- Email: [email protected]
Expression of major histocompatibility complex class I and class II antigens in canine masticatory muscle myositis
Neuromuscular Disorders 17 (2007) 313–320 www.elsevier.com/locate/nmd
Expression of major histocompatibility complex class I and class II antigens in canine masticatory muscle myositis Orlando Paciello a
a,*
, G. Diane Shelton b, Serenella Papparella
a
Department of Pathology and Animal Health – Unit of Anatomic Pathology, University of Naples Federico II, 80137 Naples, Italy b Department of Pathology, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0709, USA Received 21 December 2006; received in revised form 9 January 2007; accepted 19 January 2007
Abstract Studies in human immune-mediated inflammatory myopathies have documented expression of major histocompatibility complex class I (MHC class I) and class II (MHC class II) antigens on muscle fiber membranes in the presence or absence of cellular infiltration. Here we evaluate the presence and distribution of these antigens in canine masticatory muscle myositis, an immune-mediated inflammatory myopathy. Twelve samples of temporalis and masseter muscles from dogs with a clinical diagnosis of canine masticatory muscle myositis were examined by immunohistochemistry and double-immunofluorescence confocal microscopy. MHC class I and class II antigens were expressed in muscle fibers independent of inflammatory cell infiltration. Furthermore MHC class I and class II antigens were expressed on the sarcolemma and co-localized with dystrophin. Our results suggest that MHC class I and class II expression in canine masticatory muscle myositis may play a role in the initiation and maintenance of the pathological condition, rather than just a consequence of a preceding local inflammation. 2007 Elsevier B.V. All rights reserved. Keywords: Canine masticatory muscle myositis; Inflammatory myopathies; MHC class I; MHC class II; Dog
1. Introduction Canine masticatory muscle myositis (CMMM) is an inflammatory myopathy (IM) selectively involving the muscles of mastication (temporalis, masseter, digastricus, and pterygoid muscles) [1–3]. This muscle group contains type 2M fibers, a unique fiber type that is not present in limb muscles. While type 2M fibers have histochemical staining properties similar to those defined for type 2C fibers (i.e., acid stable following preincubation in acid media at pH 4.5 and 4.3), these fibers posses a unique myosin isoform, heavy chain and light chains [4]. Although type 2M fibers are also found in the masticatory muscles of other carnivores [2,3], an inflamma*
Corresponding author. Tel.: +39 0812536466; 0812536186. E-mail address: [email protected] (O. Paciello).
fax:
+39
0960-8966/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2007.01.012
tory myopathy selectively affecting this muscle group has only been identified and fully characterized in the dog [2]. CMMM can affect all breeds of dogs with no age or sex predilection [1,2]. Clinical presentations include an acute painful form with swelling of the masticatory muscles, jaw pain, trismus, and ocular signs (such as exophthalmus, conjunctivitis, and blindness), and a chronic form with progressive muscle atrophy (Fig. 1A and B) with or without restricted jaw mobility. Both forms are characterized histologically by myositis with cellular infiltration and myonecrosis [1,2] (Fig. 1B and C). Although the trigger(s) for CMMM have not yet been established, it is well known that this is an immune-mediated disease. CMMM is very responsive to corticosteroid therapy and circulating autoantibodies directed against type 2M fibers are found in most cases [3,5]. Although the clinical presentations are distinctly
314
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
Fig. 1. Clinical and histological features of canine masticatory muscle myositis (CMMM). (A) Marked atrophy of the temporalis muscles with an evident occipital crest (arrows) is a common clinical presentation of CMMM (case #1). (B) In addition to atrophy of this muscle group, there may be inability to open the jaw (case #9). (C) Diffuse infiltration of mixed mononuclear cells into masticatory muscles, with invasion of non-necrotic fibers and myonecrosis, is typical of acute CMMM (case #1). (D) In chronic cases, perimysial (asterisk) and endomysial fibrosis (not shown), and variability in myofiber size may be found with no detectable inflammatory cells (case #9). (Temporalis muscle, H&E stain, Original magnification 20· for both C and D.)
