Dermatomyositis

DM is a rare multisystemic autoimmune disease that affects children and adults of both genders and all ethnic groups (Table 1). It primarily involves skin and skeletal muscle. Cutaneous manifestations may precede the onset of myositis by several months or up to 2 years and more; Gottron's papules, heliotrope rash, and macular erythemas are the most typical manifestations.⁵ Skin lesions can be worsened by UVA and UVB light; this increased photosensitivity may be due to a polymorphism in tumor necrosis factor-α (TNF-α)-308A allele, detected with high frequency in adult and juvenile DM Caucasian patients (reviewed in ref. 5). Muscle weakness can vary from mild to severe (quadriparesis). Clinical manifestations other than those involving muscle tissue can occur: subcutaneous calcifications, joint contractures, dysphagia, fever, malaise, weight loss, arthralgia, Raynaud's phenomenon, tumor.^1,2 DM diagnosis is confirmed by muscle biopsy (see paragraph regarding histopathology).

Table 1

Idiopathic inflammatory myopathies: clinical features.

Polymyositis

PM, as a difference with DM, has less distinguished clinical features (Table 1).^1,2 However, PM can be suspected in all cases presenting as a subacute proximal myopathy without evidence of inherited transmission. Incidence and prevalence are reported to be similar to those of DM, but PM is extremely rare in infancy. Female to male ratio is 3:1. The clinical course of PM is usually subacute. In the typical affected adult patient anamnesis is negative for: cutaneous symptoms, involvement of ocular and facial muscles, presence of hereditary muscular diseases and exposure to myotoxic drugs or toxins. Onset of the disease can be difficult to ascertain because a subclinical disease may persist over months before the patients refer to the physician. Apart from cutaneous alterations, the degree of severity and distribution of muscle weakness and wasting are similar to those described for DM, except for myalgia and muscle tenderness, which are less frequent than in DM.

Inclusion Body Myositis

IBM has clinical-pathological features well differentiated from PM or DM (Table 1).^1,3 IBM is tipically a chronic evolutive muscle disorders whose onset is usually after the age of 50. Because onset is extremely insidious and disease course so slow, the time of beginning and the incidence of the disease is very difficult to establish. IBM is more frequent in males (male to female ratio 3:1) and in whites than in blacks. Muscle weakness and atrophy affect more frequently distal muscles: deficit of the foot extensors might be evident in more than 50% of the cases and represent the clue of early diagnosis. Selective involvement of triceps, biceps, ileopsoas and quadriceps is frequently evident and responsible for sudden falls of these patients. A noticeable evidence of asymmetric involvement of muscles is a typical feature of IBM. Tendon reflexes are usually lost and because of distal atrophy and weakness a neurogenic disease can be misdiagnosed. Though IBM is considered an acquired IM, familial cases have been described, some associated with leukoencephalopathy. An empyrical criterion to suspect IBM is the unresponsiveness to immunosuppressive therapy of suspected PM patients.

Major Histocompatibility Complex (MHC)

As for other autoimmune diseases, a strong association between human leukocyte antigen (HLA) genes and all clinical forms of IIM has been found (Table 2). At first the genetic marker associated with IIM was HLA B8 (studied in patients with juvenile DM), then the studies were extended including a large number of patients and the major genetic risk factors for the development of myositis were identified in HLA DRB1^*0301 and DQA1^*0501 in whites.⁷ DRB1^*0301 is a common genetic risk factor for familial and sporadic IIM, but contributes in a lesser extent in the familial IIM; while, the unique genetic risk factor to familial IIM is homozygosity at the HLA-DQA1 locus.⁸ Hausmanowa-Petrusewicz et al⁹ reported that the HLA-DRB1^*0301; DQA1^*0501 haplotype was found to be significantly increased in Polish IIM population as a whole and in those IIM patients positive for anti-synthetase, anti-PM-Scl, and anti-Ku autoantibodies. Other groups observed that HLA-DRB1^*0301 (DR3), DQA1^*0501, and DQB1*0201 (DQ2) alleles were each increased in white patients with myositis, especially those with PM, and most strikingly in those with myositis-specific autoantibodies. In other ethnic groups, except the Japanese, only frequencies of HLA-DQA1^*0501 and the structurally similar DQA1^*0401 alleles were significantly increased and most significantly associated with anti-Jo-1, anti-PL-12, and other autoantibodies, compared with IIM patients without autoantibodies. HLA-DQA1^*0102 and ^*0103 alleles predominated in those IIM patients, including Japanese, positive for myositis-specific antibodies but negative for HLA-DQA1^*0501 and ^*0401. A negative association of the HLA-DR2 alleles (DRB1^*1501 and ^*1503) with PM but not with DM was found.¹⁰

