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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Inclusion body myositis (IBM) is a chronic inflammatory myopathy. The muscle histology is characterized by infiltration of T cells, which invade and apparently destroy muscle fibres. This study was performed to investigate whether predominant clones of muscle-infiltrating T cells are identical in different muscles and whether they persist over time in IBM. By reverse transcriptase-polymerase chain reaction, 25 T-cell receptor (TCR) variable β (Vβ) chain families and the complementarity-determining region 3 (CDR3) of the TCR were analysed in two different muscle biopsies of four patients with IBM. In two of the patients, the muscle biopsies were obtained from different muscles at one time point, whereas in two patients, the second biopsy was obtained 9 years after the first biopsy. T cells expressing predominant Vβ families were analysed for clonality by fragment length analysis of the CDR3. Predominant Vβ families were analysed by DNA sequencing to identify identical clones. Immunohistochemical staining of Vβ families was performed to study the distribution of T cells expressing identified predominant Vβ families. The muscle-infiltrating lymphocytes showed restricted expression of TCR Vβ families. DNA sequencing proved that clonally expanded T cells were identical in different muscles and persisted 9 years after the first biopsy. Immunohistochemical analysis with Vβ family-specific antibodies demonstrated the endomysial localization of these T cells in inflammatory cell infiltrates. Our results show that in IBM there is clonal restriction of TCR expression in muscle-infiltrating lymphocytes. Identical T-cell clones predominate in different muscles, and these clones persist for many years. These results indicate an important, continuous, antigen-driven inflammatory reaction in IBM.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Inclusion body myositis (IBM) is a chronic, progressive, inflammatory muscle disease mainly affecting individuals older than 50 years [1]. Clinically, IBM is characterized by proximal and distal muscle weakness and wasting, frequently accompanied by dysphagia [2, 3]. IBM, after several years of slowly progressive course, is a severely disabling disorder and there is no curative treatment. Muscle morphology is characterized by inflammatory cell infiltrates, which invade and apparently destroy non-necrotic muscle fibres [4–6]. In addition, there are rimmed vacuoles and congophilic inclusions in muscle fibres [7]. Deposits of 15–18 nm of tubulo-filaments are present in cytoplasm and nuclei. Accumulation of various proteins have been described in association with the rimmed vacuoles [8].

In spite of resistance to immunosuppressive treatment, there is evidence that the inflammatory reaction is important in IBM [9]. The inflammatory cells are mainly composed of T cells and macrophages, and non-necrotic muscle fibres are invaded by inflammatory cells, most of which are CD8+ T cells [4]. Muscle fibres invaded by T cells are several times more frequent than fibres displaying other pathologic alterations such as congophilic inclusions [10]. A large proportion of the autoinvasive CD8+ T cells are human leucocyte antigen-DR (HLA-DR)-positive suggesting that they are activated [11]. All invaded muscle cells express HLA class I antigen on their surface, which is consistent with a major histocompatibility complex (MHC) class I-restricted cytotoxic cell-mediated attack on muscle fibres. Inflammatory cell-infiltrates in IBM exhibit a restricted expression of the variable α/β (Vα/β) chain families of the T-cell receptor (TCR) [12–14], and there are clonal expansions of muscle-infiltrating T cells [15–17].

If specific antigens in muscle trigger the inflammatory reaction, it would be expected that T cell-expressing identical TCRs are expressed in all muscles and also persist over time. In this study, we investigated the predominant T-cell clones of muscle-infiltrating lymphocytes in different muscles obtained at one time point and in repeat muscle biopsies obtained 9 years apart. The results show that the TCR expression is restricted and that predominant T-cell clones are identical in different muscles and persist for many years.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients.  Muscle specimens from four patients with IBM were studied. The patients had progressive muscle weakness and the diagnostic changes of definite IBM, according to Griggs et al. [18]. A summary of clinical data is summarized in Table 1. The muscle tissue specimens were obtained by open biopsy and immediately frozen in a mixture of propane and propylene chilled by liquid nitrogen and kept at −85 °C until analysed. Specimens from two different muscles were obtained at the same time point in patient 1 and 2. In patient 3 and 4, the two muscle biopsies were obtained with a time interval of 9 years.

