also showed focal expression of MHC-I in scattered or small grouped non-necrotic fibers along sarcolemma. The staining intensity of MHC-I ranged from weakly positive to positive (1+to 2+) in this group as in PM.Detection of CD8/MHC-I complex in the 5 groups of patientsBy means of co-localization of CD8 and MHC-I, we identified typical CD8/MHC-I complex in only 4 out of 20 cases (20%) of PM and 3 of 5 cases (60%) of IBM with mononuclear cell infiltration in muscle biopsies. There was no CD8/MHC-I complex found either in DM or in dystrophies with muscle inflammation. Just as the definition implies, no CD8/MHC-I complex was found in the 10 cases of steroid-responsive necrotizing myopathies without inflammation.Conclusions1. Detection of MHC-I molecule cannot be used as a reliable diagnostic test to differentiate PM from dystrophies with secondary inflammation or steroid-responsive necrotizing myopathy, but it may be helpful for the diagnosis of DM as suggested by previous studies.2. Given the infrequency of the complex and requirement for double immunofluorecent labeling detection, we believe that CD8/MHC-I complex, although showing high specificity for PM/IBM, is neither a sensitive nor an easy-to-handle diagnostic test for PM. BackgroundThe idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of autoimmune diseases of skeletal muscle characterized by progressive muscle weakness associated with inflammatory cells infiltration within the muscle. On the basis of well-defined clinical and histopathological features, IIMs can be subdivided broadly into dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM).DM has been wildly modeled as an autoimmune disease largely mediated by the adaptive immune system, including a local humorally mediated response with B cells and CD4+ T helper cells infiltration, antibody and complement mediated injury of capillaries, and perifascicular atrophy of muscle fibers caused by ischemia. Although PM and IBM have both traditionally been thought of as CD8+T-cell mediated disorders, there is also increasing evidence for a humoral immune component as shown by the presence of abundant plasma cells in the inflammatory infiltrates. In addition, microarray studies have consistently demonstrated a surprising abundance of immunoglobulin transcripts in muscle of PM and IBM. Through the analysis of the sequences of the immunoglobulin heavy chain transcripts in muscle, Bradshaw et al further demonstrated that the B cells and plasma cells present in DM, PM and IBM are antigen driven. These findings suggest a common local antigen-driven humoral response present in the IIMs and imply a pathogenic role for B cells and/or plasma cells not only in DM but also in PM and IBM.Clinically, the frequent presence of autoantibodies in polymyositis and dermatomyositis also indicates a role of B cells in these diseases. A further support for a pathogenic role of B cells in IIMs is the beneficial effect of B cell depletion therapy with rituximab in resistant cases of patients with dermatomyositis and polymyositis as described in one open pilot study and several case reports.B-cell-activating factor (BAFF) belonging to TNF superfamily member, has been believed to play a crucial role in B cell maturation and survival, plasma cell survival, autoantibody production, immunoglobulin-class switch recombination as well as in T cell co-stimulation and certain Thl-associated inflammatory responses. The involvement of BAFF in the pathogenesis of autoimmune diseases is well illustrated by BAFF overexpression in mice models, which leads to autoimmune disease mimicking rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjogren's syndrome (pSS).An increasing body of evidence supports a pivotal role of BAFF in the pathogenesis of many autoimmune diseases. Elevated BAFF levels have been reported in patients with a variety of various autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjogren's syndrome (pSS) and immune thrombocytopenia (ITP), indicating a role for BAFF in these pathologies, However, to date, there are limited data regarding the expression of BAFF in the idiopathic inflammatory myopathies.Steroids has been wildly recognized as the most appropriate first-line treatment for IIMs, in which DM and PM showed favorable response, while IBM always demonstrated poor response to steroids. The mechanism underlying the discrepant response to steroids between DM, PM and IBM has been poorly understood. Further, the impact of adequate dose of prednisone on BAFF expression in IIMs patients is still unclear.In this study, we first investigated the expression of BAFF in peripheral blood as well as muscle biopsies in untreated patients with DM, PM and IBM and then to assess the effect of prednisone on BAFF expression in patients with DM and PM.Material and methodsPatients and controlsTwenty one active IIMs patients (16 females and 5 males, age range 12-67 years, median 46 years), which included 10 DM,9 PM and 2 cases of IBM, were enrolled prospectively in the study during 8 months period from October 2008 to May 2009 at the Neuromuscular Institute of Qilu Hospital affiliated to Shandong University. In all IIMs patients, diagnosis was established using standard diagnostic criteria which were based on a combination of clinical data and morphological analysis of open skeletal muscle biopsies. Overlap-syndromes, collagen vascular diseases, malignancy-associated IIMs, and evidence of lymphoma, HIV or hepatitis C virus infection were excluded by clinical and laboratory studies at the moment of the study. None of the patients had received immunomodulatory therapy or immunosuppressive treatment for at least 6 months prior to muscle biopsy. The normal control group consisted