Recently, HLA I and II haplotypes have been analyzed in a cohort of s-IBM patients:¹¹ the previously mentioned association with B8 and DR3^7,8,10 was detected and a new HLA association, A^*03, DQ5/DQB1^*05, was observed.¹² Three hypothesis about the cause of HLA association with s-IBM were put forward by the authors: (1) s-IBM is caused by a viral infection (even if in s-IBM it appears unlikely); (2) s-IBM is an autoimmune disease (putative antigens can be auto and viral antigens); (3) s-IBM is due to genes, so far unidentified, in linkage disequilibrium with HLA alleles (for example, in MHC locus are located genes for TNF-α and β, the complement factors 2 and 4, heat shock protein 70).¹¹

The importance of HLA molecules in the pathogenesis of IIM is strongly supported by the observation that, while normal muscle fibers do not express MHC class I molecules on their surface, IIM muscle fibers are strongly positive for MHC class I and class II expression, even in cells apparently distant from cell infiltrates.^1,12,13 It remains to be elucidated whether MHC molecule expression is induced by infectious agents or by proinflammatory cytokines^14,15 or by a nonspecific response to tissue injury and regeneration. Lundberg et al¹⁶ observed that in chronic PM and DM clinical symptoms persist even in the absence of inflammatory infiltrates together with an increased expression of IL-1α in the capillaries and MHC class I on muscle fibers, mainly confined to type II muscle fibers.¹⁷ The authors hypothesized that infiltrating cells might not be the primary factors of muscle damage. As observed in an animal model,¹⁸ overexpression of IL-1α and MHC class I might be sufficient to induce clinical myositis, muscle damage and eventually muscle inflammation.

In vitro myoblasts and myotubes constitutively express low levels of MHC class I and adhesion molecules such as LFA-3. After myoblast stimulation with IFN-γ or IFN-γ and TNF-α increased expression of MHC class I and de novo synthesis of MHC class II and ICAM-1 has been observed.^19-25 When myoblasts are allowed to fuse into myotubes and these cells are innervated MHC class II molecules disappear on cell surface even after IFN-γ stimulation, suggesting that MHC class II synthesis is developmentally regulated during myogenesis and that overexpression of this molecule on pathological muscle fibers might be independent by proinflammatory cytokine production.²²

In the last years, it is emerging the role of non classic MHC class I molecules, in particular of HLA-G, in IIM pathogenesis. This molecule is similar to the MHC class I (β2-microglobulin association, CD8 binding, presentation of a restricted peptide repertoire) but with peculiar characteristics: less polymorphic, highly restricted tissue distribution, seven different isoforms (membrane-associated, HLA-G1, -G2, -G3, -G4, and soluble, HLA-G5, -G6, -G7).²⁶ HLA-G is a key molecule in fetal-maternal tolerance and in the adult life protects target cells from cytotoxic T and natural killer cell attack.²⁷ Normally muscle fibers do not express HLA-G, while a highly positive signal has been observed on IIM muscle fibers, also positive for MHC class I molecules, and on many inflammatory cells.²⁸ Moreover, the authors demonstrated that in vitro IFN-γ was able to up regulate mRNA transcripts corresponding to different isoforms of HLA-G and their surface expression in cultured myoblasts isolated from control subjects and patients.²⁸ Transfection of myoblasts or muscle cell line (TE671) with HLA-G molecules (HLA-G1 and -G5) rendered these cells resistant to alloreactive lysis, reduced alloproliferation, interfered with priming of antigen-specific cytotoxic T cells or inhibited antigen-specific effector lysis.²⁹ In inflammatory myopathies and in other conditions of inflamed muscles (e.g., myoblast transplantation, vaccination) HLA-G might be a muscle cell effort to protect themselves from immune cell-mediated attack.³⁰