Table 1.  Clinical and morphological data on the inclusion body myositis (IBM) patients included in the study
PatientGender (M/F)Age (years)Duration of symptoms (years)Main clinical findingsCK*TreatmentDegree of muscle inflammationHLA
  • *

    Creatine kinase; µkat/l, normal value <3.5.

  • HLA, human leucocyte antigen.

1M674Weakness of elbow flexion and long finger flexors. Quadriceps muscle atrophy10NoneBiceps: moderate inflammation. Quadriceps femoris: severe inflammation HLA-A*02 B*08,51 DRB1*03,11
2M605Severe weakness of long finger flexors. Moderate weakness in knee extension, hip flexion and neck flexion15NoneDeltoid: moderate inflammation. Quadriceps femoris: moderate inflammationHLA-A*03,33 B*14,51 DRB1*01,03
3F606Weakness in knee extension, hip flexion and finger flexion. Swallowing difficulties3.0NoneDeltoid: moderate inflammationHLA-A*03,26; B*07,51 DRB1*01,13
  6915Severe weakness in knee extension, hip flexion and finger flexion. Moderate to severe swallowing difficulties1.4Prednisone since age 60. Dosage: 15 mg/2 days since age 66. Methotrexate 5 mg/week since age 68Deltoid: slight inflammation 
4M732Weakness in knee extension, hip flexion and finger flexion14NoneGastrocnemius: severe inflammationHLA-A*03,29; B*07,44 DRB1*11,13
  8211Severe weakness in knee extension, hip flexion and finger flexion. Moderate proximal weakness in upper extremities4.5Prednisone since age 73. Dosage: 10 mg/3 days since age 80Deltoid: severe inflammation 

Polymerase chain reaction and fragment analysis.  Total RNA from muscle tissue was extracted by using the SV Total RNA Isolation System (Promega, Madison, WI, USA). First strand cDNA was synthesized by Ready To Go you-prime first-strand beads with molony murine leukaemia virus (M-MuLV) reverse transcriptase and the oligonucleotide pd(T)12−18 as primer (Pharmacia Amersham Biotech, Uppsala, Sweden). Polymerase chain reaction (PCR) analysis of the complementarity-determining region 3 (CDR3) using 25 different Vβ family-specific primers was performed as previously described [16]. Predominant Vβ families in both muscles from each case were selected for further analysis of the CDR3 region by fragment analysis using a Cβ primer conjugated to the fluorescent dye 6-FAM (KEBO Laboratory, Stockholm, Sweden) as previously described [16]. The labelled PCR products were loaded on a 5.75% polyacrylamide-sequencing gel (Long Ranger Singel Packs, FMC Bioproducts, Rockland, Maine, UK) and run on an automated DNA sequencer (Model 377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA) to separate CDR3 fragments of various length. The results were analysed by GeneScanTM Analysis software (Perkin Elmer, Foster City, CA, USA). As the positions of the Vβ and the Cβ primers were fixed, the length distribution observed in the PCR products only depended on the size of the CDR3 region, which was set according to Moss [19].

Cloning and sequencing.  Predominant PCR products of the different Vβ families were further analysed by sequencing to identify identical clones. The PCR fragments were sequenced directly or after subcloning with TOPO TA Cloning Kit, Dual Promoter (Invitogen BV, Groningen, The Netherlands). The plasmid inserts of randomly selected colonies were amplified by PCR and then purified using Micro Spin columns (Pharmacia Amersham Biotech). Automatic sequencing was performed with a 377 DNA Sequencer (Applied Biosystems) using ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

Immunohistochemical staining of Vβ families.  Cryostat sections were fixed in cold acetone for 5 min, washed in phosphate-buffered saline (PBS) and treated with 0.3% H2O2 for 5 min. They were then incubated in PBS with 4% PBS/bovine serum albumin (BSA) for 10 min followed by incubation with a mouse monoclonal antibody to Vβ8 or Vβ5.1 (Beckman-Coulter, Bromma, Sweden) diluted 1 : 200 in 1% PBS/BSA for 1 h. After rinsing in PBS, the sections were treated with a biotinylated rabbit antimouse secondary antibody for 30 min (Dako, Glostrup, Denmark). The binding of biotinylated antibodies was detected following stepwise incubation with avidin–biotin–peroxidase complexes (Dako) and 3,3′-diaminobenzidine tetrahydrochloride as a chromogen.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