different, histologic and immunophenotypic similarities in muscle biopsy specimens between human dermatomyositis (DM) and CMMM have been described [6–8]. Although an angiopathy has not yet been identified in CMMM, perivascular accumulations of inflammatory cells are common [6]. In human IMs, expression of MHC class I and class II molecules have been of interest because they may be associated with a drastically changed potential to participate in immunological reactions related to T cell activation and anergization [7–9]. Muscle fibers do not normally express MHC class I and class II antigens, but in human IM, widespread over-expression of MHC class I and class II has been found even in areas remote from the inflammation [9,10]. These findings suggest that muscle cells may play an active role in the immune response [7] and are of critical importance in pathogenesis. In addition, localization of MHC class I and class II molecules may be important in the diagnosis of inflammatory myopathies (IM) as cellular infiltrates may be missed on individual biopsy specimens [9–11]. Considering the potentially important roles of MHC class I and class II expression on muscle cells in the pathogenesis of disease [7–9] and their possible use in the diagnosis of IMs in both animals and humans, we investigated MHC class I and class II expression in canine MMM using confocal
laser scanning microscopy in parallel with conventional immunohistology. 2. Materials and methods 2.1. Animal groups Biopsies (n = 12) from the temporalis and masseter muscles of dogs with a clinical diagnosis of CMMM were examined. Dogs were further divided into three groups according to the clinical presentation and on the basis of the presence or absence of cellular infiltrates. The clinical data is shown in Table 1. Group 1 (dogs 1–4): Acute onset of clinical signs with swollen and painful masticatory muscles and detectable infiltration of inflammatory cells in the muscle biopsy. The serum CK concentration was mildly elevated in 3 dogs (dogs 1–3) and normal in dog 4. Group 2 (dogs 5–7): Chronic disease with atrophy and muscle pain, and detectable clusters of infiltrating cells in the muscle biopsy. The serum CK concentrations were mildly elevated in all 3 dogs. These dogs may represent relapse of previously active disease, or most likely, a chronic slowly progressive course. Group 3 (dogs 8–12): Chronic stage of disease with severe masticatory muscle atrophy, no detectable masticatory muscle or jaw pain, an absence of inflammatory
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
315
Table 1 Clinical data on dogs with masticatory muscle myositis Dog #
Breed
1 2 3 4 5 6 7 8 9 10 11 12
Dalmatian German Shepherd Rottweiler Mixed German Shepherd German Shepherd Poodle Mixed Rottweiler Mixed German Shepherd Mixed
dog
dog dog
dog
Sex
Age (yr)
CK (<200 IU/L)
Clinical signs
Presence of inflammatory cells in the biopsy
M F F F M M M FS M FS M FS
3 4 8 8 3 6 9 12 7 6 8 5
300 380 260 <200 500 350 307 160 <200 <200 178 <200
Jaw and masticatory muscle pain Masticatory muscle swelling and pain Masticatory muscle swelling and pain Jaw and muscle pain Masticatory muscle atrophy, jaw and muscle pain Masticatory muscle atrophy, jaw and muscle pain Masticatory muscle atrophy, jaw and muscle pain Severe masticatory muscle atrophy and entropion Severe masticatory muscle atrophy and trismus Severe masticatory muscle atrophy Severe masticatory muscle atrophy and trismus Severe masticatory muscle atrophy and trismus
Yes Yes Yes Yes Yes Yes Yes No No No No No
M, male; F, female; FS, female spayed; CK, creatine kinase.
cells in the muscle biopsy and endomysial and perimysial fibrosis. The serum CK concentrations were normal in all dogs in this group. Group 4 (Normal and neuromuscular disease controls). Normal control (n = 3): Temporalis muscles obtained at necropsy from dogs without any known neuromuscular disease were used as controls. Neuromuscular disease controls (n = 6): Archived muscle biopsy specimens stored at 80 C from the temporalis and biceps femoris muscles of dogs with other histologically confirmed neuromuscular diseases were used as controls. Cases were selected based on the presence of generalized muscle disease affecting both the masticatory and limb muscles, and with clinical presentations including muscle atrophy, weakness, and myalgia. Cases included immune-mediated polymyositis (CPM) (2), dystrophin deficient muscular dystrophy (1), hypothyroid myopathy (1), and peripheral neuropathy with muscle atrophy in a neurogenic pattern with fiber type grouping consistent with chronic denervation (2).