Besides MHC class I and II molecules, costimulatory molecules are necessary to stimulate T lymphocytes. Three different costimulatory pathways have been discovered: the B7-1/B7-2 (CD80/CD86) and their receptors CD28/CTLA-4, the best characterized; the inducible costimulatory ligand (ICOSL) and its receptor ICOS (a T cell specific costimulatory molecule homologous to CD28/CTLA4); the receptor PD-1 (programmed death gene 1), which interacts with two novel B7 family members, PD-L1 (B7-H1) and PD-L2 (B7-DC). All these coreceptors can enhance or attenuate T cell activation.³¹

Muscle fibers do not express constitutively or under pro-inflammatory stimuli, detectable levels of CD80/CD86 molecules both in vivo and in vitro.^32-35 Nevertheless, they are able to activate antigen-specific T cell response.³⁶ It is not yet clear whether they are able to prime naïve T cells. Other molecules have been postulated to be expressed on muscle fibers such as a yet unidentified B7-related protein (BB-1) that interacts with CD28/CTLA4 and stimulates T lymphocytes.^34,35 Recently, important advances in the field of muscle capacity to stimulate an immune response have been obtained by analyzing the costimulatory pathways alternative to CD80/CD86-CD28/CTLA4 pathway. ICOSL was expressed at low levels on muscle fibers and to be up regulated in IIM muscle tissue, in particular in PM, on the muscle fibers surrounded and invaded by T lymphocytes ICOS⁺³⁷ (a marker of T cell activation).³⁸ In DM, a strong positivity for ICOSL was observed on endothelial cells of blood vessels.³⁷ Furthermore, ICOSL was observed on cultured myoblasts in basal condition and enhanced after TNF-α stimulation. Cocultures of MHC class II⁺ myoblasts with CD4⁺ T cells together with superantigen demonstrated that ICOSL is active since it modulates Th1 and Th2 cytokine synthesis by activated T cells.³⁷ These observations paralleled those of B7-H1 molecule. This protein was expressed in IIM muscle biopsies and not in normal or nonmyopathic muscle tissues; it was localized to areas of strong inflammation either on muscle and mononuclear cells.³⁹ In vitro myoblasts became positive for B7-H1 only after IFN-γ stimulation. Anti-B7-H1 monoclonal antibody strongly augmented the Th1 and Th2 cytokines in cocultures of IFN-γ stimulated myoblasts, CD4⁺ or CD8⁺ T cells and superantigens.³⁹ The authors speculate that B7-H1 could interfere with the activity of cytotoxic T cells and, hence, this expression is another effort of muscle fiber to protect itself from the autoimmune attack.³⁹

T Cell Receptor (TCR)

T lymphocytes recognize the antigen, presented by the MHC class I or II, via T cell receptor (TCR), a heterodimer composed by two chains, α/β or γ/δ (less frequent), encoded by different gene families combined to form the variable (V), diversity (D), joining (J) and constant (C) regions.^40,41 The contact point between TCR and the antigen-MHC complex lies in the complementarity-determining region 3 (CDR3), composed by the V-(D)-J combination. If TCR recognizes an antigen the amino acid sequence of the CDR3 region should be conserved in the recruited T cells.^40,41 In PM and s-IBM patients, but not in DM, T lymphocytes with a restricted TCR repertoire are recruited from the blood stream to the muscle tissue. Sequence analysis of the TCR families revealed a restricted use of Jβ genes and a CDR3 consensus motif. These data are suggestive of the presence of a conventional antigen on muscle fibers, which attracts specifically CD8⁺ T cells.^42-50 In selected s-IBM patients the TCR repertoire has been analyzed in sequential muscle biopsies during a period of 19-22 months.⁵¹ A persistent clonal expansion of CD8⁺ T cells with the same TCRBV families and a persistent CDR3 amino acid sequence were observed, supporting the hypothesis that endomysial T cells are recruited by a continuous presentation by muscle fibers of the same antigen(s), even in the late stages of the disease.⁵¹ Analysis of peripheral T cells from IIM patients and age-matched controls showed in the patients a more frequent CD8⁺ T cell clonal expansion than CD4⁺ T cells. The expanded T cells persisted as large populations over time and some of the expanded clones were found in the affected muscles from the same patients.⁵² These results provide the evidence that a local autoimmune reaction can directly influence the periphery. Moreover, to have the possibility to isolate pathological T cell clones from the periphery will be of great help in understanding the evolution of the disease and the efficacy of specific therapeutic treatments. Benveniste et al⁵³ demonstrated that TCR repertoire was perturbated in the peripheral blood of PM patients but not of DM patients. Analysis of TCR repertoire in the periphery might be useful in differential diagnosis between PM and DM. However, a study like this does not allow proving that clonally expanded T cells are those that invade the single nonnecrotic muscle fibers. The use of a laser microdissector has overcome this problem. Hofbauer et al⁵⁴ combined CDR3 spectratyping analysis with single cell PCR performed on cells localized in direct contact with the muscle fiber and isolated by laser microdissector. With this approach they were able to identify and track autoaggressive T lymphocytes. It is accepted that in PM/IBM specific antigens, presented by muscle fibers, recruit T cells, what is the antigen remains a mistery.