A restricted usage of Vβ families in muscle-infiltrating lymphocytes was found in all eight examined muscle biopsies. The results from patient 1 are illustrated in Fig. 1. One or more Vβ family predominated in both muscle samples of each patient, and these were chosen for analysis of oligoclonal expansions by fragment size analysis. The results of the selected and analysed Vβ families in patient 1 (Vβ2, 4, 6, 8, 14 and 18) are illustrated in Fig. 2. In this patient, the Vβ8 family showed an oligoclonal pattern in both muscles, whereas the other Vβ families showed a polyclonal pattern. In patients 2–4, a similar variability was observed. We then characterized the predominant CDR3 of TCR Vβ8 in patients 1, 2 and 4, the Vβ5.1 in patients 2 and 3 and Vβ6 in patient 3 by DNA sequencing. The results show that identical T-cell clones were present in both investigated muscles of all four patients. The deduced amino acid sequences of the identified clones are presented in Fig. 3. To study the localization of the investigated T cells within the tissue, we performed immunohistochemical analysis using Vβ family-specific antibodies. We found that Vβ8-expressing T cells of patient 1 and 4 and the Vβ5.1-expressing T cells of patient 2 and 3 were present at high frequency in endomysial T-cell infiltrates (Fig. 4).

image

Figure 1. Reverse transcriptase-polymerase chain reaction analysis of T-cell receptor variable β (Vβ) family usage in muscle-infiltrating lymphocytes from patient 1 with inclusion body myositis and in peripheral blood lymphocytes (PBL) from a healthy control. The different Vβ families are indicated above each lane. There is a restricted but variable usage of the different Vβ families. Some of the Vβ families appear as strong bands in both muscles, e.g. Vβ2, 4, 6, 8, 14 and 18.

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image

Figure 2. Spectratyping of predominant variable β (Vβ) families in patient 1. Vβ8 shows only one peak in both muscles, indicating that the Vβ8-expressing T cells in both muscles include one predominant clone.

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image

Figure 3. Results from DNA sequencing of reverse transcriptase-polymerase chain reaction amplified fragments of predominant variable β families. Direct sequencing and/or subcloning before sequencing shows that there are identical T-cell clones in the different muscle specimens in each patient. The results are given as deduced amino acid sequences of the complementarity-determining region 3 (CDR3). In patient 3 and 4, there are 9 years between the 1st and 2nd biopsy in each case. The figure includes only clones that were identified in two different muscles or at two different occasions.

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image

Figure 4. Haematoxylin and eosin (HE) staining (A and C) and immunohistochemical staining of the T cells expressing T-cell receptor variable β 8 (Vβ8) chain (B and D) that were found to be predominant and oligoclonal in patient 1 (A and B) and 4 (C and D). The analysis shows that the Vβ8-expressing T cells are located in endomysial inflammatory cell infiltrates. Bars correspond to 10 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this study, we have demonstrated that muscle-infiltrating T cells show restricted Vβ TCR expression and expansion of T-cell clones that are identical in different muscles and persist over very long time in patients with IBM.

TCR expression of muscle-infiltrating T cells has previously been studied in single muscle biopsies in IBM and polymyositis (PM) by analysis of the TCR Vβ repertoire and the CDR3. Results from such studies have demonstrated restricted usage of Vβ families [12–14, 20, 21] and clonal expansions of T cells in muscle in IBM and PM [15–17, 22]. The preferentially expressed Vβ families varied between different studies and also between individual patients. However, Vβ1, 3, 5.1, 5.2, 6 and 8 were reported as being frequently expressed [12–17, 22]. The pattern of Vβ family expression in muscle-infiltrating lymphocytes is different from peripheral blood [12]. Such restricted Vβ family expression and clonal expansions of T cells in muscle in IBM and PM indicate an antigen-driven immune response with a limited number of antigens presented by MHC I-expressing muscle cells. In addition, Bender et al. [17] showed that the autoinvasive T cells were clonally restricted, whereas the noninvasive interstitial T cells were clonally diverse in IBM, providing further evidence for an antigen-driven MHC-restricted immune response.