2.2. Muscle biopsies Muscle biopsy specimens were frozen in isopentane pre-cooled in liquid nitrogen and stored at 80 C until further processed. Sections (8 lm) were stained by the following histological and histochemical techniques: haematoxylin and eosin (HE), Engel trichrome [12], and the myofibrillar ATPase reactions at pH 9.4 and 4.3. Biopsy samples were sectioned at several levels and then stained by the ATPase reaction to verify the selective involvement of type 2M fibers.
2.3. Immunohistochemical examination Frozen muscle specimens were sectioned (6 lm), fixed in acetone at 4 C for 5 min, then blocked for endogenous peroxidase in 0.3% H2O2 in methanol for 20 min. Muscle sections were then incubated with two carefully selected and well characterized mouse monoclonal antibodies against canine MHC class I (1:100, clone H58A, VMRD, Inc., USA) [13] and canine MCH class II (1:50, clone H34A, VMRD, Inc., USA) [13] for 2 h at room temperature. Slides were washed three times with PBS, then incubated with biotinylated anti-mouse and labeled streptavidin biotin (LSAB kit; DakoCytomation, Denmark) for 30 min, followed by incubation with streptavidin conjugated to horseradish peroxidase (LSAB Kit; DakoCytomation, Denmark). Color development was obtained following 5–20 min of diaminobenzidine (DakoCytomation, Denmark) treatment. Sections were counterstained with Mayer’s haematoxylin. In the corresponding negative control section, the primary antibody was either omitted or replaced with normal mouse serum in place of the monoclonal antibody. 2.4. Immunofluorescence and confocal laser scanning microscopy Muscle sections were air dried, blocked with normal mouse serum diluted 1:10, then rinsed in 0.01 M PBS (pH 7.4) containing 0.2% Triton X-100 and 0.1% bovine serum albumin. Sections were then incubated for 24 h at 4 C with mouse primary monoclonal antibodies against the carboxy-terminus of dystrophin, against spectrin as a control for membrane integrity, and against desmin to identify regenerating fibers. All primary antibodies were obtained from Novocastra Laboratories Ltd, and diluted 1:50 in 1:5 normal goat serum. The sections were then washed in PBS followed by incubation with goat anti-mouse IgG-Fab fragment conjugated to TRITC
316
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
primary antibody against canine MHC class I and II (diluted 1:50 in 1:5 normal rabbit serum, antibody details are above) for 24 h at 4 C. After rinsing in PBS, the sections were treated with affinity-purified rabbit anti- mouse IgG-Fab conjugated to FITC fluorochrome (1:50; Jackson Laboratories), for 1 h at room temperature. Finally, the sections were rinsed with PBS and mounted with glycerin diluted with PBS 1:1. Specificity of the immunoreactivity in double immunolabeling using monoclonal antibodies raised in the same species (Fab fragment method) was tested by omitting the primary antibody in the second staining and using testing procedures recommended by Negoescu et al. [15]. 2.5. Scanning and photography Fig. 2. Temporalis muscle biopsy from a healthy control dog demonstrating that MHC reactivity is present only in endothelial cells (arrows). (Avidin-biotin-peroxidase complex method with Mayer’s hematoxylin counterstain, Original magnification 20·).
fluorochrome (1:50, Jackson Laboratories) for 1 h at room temperature. To avoid antibody cross-reaction in double immunofluorescence staining utilizing two primary mouse antibodies, fluorochrome conjugated Fab fragments were used (Jackson Laboratories) according to the method of Wessel and McClay [14]. The sections were again rinsed in PBS and incubated with the second
For scanning and photography, a confocal laser scanning microscope LSM-510 (Zeiss, Go¨ttingen, Germany) was used. FITC was irradiated at 488 nm and detected via a 505–560 nm band pass filter. TRITC was irradiated at 543 nm and detected with a 560 long pass filter. Two-channel frame-by-frame multi-tracking was used to avoid cross-reacting signals. The different frames were scanned separately, with appropriate installation of the optical path for excitation and emission of each scan according to the manufacturer’s instructions.