Myositis-Specific Autoantibodies

Most of IIM patients' sera, approximately 50%, are positive for myositis-specific autoantibodies (MSAs). The targeted antigens are not specific for muscle tissue, the majority of them are aminoacyl-tRNA synthetases, components of the signal recognition particle, translation factors, components of a nucleosome remodelling complex (for a comprehensive review see ref. 55). MSAs are associated with specific clinical characteristics, for example anti-Jo-1 (anti-histidyl tRNA synthetase, HisRS) antibodies and the antisynthetase syndrome (DM or PM, idiopathic interstitial lung disease, arthritis and Raynaud phenomenon).⁵⁵ The exact role of MSAs in IIM immunopathogenesis is still unknown. Nagaraju et al¹⁸ demonstrated that conditional overexpression of MHC class I molecules in the skeletal muscles of young mice was able to induce an inflammatory disease, limited to skeletal muscles, self-sustaining, more severe in females, and often accompanied by autoantibodies, including, in some mice, anti-Jo-1 autoantibodies (the most frequent antibodies in IIM patients, 15-25% of cases). The authors suggested that an apparently non specific event, such as the up-regulation of MHC class I in a tissue, might generate a highly specific autoimmune disease and that specific autoantibodies derive not from the specificity of the stimulus, but from the context, location, and probably the duration of the stimulus.

The majority of autoantigens, including the aminoacyl-tRNA synthetases, targets of the immune attack in different systemic autoimmune diseases, have in their sequence the cleavage site recognized by granzyme B, a highly specific protease released by activated immune cells that cuts target molecule after aspartate residues.^56-58 Nonautoantigens are refractory to granzyme B cleavage.⁵⁷ The HisRS is cleaved by granzyme B in the N-terminal domain and the presence of anti-Jo-1 antibodies inhibited the granzyme B cleavage, suggesting that the immunodominant epitope and the cleavage site are very close.⁵⁸ HisRS and asparaginyl-tRNA synthetase (AsnRS) have chemoattractant properties versus CCR3- and CCR5-expressing cells, and can recruit immature dendritic cells.⁵⁹ Moreover, T cells isolated from peripheral blood of PM patients, positive for anti-Jo-1 antibodies, and from control subjects proliferated in response to Jo-1 full-length, or peptides, in the presence of dendritic cells with a predominant response versus the N-terminal domain (the dominant B cell epitope). This response was MHC class II dependent.⁶⁰ Altogether these results suggest an active role of aminoacyl-tRNA synthetase in initiating and perpetuating the immune response within IIM muscle tissue. A still unknown event (e.g., viral infection) in the appropriate host might damage muscle tissue, aminoacyl-tRNA synthetases might undergo conformational changes becoming susceptible to granzyme B cleavage and be released in the microenvironment. The fragmented aminoacyl-tRNA synthetase might recruit mononuclear cells initiating a cascade of immune events such as antigen presentation to T lymphocytes, production of B cell stimulating cytokines that results in autoantibody synthesis, further muscle damage via release of cytotoxic enzymes.^58,59