Specific stimulation of naive T cells requires TCR interaction with an antigen presented by MHC class I or II, interaction between CD28 on T cells with costimulatory molecules belonging to the B7 family and other factors. Mature muscle cells do not express B7-1/B7-2 neither normally nor in inflammatory myopathies [23], but another functional costimulatory molecule (BB-1) has been demonstrated to be expressed by muscle fibers in inflammatory myopathies including IBM [24, 25]. This finding indicates that muscle cells may act as antigen-presenting cells to T cells with specific TCR rearrangements.

Because there is no efficient immunosuppressive treatment of IBM, the concept of IBM as an autoimmune disease has been debated. The importance of the inflammatory reaction in the pathogenesis of IBM is supported by several observations. Non-necrotic muscle fibres invaded by T cells are several-fold more frequent than fibres displaying other pathologic alterations such as congophilic inclusions [10]. Upregulation of MHC class I is consistently found in IBM muscle biopsies irrespective of presence or absence of inflammatory cell infiltration [26]. In addition, several genetic studies on IBM have shown a strong association with the HLA-DR3 haplotype [27–29], supporting the concept that autoimmunity is important in the pathogenesis of IBM.

The mechanisms by which the T cells destroy the muscle fibres are not known. There are two major pathways of T-cell-mediated cell death. One is the perforin/granzyme pathway and the other is the Fas/Fas ligand pathway [30]. Both mechanisms may act by induction of apoptosis. In polymyositis and IBM, perforin-positive cells have been demonstrated in association with invasion of non-necrotic muscle fibres [31, 32]. Fas is not normally expressed by muscle fibres but is expressed by muscle fibres in IBM and PM [33, 34]. However, apoptosis of muscle cells has not been shown to be of significance in IBM and PM [33, 35–37], although a recent study indicates that apoptosis occurs in muscle cells in inflammatory myopathies [38]. Cytokines that are expressed or induced by the inflammatory cells may also be important in the pathogenesis of muscle fibre atrophy and destruction [39–41].