Fig. 3. Expression of both MHC class I and MHC class II was present on muscle fibers in CMMM in the presence or absence of cellular infiltration. In biopsies with obvious cellular infiltrations (case #5), there was strong immunoreactivity of MHC class I (A) and MHC class II (B) on muscle fibers (arrows), inflammatory cells (arrow heads), and endothelial cells. In biopsies without obvious cellular infiltration (case #12), many muscle fibers showed less intense but still obvious immunoreactivity for MHC class I (arrows, C) and MHC class II (arrows, D). (Avidin-biotin-peroxidase complex method with Mayer’s hematoxylin counterstain, Original magnifications 20· for all figures.)
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
2.6. Scoring of immunoreactivity Expression of MHC class I and class II was assessed as follows: 0, negative staining of muscle fibers; 1+, positive staining of endothelial cells and 1–25% positively stained fibers; 2+, positive staining of endothelial cells and 26–50% positively stained fibers; 3+, positive staining of endothelial cells and 51–70% positively stained fibers; 4+, positive staining of endothelial cells and 71–100% positively stained fibers.
3. Results 3.1. Normal control tissue The temporalis muscle specimens were histologically normal without obvious cellular infiltrations in all healthy control dogs. MHC class I and class II expression was restricted to endothelial cells of arterioles, venules, and capillaries. No positivity was observed on muscle fibers (Fig. 2). 3.2. MHC class I and class II expression in CMMM and controls Group 1 (dogs 1–4): Acute onset of clinical signs with swollen and painful masticatory muscles and detectable infiltration of inflammatory cells in the muscle biopsy. Mononuclear cell infiltrates having an endomysial, perimysial, and perivascular distribution were present within muscle biopsies from all 4 dogs as previously described (6). Type 2M fibers constituted approximately 80% of the total fiber number. The expression of both MHC class I (Fig. 3a, score 4+) and MHC class II (Fig. 3b, score 3+ to 4+) was evident on the sarcolemma of the majority of muscle fibers (Fig. 3). Immunoreactivity was sometimes noted in the cytoplasm. In addition, inflammatory cells and endothelial cells strongly expressed both MHC class I and MHC class II. No difference in staining intensity between MHC class I and MHC class II was observed. Group 2 (dogs 5–7):Chronic disease with atrophy and muscle pain, and only small detectable clusters of infiltrating cells in the muscle biopsy. In this group, muscle biopsies showed only rare, small groups of lymphocytes. Type 2M fibers constituted approximately 40– 50% of the muscle fiber population. Sarcolemmal immunostaining for MHC class I (Fig. 3c, score 1+ to 3+) and MHC class II (Fig. 3d, score 1+ to 3+) was present not only on the sarcolemma of fibers close to areas of cellular infiltration, but also on the sarco-
317
lemma of fibers distant from the cellular infiltrates. Staining for MHC class I and MHC class II was also present in endothelial cells of capillaries, arterioles, and venules in all biopsies. Group 3 (dogs 8–12): Chronic stage of disease with severe masticatory muscle atrophy, no detectable masticatory muscle or jaw pain, and an absence of inflammatory cells in the muscle biopsy. In this group, type 2M fibers constituted 20–30% of the muscle fiber population. Sarcolemmal immunostaining for MHC class I (Fig. 4A, B and C, score 1+ to 3+) and class II (Fig. 4D, E and F; score 1+ to 