Cytokines and Chemokines

Cytokines play a crucial role in inflammatory reaction. These molecules are soluble, short-lived proteins produced, constitutively or under proper stimulation, by several cell types.⁶¹ In muscle biopsies of patients with IIM several cytokines can be amplified or immunolocalized: interleukin (IL)-1α and 1β, IL-2, IL-6, IL-10, TNF-α, IFN-γ, TGF-β e GM-CSF.^62-67 Some of them might play an important role in the pathogenesis of IIM, in particular IL-1α, TNF-α and TGF-β. IL-1α was mainly expressed in endomysial capillaries, in perifascicular arterioles and venules, even in the absence of inflammation,¹⁶ and in in vitro experiments it influenced MHC expression on cultured human myoblasts and myotubes, suggesting that an altered muscle metabolism can cause an eventually immune response.⁶⁸ TNF-α, an important mediator of inflammation and cellular immune responses, was occasionally expressed in mononuclear cells and on muscle fibre membranes.^66,67,69 A proportion of TNF-α positive fibers were also positive for the developmental form of myosin heavy chain, indicating that TNF-α might implicated also in the regenerative process and that muscle fibers can be the target of infiltrating cells, but also an active player in the immune response.⁶⁹ In muscle fibers of juvenile DM (JDM) patients TNF-α was higher expressed in those patients positive for TNF-α-308A allele than in JDM patients negative for the allele. It has been hypothesized that TNF-α-308A allele influencing the overproduction of the cytokine in response to the stimulus, contributes to the chronicity of the disease and, if not treated, to the formation of calcifications (for a comprehensive review, see ref. 70). TGF-β1 was immunolocalized in extracellular matrix of IIM muscle biopsies and never in correspondence of mononuclear cell infiltrates.⁷¹ TGF-β1, linked to the extracellular matrix, might contribute to the recruitment of mononuclear cells within the muscle, since it increases the adhesiveness of endothelial cells for leukocytes, inhibits E-selectin expression in endothelial cells⁷² and induces the chemokine monocyte chemoattractant protein (MCP)-1/CCL2 synthesis.⁷³

Chemokines are chemotactic cytokines that regulate leukocyte migration into inflamed area, as well as homeostatic trafficking of lymphocytes and dendritic cells. Their primary structure is characterized by the presence of four conserved cysteine residues. The largest and best-characterized families are the α-chemokines (CXCL) and the β-chemokines (CCL).⁷⁴ Several chemokines (CCL2, CCL3, CCL5, CCL9, CXCL8, CXCL9, CXCL10) have been detected in IIM muscle tissue localized in correspondence of infiltrating inflammatory cells and in the extracellular matrix with a pattern of distribution related to the different pathogenetic processes underlying the three IIM forms.^75-77 Chemokine synthesis and storage in the extracellular matrix can act as a microenviromental factor amplifying lymphocyte activation and migration, thereby maintaining the autoimmune attack and tissue degeneration.

Muscle cells might be actively involved in chemokine synthesis and release in the inflamed area. It has been demonstrated that CXCL8, CCL5 were constitutively expressed by cultured myoblasts and enhanced after pro-inflammatory stimulus;⁷⁸ CCL2, CXCL9, CXCL10 were induced by IFN-γ or TNF-α stimulus.^77,78 These data further support the hypothesis that muscle cells are not only the target of the immune-mediated attack but that they may directly release cytokines/chemokines necessary to initiate and perpetuate immunocompetent cell recruitment.

Mechanisms of Muscle Cell Damage

Degeneration and necrosis of muscle fibers by CD8⁺ T lymphocytes in PM and s-IBM is predominantly mediated by release of cytotoxic enzymes: perforin and granzymes.