In chronic inflammatory diseases like IBM, it is probable that the T-cell usage eventually becomes more diverse. In experimental allergic encephalomyelitis, there is initially a restricted T-cell usage, which eventually becomes more diverse, although the initial pathogenic T-cell clones remain and predominate in the inflammatory cell infiltrate [42]. We found that the predominant T-cell clones in muscle-infiltrating lymphocytes persisted for at least 9 years. This is in accordance with a report where three IBM patients were followed for up to 22 months [43]. This finding strongly indicates that a limited number of important antigens continuously drive the inflammatory reaction in IBM leading to progressive muscle destruction and weakness. Identification of the antigens recognized by these T-cell clones would give important insights in the pathogenesis of IBM.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This study was supported by grants from the Swedish Medical Research Council (Proj. no. 7122), King Gustav V 80th Anniversary Fund, the Swedish Rheumatism Association and the Göteborg Medical Society. Professor Lennart Rydberg is acknowledged for valuable help with HLA typing.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Oldfors A, Fyhr IM. Inclusion body myositis: genetic factors, aberrant protein expression, and autoimmunity. Curr Opin Rheumatol 2001;13: 46975.
  • 2
    Lindberg C, Persson L, Björkander J, Oldfors A. Inclusion body myositis – clinical, morphological, physiological and laboratory findings in 18 cases. Acta Neurol Scand 1994;89: 12331.
  • 3
    Oldfors A & Lindberg C. Inclusion body myositis. Curr Opin Neurol 1999;12: 52733.
  • 4
    Arahata K, Engel AG. 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. Ann Neurol 1984;16: 193208.
  • 5
    Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies. III: immunoelectron microscopy aspects of cell-mediated muscle fiber injury. Ann Neurol 1986;19: 11225.
  • 6
    Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in myopathies. IV: Cell-mediated cytotoxicity and muscle fiber necrosis. Ann Neurol 1988;23: 16873.
  • 7
    Mendell JR, Sahenk Z, Gales T, Paul L. Amyloid filaments in inclusion body myositis. Novel findings provide insight into nature of filaments. Arch Neurol 1991;48: 122934.
  • 8
    Askanas V, Engel WK. Inclusion-body myositis and myopathies: different etiologies, possibly similar pathogenic mechanisms. Curr Opin Neurol 2002;15: 52531.
  • 9
    Dalakas MC. Myosites a inclusions: mecanismes etiologiques. Rev Neurol (Paris) 2002;158: 94858.
  • 10
    Pruitt JN, Showalter CJ, Engel AG. Sporadic inclusion body myositis: counts of different types of abnormal fibers. Ann Neurol 1996;39: 13943.
  • 11
    Engel AG, Arahata K. Monoclonal antibody analysis of mononuclear cells in myopathies. II: phenotypes of autoinvasive cells in polymyositis and inclusion body myositis. Ann Neurol 1984;16: 20915.
  • 12
    Fyhr IM, Moslemi AR, Tarkowski A, Lindberg C, Oldfors A. Limited T-cell receptor V gene usage in inclusion body myositis. Scand J Immunol 1996;43: 10914.
  • 13
    Lindberg C, Oldfors A, Tarkowski A. Restricted use of T cell receptor V genes in endomysial infiltrates of patients with inflammatory myopathies. Eur J Immunol 1994;24: 265963.
  • 14
    O'Hanlon TP, Dalakas MC, Plotz PH, Miller FW. The αβ T-cell receptor repertoire in inclusion body myositis – diverse patterns of gene expression by muscle-infiltrating lymphocytes. J Autoimmun 1994;7: 32133.
  • 15
    Fyhr IM, Moslemi AR, Mosavi AA, Lindberg C, Tarkowski A, Oldfors A. Oligoclonal expansion of muscle infiltrating T cells in inclusion body myositis. J Neuroimmunol 1997;79: 1859.
  • 16
    Fyhr IM, Moslemi AR, Lindberg C, Oldfors A. T cell receptor beta-chain repertoire in inclusion body myositis. J Neuroimmunol 1998;91: 12934.
  • 17
    Bender A, Behrens L, Engel AG, Hohlfeld R. T-cell heterogeneity in muscle lesions of inclusion body myositis. J Neuroimmunol 1998;84: 8691.
  • 18
    Griggs RC, Askanas V, DiMauro S et al. Inclusion body myositis and myopathies. Ann Neurol 1995;38: 70513.
  • 19
    Moss PAH, Bell JI. Sequence analysis of the human alpha beta T-cell receptor CDR3 region. Immunogenetics 1995;42: 108.
  • 20