3+) was present, but was limited to a lesser number of muscle fibers than in dogs in Groups 1 and 2. Furthermore, sarcolemmal staining was less intense than in CMM with obvious cellular infiltrates. Endothelial and interstitial cells were always positive. Group 4 (Neuromuscular disease controls): Muscle biopsies from 3 healthy dogs and from 6 dogs with other histologically confirmed neuromuscular diseases including polymyositis, dystrophin deficient muscular dystrophy, hypothyroid myopathy, and peripheral neuropathies were similarly tested. The staining pattern for MHC class I and class II antigens on muscle fibers from dogs with dystrophin deficient muscular dystrophy (not shown), hypothyroid myopathy (Fig. 5A and B), and peripheral neuropathy (not shown) was similar to control biopsies from healthy dogs, with no expression of either MHC class I or class II on the muscle fibers. The staining pattern for MHC class I and MHC class II in polymyositis was similar to the expression in CMMM, with both antigens expressed on muscle fiber membranes. MHC class I and MHC class II reactivity was also occasionally noted in the cytoplasm. Both normal and morphologically changed fibers expressed MHC class I and MHC class II antigens independently of the relation to foci of cellular infiltrates. Inflammatory cells and endothelial cells also expressed MHC class I and MHC class II antigens. No difference in staining pattern between PM and CMMM was recorded (Fig. 5C and D, score 4+). 3.3. Confocal laser scanning microscopy Immunofluorescence labeling gave a staining pattern superimposable to that of the immunohistochemical stainings in all groups (not shown). All muscle fiber membranes were positive using a monoclonal antibody against the carboxy-terminus of dystrophin, a membrane protein. Double-immunofluorescence staining analyzed by confocal laser scanning microscopy demonstrated a patchy, uneven distribution of co-localization of MHC class I and dystrophin, and MHC class II and dystrophin (Fig. 4). Double stained fibers were present in areas with or without inflammatory infiltrates. The pattern of co-localization of MHC class I
318
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
Fig. 4. Double immunofluorescence staining analyzed by confocal laser scanning microscopy showed co-localization of both MHC class I and class II with dystrophin. (A and D) Dystrophin localization to the muscle fiber membrane (red fluorescence). (B and E) Localization of MHC class I (B) and MHC class II (E) to the muscle fiber membrane (green fluorescence). Overlapping images demonstrated patchy areas of co-localization of MHC class I and dystrophin (C, yellow color), and MHC class II and dystrophin (F, yellow color). (Original magnifications 100· for all figures.)
Fig. 5. Immunofluorescence staining of MHC class I and class II in neuromuscular disease controls. MHC class I (A) and MHC class II (B) expression in a dog with hypothyroid myopathy was restricted to endothelial cells of arterioles, venules, and capillaries. Expression of MHC class I (C) and MHC class II (D) in a dog with polymyositis was similar to CMMM, with both antigens present on muscle fibers, inflammatory cells, and endothelial cells. (Original magnifications 40· for all figures.)
and spectrin, and MHC class II and spectrin was similar to that of dystrophin. Small numbers of regenerating muscle fibers were identified using the antibody against desmin. These fibers were positive for MHC class I but in all cases negative for MHC class II (not shown).