of 10 adult healthy volunteers (6 females and 4 males, age range 21-65 years, median 45 years).Treatment regimenAt the beginning of been recruited in this study, all IIMs patients except 2 cases of IBM received empirical prednisone mono-therapy with the initial dose 1mg/kg/d, when strength improved and CK levels declined 4-6 weeks later, tapering the steroid dose by 5mg/day/week to reach a dose of 25-30mg/day, and then converting to an alternate-day regimen. Clinical response evaluation was made at the start of the first dose reduction about 4-6 weeks after treatment initiation.Preparation of plasma and peripheral blood mononuclear cells (PBMCs)Peripheral blood (15ml) was collected in EDTA-anticoagulated tubes both before the initiation of steroids treatment and the start of therapy response evaluation about 4-6 weeks later. The samples were centrifuged immediately at 3000g for 10 minutes at 18-21℃(RT) to ensure a cell-free plasma specimen. Plasma obtained from all subjects was aliquoted and stored at-80℃until determination of cytokines. Ficoll-Hypaque (TBDsciences, Tianjin, China) density gradient centrifugation was performed subsequently at RT (2000 rpm for 20 min) to separate the PBMCs. The isolated PBMCs were stored in TRIzol reagent (Invitrogen, Carlsbad, CA) at-80℃until further processing.Measurement of BAFF levels by ELISAPlasma soluble BAFF was measured by human BAFF immunoassay (R&D) according to the manufacturer's protocols. Samples were analyzed in duplicate. The lower detection limit was 62.5pg/ml (depends on the lowest standard dilution).Determination of the expression of BAFF mRNA by Real time-PCRTotal RNA was extracted from PBMCs by TRIzol reagent and reverse transcribed into cDNA using Quantscript RT Kit according to the manufacturer's instructions. Real-time quantitative PCR was performed for BAFF and the endogenous control (β-actin) on ABI StepOne PlusTM Real-Time PCR Systems using SYBR Green as double-strand DNA-specific binding dye. The amplification efficiency between the target (BAFF) and the reference control (β-actin) were relatively quantified by evaluating Ct values according to the comparative Ct (△△Ct) method. The relative quantification (RQ) (i.e., fold-change in expression) was expressed as 2-△△Ct. Muscle biopsy and Immunohistochemical studyMuscle specimens were taken from biceps brachii under local anaesthesia by open muscle biopsy for diagnostic purpose.8μm thick serial frozen sections were cut for routine hematoxylin and eosin (H&E) staining. Immunohistochemical detection of BAFF, CD3, CD4, CD8, CD20 and CD68 was performed in all IIMs cases and in control biopsies.ResultsElevated Plasma BAFF levels in untreated DM and PM patients but not in IBM patientsPlasma BAFF levels were significantly higher in patients with DM and PM (3262±896 pg/ml and 1713±463 pg/ml respectively) than in healthy controls (767±171 pg/ml, P＜0.001). Patients with DM had higher BAFF levels compared to PM (P＜0.001). Whereas plasma BAFF levels of the 2 cases of IBM patients (924 pg/ml and 785 pg/ml respectively) were found within the normal limits of controls (range from 451 pg/ml to 1092 pg/ml).Elevated BAFF mRNA expression in untreated DM and PM patientsQuantitative reverse transcription-PCR analyses to determine BAFF mRNA levels were done using the 2-△△CT method. BAFF mRNA expression values ranged from 1.87-to 4.98-fold change in DM and 1.02-to 2.45-fold change in PM relative to normal controls. The relative amount of BAFF mRNA in untreated DM and PM patients were 3.26-fold and 1.82-fold respectively of that of healthy controls (P＜0.001).Correlation of plasma BAFF with its mRNA level in DM and PM patientsWe observed that plasma BAFF correlated with its mRNA levels in active DM and PM patients (r=0.74 and r=0.91 respectively, P＜0.001). Decreased expression of BAFF in DM and PM patients after prednisone treatmentAfter administration of prednisone (1mg/kg/d) for 4 to 6 weeks, plasma BAFF levels in DM and PM patients were significantly decreased (932±422 pg/ml,658±220 pg/ml respectively) compared with pretreatment (3262±896 pg/ml and 1713±463 pg/ml respectively, P＜0.001. Similar results were found on BAFF mRNA levels. After administration of prednisone, the relative amounts of BAFF mRNA in DM and PM were 0.61 fold and 0.57 fold of that of untreated patients respectively (P＜0.001).Changes of BAFF correlated with clinical responsesAfter administration of adequately dosed prednisone, all DM and PM patients had reduced plasma BAFF level. Among them,15 of 19 (78.9%) cases of patients showed favorable clinical response. However, there were also 4 patients (2 PM and 2 DM) who had reduced plasma BAFF levels but did not show favorable clinical response to steroids.BAFF did not expressed in the muscle biopsies of IIMsTo test whether BAFF expression was up-regualted in the muscle tissue, the main target of autoimmunity in PM and DM, as in peripheral PBMCs, we investigated BAFF protein expression on muscle biopsies of DM and PM patients. Contrast to the above hypothesis, complete absence of BAFF expression was identified not only in myofibers but also in infiltrated inflammatory cells (including T and B lymphocytes) and endothelial cells.Conclusions1. Our results suggest that BAFF expression is increased in untreated DM and PM patients with active disease, and can be inhibited by sustained adequate dosed steroids. Steroids including prednisone may play its role in treatment of DM and PM partly through regulating BAFF expression, which to some extent could be the possible mechanism underlying the discrepant response to steroids between DM, PM and IBM.2. Blocking the expression of BAFF in peripheral blood may be a potential treatment target for DM and PM.