    Mantegazza R, Andreetta F, Bernasconi P et al. Analysis of T-cell receptor repertoire of muscle-infiltrating T-lymphocytes in polymyositis. Restricted V-alpha/beta rearrangements may indicate antigen-driven selection. J Clin Invest 1993;91: 28806.
  • 21
    O'Hanlon TP, Dalakas MC, Plotz PH, Miller FW. Predominant TCR-αβ variable and joining gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies. J Immunol 1994;152: 256976.
  • 22
    Bender A, Ernst N, Iglesias A, Dornmair K, Wekerle H, Hohlfeld R. T cell receptor repertoire in polymyositis: clonal expansion of autoaggressive CD8+ T cells. J Exp Med 1995;181: 18638.
  • 23
    Bernasconi P, Confalonieri P, Andreetta F, Baggi F, Cornelio F, Mantegazza R. The expression of co-stimulatory and accessory molecules on cultured human muscle cells is not dependent on stimulus by pro-inflammatory cytokines: relevance for the pathogenesis of inflammatory myopathy. J Neuroimmunol 1998;85: 528.
  • 24
    Murata K, Dalakas MC. Expression of the costimulatory molecule BB-1, the ligands CTLA-4 and CD28, and their mRNA in inflammatory myopathies. Am J Pathol 1999;155: 45360.
  • 25
    Behrens L, Kerschensteiner M, Misgeld T, Goebels N, Wekerle H, Hohlfeld R. Human muscle cells express a functional costimulatory molecule distinct from B7.1 (CD80) and B7.2 (CD86) in vitro and in inflammatory lesions. J Immunol 1998;161: 594351.
  • 26
    Dahlbom K, Lindberg C, Oldfors A. Inclusion body myositis: morphological clues to correct diagnosis. Neuromuscul Disord 2002;12: 8537.
  • 27
    Koffman BM, Sivakumar K, Simonis T, Stroncek D, Dalakas MC. HLA allele distribution distinguishes sporadic inclusion body myositis from hereditary inclusion body myopathies. J Neuroimmunol 1998;84: 13942.
  • 28
    Garlepp MJ, Laing B, Zilko PJ, Ollier W, Mastaglia FL. HLA associations with inclusion body myositis. Clin Exp Immunol 1994;98: 405.
  • 29
    Rider LG, Gurley RC, Pandey JP et al. Clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy. Arthritis Rheum 1998;41: 7109.
  • 30
    Lowin B, Hahne M, Mattmann C, Tschopp J. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 1994;370: 6502.
  • 31
    Orimo S, Koga R, Goto K et al. Immunohistochemical analysis of perforin and granzyme a in inflammatory myopathies. Neuromuscul Disord 1994;4: 21926.
  • 32
    Goebels N, Michaelis D, Engelhardt M et al. Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis. J Clin Invest 1996;97: 290510.
  • 33
    Behrens L, Bender A, Johnson MA, Hohlfeld R. Cytotoxic mechanisms in inflammatory myopathies. Co-expression of Fas and protective Bcl-2 in muscle fibres and inflammatory cells. Brain 1997;120: 92938.
  • 34
    Fyhr IM, Oldfors A. Upregulation of Fas/Fas ligand in inclusion body myositis. Ann Neurol 1998;43: 12730.
  • 35
    Fyhr IM, Lindberg C, Oldfors A. Expression of Bcl-2 in inclusion body myositis. Acta Neurol Scand 2002;105: 4037.
  • 36
    Hutchinson DO. Inclusion body myositis: abnormal protein accumulation does not trigger apoptosis. Neurology 1998;51: 17425.
  • 37
    Schneider C, Gold R, Dalakas MC et al. MHC class I-mediated cytotoxicity does not induce apoptosis in muscle fibers nor in inflammatory T cells: studies in patients with polymyositis, dermatomyositis, and inclusion body myositis. J Neuropathol Exp Neurol 1996;55: 12059.
  • 38
    Sugiura T, Murakawa Y, Nagai A, Kondo M, Kobayashi S. Fas and Fas ligand interaction induces apoptosis in inflammatory myopathies: CD4+ T cells cause muscle cell injury directly in polymyositis. Arthritis Rheum 1999;42: 2918.
  • 39
    De Bleecker JL, De Paepe B, Vanwalleghem IE, Schröder JM. Differential expression of chemokines in inflammatory myopathies. Neurology 2002;58: 177985.
  • 40
    Lundberg IE & Nyberg P. New developments in the role of cytokines and chemokines in inflammatory myopathies. Curr Opin Rheumatol 1998;10: 5219.
  • 41
    Oldfors A, Lindberg C. Inclusion body myositis. In: ChandraP, ed. Advances in Clinical Neurosciences. Ranchi: The Catholic Press, 2001, 31533.
  • 42
    Kim G, Tanuma N, Kojima T et al. CDR3 size spectratyping and sequencing of spectratype-derived TCR of spinal cord T cells in autoimmune encephalomyelitis. J Immunol 1998;160: 50913.
  • 43
    Amemiya K, Granger RP, Dalakas MC. Clonal restriction of T-cell receptor expression by infiltrating lymphocytes in inclusion body myositis persists over time: studies in repeated muscle biopsies. Brain 2000;123: 20309.