4. Discussion In this study we demonstrate that muscle fibers in the focal canine inflammatory myopathy CMMM and in the generalized inflammatory myopathy PM, express both MHC class I and MHC class II antigens in the
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
presence or absence of cellular infiltration. Neither healthy muscle or muscle from other non-inflammatory myopathies such as dystrophin deficient muscular dystrophy and hypothyroid myopathy, or in cases of peripheral neuropathy with neurogenic atrophy, showed obvious expression on muscle membranes. This supports the contention that these molecules are induced and not constitutively expressed on muscle fibers. This is also consistent with the findings that under inflammatory conditions with expression of pro-inflammatory cytokines, human muscle cells can express MHC class I as well as MHC class II molecules [16]. Although we did not specifically address the question of whether type 2M fibers specifically expressed MHC antigens in this study, there was an obvious trend of decreased expression of MHC antigens on muscle membranes with loss of type 2M fibers in the acute and chronic groups. In vivo, under physiologic conditions, myofibers do not normally express MHC class I and class II antigens on the cell surface [17], although both antigens are upregulated in various human inflammatory myopathies [17–19]. In dogs, expression of MHC class I has been previously described in a case of canine polymyositis [20], and in a case of myositis associated with neosporosis [21]. Expression of MHC class II has been demonstrated in both CMMM and in canine polymyositis [6]. Expression of these antigens indicates that the muscle fibers are immunologically activated, but the mechanism underlying their expression as well as the consequences are as yet unknown. In both human [16] and canine IMs [5], there is evidence for the presence of antigen processing and presentation within muscle. In a recent microarray study of IMs in dogs, several genes associated with macrophage and dendritic cell function were up-regulated in both CMMM and CPM [5]. These include genes important in activation, migration, and antigen processing, and antigen presentation including cathepsin precursors and cathepsin S proteases, MHC class II-associated invariant chain, MHC class I (DLA-A, beta 2 microglobulin), and MHC class II (DLA-DR, DQ). If DLA-DR and DLA-DQ are expressed on the surface of canine muscle fibers in vivo, the muscle fibers could theoretically present not only viral or bacterial antigens but also muscle autoantigens or alloantigens to CD4+ T cells. The expression of HLA-DR on the surface of muscle fibers in human inflammatory lesions has been inconsistent [9,19]. Whether the expression of MHC on the muscle fiber membranes switches the activation or the anergy in T lymphocytes could depend on the presence of other co-stimulatory molecules such as the B7 family [22,23]. The co-stimulatory molecules CD80 and CD86 were both identified in canine IMs using monoclonal antibodies [5]. MHC expression could be an earlier event than leukocyte infiltration in initiating or maintaining pathological condition in MMM.
319
We also demonstrated that MHC expression can occur independently of the presence of inflammatory cell infiltration. This finding is of diagnostic importance, since cellular infiltrations in inflammatory myopathies can have a patchy or multifocal distribution and be missed on individual biopsy specimens, whereas MHC class I staining is diffuse throughout the sample. Several authors [10,11] suggested that localization of MHC class I is a valid test for the diagnosis of idiopathic inflammatory myopathies when inflammatory cells may be undetectable during short term use of immunosuppressive agents. The serum CK concentration was within the reference range in 6 of 12 dogs with CMMM, in which 5 dogs had the chronic form. Even in dogs with acute CMMM, it is not unusual to find the CK value within the reference range or only mildly elevated (2–3X normal). The amount of muscle mass involved in CMMM is small compared to the entire body mass, so only mild elevations would be expected even with acute disease. In dogs with chronic CMMM, muscle is replaced by fibrous tissue and CK concentrations are usually normal. Although the CK concentration is a sensitive indicator of muscle fiber injury, it is not elevated in all human patients