When cytotoxic T cells recognize the antigen via TCR, the lytic granules polarise towards the interface with the target cell (the immunological synapse), fuse with the target cell plasma membrane, and focally release soluble lytic proteins (including perforin and granzymes) to induce target cell death.⁷⁹ In DM and PM perforin and granzyme transcripts were expressed at similar levels and either CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells were perforin positive.^62,80 By confocal microscopy, in DM perforin was distributed randomly in the cytoplasm of the inflammatory T cells, while in PM the cytotoxic T cells that contacted a muscle fiber showed perforin located vectorially towards the target muscle cell. This suggests that in DM perforin distribution reflects a nonspecific T cell activation, while in PM the oriented perforin distribution reflects a specific T cell activation by an antigen present on muscle fibers.⁸⁰

The Fas-FasL process does not seem to be involved in IIM muscle degeneration. IIM muscle fibers and T lymphocytes, but not the control muscles, are Fas positive and FasL has been observed on some degenerating/regenerating fibers and on most of infiltrating CD8⁺ T cells, however, apoptotic signs are absent.^81-86 The resistance to Fas-mediated cell death seems to be due to the expression of anti-apoptotic molecules heterogeneously expressed in muscle fibers: Bcl-2, Fas-associated death domain-like IL-1-converting enzyme inhibitory protein (FLIP), which inhibits Fas-mediated death signaling, and human inhibitor of apoptosis (IAP)-like protein (hILP), which inhibits the activity of caspases, all proteins that play an important role in initiating and maintaining the apoptotic process.^81,83,87

Gene Expression Profiles

As yet a limited experience on gene expression profile of IIM muscle biopsies by microarray technology has been reported. The study from Greenberg et al⁸⁸ showed that muscle tissues from IIM patients expressed genes different from the normal muscles and that these gene profiles were diverse among the different IIM forms. With this approach the authors had the possibility to make diagnosis in two patients for whom the muscle biopsy did not show the classical pathological alterations. Several genes (MHC class I and II, cytokines, chemokines, granzyme proteases, adhesion molecules, matrix metalloproteinases) are overexpressed confirming previous data obtained with other approaches (PCR and/or immunohistochemistry). On the other hand, the molecular approach revealed some unexpected results: for example, keeping in mind the histopathology of the single IIM forms, a number of immunoglobulin genes were more abundantly expressed in PM/IBM than in DM, while IFN-inducible genes were more expressed in DM than PM/IBM, the latter result resembles that observed in JDM, where a viral antigen as triggering factor has been hypothesized;⁸⁹ genes reported as relevant for IBM pathogenesis are also significantly overexpressed in PM and DM, suggesting that in IBM the abnormal accumulation of different proteins might be due to post-transcriptional defects.

IBM-Specific Genetics

A characteristic feature of IBM muscle is the abnormal accumulation, aggregation, and misfolding of several proteins, a scenario similar to that observed in Alzheimer disease brain.^3,6 The major accumulated proteins include: amyloid-β precursor protein (AβPP) and amyloid-β; phosphorylated tau in the form of paired helical filaments (PHFs); presenilin-1. Since in s-IBM the disease onset is usually after age 50 years, it has been hypothesized that the abnormal protein accumulation might be due to a defective processing related to muscle fiber aging. The abnormally processed proteins might then make the muscle fiber a “foreign” to be attacked by the immune system. For h-IBM, in which no signs of inflammation are observed, responsible for protein accumulations might be a genetic defect. Candidate genes are: UDP-N-acetylglucosamine-2 epimerase/N-acetylmannosamine kinase (GNE) gene, myosin heavy chain IIa gene, transthyterin (for a comprehensive review see refs. 3,6). GNE is a bifunctional enzyme catalyzing the first two steps in the synthesis of N-acetylneuraminic (sialic) acid. Any dysregulation of sialic acid biosynthesis and distribution could lead to severe abnormalities of glycoconjugate biosynthesis. Missense mutations were identified in h-IBM Iranian Jews, Japanese and few other ethnic groups. The mutations might induce an uncorrect sialation/glycation of one or several muscle proteins, causing their misfolding and eventually abnormal processing. A missense mutation in the myosin heavy chain IIa gene has been reported in Swedish h-IBM; the observation of an overexpression of myosin heavy chain IIa protein in the IBM vacuoles suggests that mutations in myosin heavy chain IIa gene might influence the formation of vacuoles. Transthyterin binds β-amyloid preventing its fibrillar amyloidogenesis; a transthyterin mutation (Val122Ile), found in a patient with h-IBM and cardiac amyloidosis, might cause the abnormal β-amyloid deposits and amyloidogenesis.⁶