with polymyositis and dermatomyositis [24]. In the two cases of CPM used as controls, the serum CK concentrations were elevated at 1800 and 1010 IU/L (5–10X normal), respectively. Multifocal areas of mixed mononuclear cell infiltrations were present involving multiple muscle groups, and both dogs showed clinical evidence of muscle weakness and stiffness in addition to generalized muscle atrophy. While not specifically addressed in this study, similar to human IMs, the serum CK concentration is not always elevated in cases of canine PM (Shelton, unpublished observations). A normal CK can occur in canine PM when the pathological process is concentrated in the perimysium and fiber necrosis is minimal. Although the data is conflicting, muscle cells, as well as invading lymphocytes and endothelial cells, secrete pro-inflammatory cytokines such as IL-1a, IL-1b, TNF-a, IL-6, IL-2 and IFN-c, but not TGF-b [23]. It is unlikely that induction of MCH expression in IMs occurs in response to local lymphocyte products in muscle without obvious cellular infiltration [25,26]. It is theoretically possible that muscle fibers create a local environment for chronic inflammation by secretion of pro-inflammatory cytokines. Reports have documented an increased expression of pro-inflammatory cytokines such as IL-1a in capillary endothelial cells in IM, and IL-1a has been found in muscle tissue of patients with clinical signs of myositis in the absence of detectable infiltration of inflammatory cells [27]. Pro-inflammatory cytokines have recently been shown to influence MHC expression on human myoblasts and myotubes in
320
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
in vitro experiments [16]. It is theoretically possible that MHC class I and MHC class II are at least partially regulated in response to IL-1a [26]. In the recent microarray study of CMMM and CPM, there was increased expression of several pro-inflammatory cytokine genes in both CMMM and CPM [5]. In conclusion, this study documents the muscle membrane expression of both MCH class I and class II molecules in CMMM and CPM, in the presence or absence of cellular infiltrations. These findings suggest that these glycoproteins may play a crucial role in the pathogenesis of CMMM and PM, and possibly other idiopathic inflammatory myopathies. Further, using double-immunofluorescence staining and confocal laser microscopy, we demonstrate for the first time that MHC class I and class II molecules are expressed on the sarcolemma and co-localize with dystrophin in the presence or absence of cellular infiltrates. Thus, expression of MHC class I and class II molecules in CMMM and CPM is not an artifact caused by shedding of molecules from the contacting inflammatory cells. As suggested by other authors [6,8], this study provides further support for the role of canine IMs as an animal model for the study of many aspects of human IMs. References [1] Podell M. Inflammatory myopathies. Vet Clin North Am Small Anim Pract 2002;32:147–67. [2] Shelton GD, Cardinet G, Bandman E. Canine masticatory muscle disorders: a study of 29 cases. Muscle Nerve 1985;10:783–90. [3] Shelton GD, Cardinet G, Bandman E, Cuddon P. Fiber typespecific autoantibodies in a dog with eosinophilic myositis. Muscle Nerve 1985;8:783–90. [4] Shelton GD, Bandmann E, Cardinet III GH. Electrophoretic comparison of myosins from masticatory muscles and selected limb muscles in the dog. Am J Vet Res 1985;46:493–8. [5] Shelton GD, Hoffman EP, Ghimbovschi S, et al. Immunopathogenic pathways in canine inflammatory myopathies resemble human myositis. Vet Immunol Immunopathol 2006;113:200–14. [6] Pumarola M, Moore PF, Shelton GD. Canine inflammatory myopathy: analysis of cellular infiltrates. Muscle Nerve 2004;29:782–9. [7] Dalakas MC, Hohlfeld R. Polymyositis and dermatomyositis. Lancet 2003;362:1762–3. [8] Dalakas MC. From canine to man: on antibodies, macrophages, and dendritic cells in inflammatory myopathies. Muscle Nerve 2004;29:753–5. [9] Englund P, Lindroos E, Nennesmo I, Klareskog L, Lundberg IE. Skeletal muscle fibers express major histocompatibility complex class II antigens independently of inflammatory infiltrates in inflammatory myopathies. Am J Pathol 2001;159:1263–73. [10] Topaloglu H, Muntoni F, Dubowitz V, Sewry C. Expression of HLA class I antigens in skeletal muscle is a diagnostic marker in juvenile dermatomyositis. J Child Neurol 1997;12:60–3.
[11] Van der Pas J, Hengstman GJ, ter Laak HJ, Borm GF, van Engelen BG. Diagnostic value of MHC class I staining in idiopathic inflammatory myopathies. J Neurol Neurosurg Psychiatry 2004;75:136–9. [12] Engel WK, Cunningham GG. Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections. Neurology 1963;13:919–23. [13] Davis WC, Marusic S, Lewin HA, et al. The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species. Vet Immunol Immunopathol 1987;15:337–76. [14] Wessel GM, McClay DR. Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies. J Histochem Cytochem 1986;34:703–6. [15] Negoescu A, Labat-Moleur F, Lorimier P, et al. F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J Histochem Cytochem 1994;42:433–7. [16] Wiendl H, Hohlfeld R, Kieseier BC. Immunobiology of muscle: advances in understanding an immunological microenvironment. Trends Immunol 2005;26:373–80. [17] Karpati G, Pouliot Y, Carpenter S. Expression of immunoreactive major histocompatibility complex products in human skeletal muscles. Ann Neurol 1988;23:64–72. [18] Appleyard ST, Dunn MJ, Dubowitz V, Rose ML. Increased expression of HLA ABC class I antigens by muscle fibres in Duchenne muscular dystrophy, inflammatory myopathy, and other neuromuscular disorders. Lancet 1985;1:361–3. [19] Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibility complex class I antigen expression, immunolocalization of interferon subtypes, and T cell-mediated cytotoxicity in myopathies. Hum Pathol 1989;20:224–31. [20] Morita T, Shimada A, Yashiro S, et al. Myofiber expression of class I major histocompatibility complex accompanied by CD8+ T-cell-associated myofiber injury in a case of canine polymyositis. Vet Pathol 2002;39:512–5. [21] Paciello O, D’Orazi A, Borzacchiello G, Martano M, Restucci B, Maiolino P, Papparella S. Expression of Major Histocompatibility Complex Class I and II in a case of Neospora caninum myositis in a dog. Acta Myol 2004;23:151–3. [22] Figarella-Branger D, Civatte M, Bartoli C, Pellissier JF. Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies. Muscle Nerve 2003;28:659–82. [23] Marino M, Scuderi F, Mannella F, Bartoccioni E. TGF-b1 and IL-10 modulate IL-1b-induced membrane and soluble ICAM-1 in human myoblasts. J Neuroimmunol 2003;134:151–7. [24] Engel AG, Hohlfeld R. The polymyositis and dermatomyositis syndromes. In: Myology. McGraw-Hill; 2004. [25] Isenberg DA, Rowe D, Shearer M, Novick D, Beverley PC. Localization of interferons and interleukin 2 in polymyositis and muscular dystrophy. Clin Exp Immunol 1986;63:450–8. [26] Kalovidouris AE. The role of cytokines in polymyositis: interferon-gamma induces class II and enhances class I major histocompatibility complex antigen expression on cultured human muscle cells. J Lab Clin Med 1992;120:244–51. [27] Nyberg P, Wikman AL, Nennesmo I, Lundberg I. Increased expression of interleukin 1alpha and MHC class I in muscle tissue of patients with chronic, inactive polymyositis and dermatomyositis. J Rheumatol 2000;27:940–8.
Expression of major histocompatibility complex class I and class II antigens in canine masticatory muscle myositis Orlando Paciello a
a,*
, G. Diane Shelton b, Serenella Papparella
a
Department of Pathology and Animal Health – Unit of Anatomic Pathology, University of Naples Federico II, 80137 Naples, Italy b Department of Pathology, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0709, USA Received 21 December 2006; received in revised form 9 January 2007; accepted 19 January 2007
Abstract Studies in human immune-mediated inflammatory myopathies have documented expression of major histocompatibility complex class I (MHC class I) and class II (MHC class II) antigens on muscle fiber membranes in the presence or absence of cellular infiltration. Here we evaluate the presence and distribution of these antigens in canine masticatory muscle myositis, an immune-mediated inflammatory myopathy. Twelve samples of temporalis and masseter muscles from dogs with a clinical diagnosis of canine masticatory muscle myositis were examined by immunohistochemistry and double-immunofluorescence confocal microscopy. MHC class I and class II antigens were expressed in muscle fibers independent of inflammatory cell infiltration. Furthermore MHC class I and class II antigens were expressed on the sarcolemma and co-localized with dystrophin. Our results suggest that MHC class I and class II expression in canine masticatory muscle myositis may play a role in the initiation and maintenance of the pathological condition, rather than just a consequence of a preceding local inflammation. 2007 Elsevier B.V. All rights reserved. Keywords: Canine masticatory muscle myositis; Inflammatory myopathies; MHC class I; MHC class II; Dog
1. Introduction Canine masticatory muscle myositis (CMMM) is an inflammatory myopathy (IM) selectively involving the muscles of mastication (temporalis, masseter, digastricus, and pterygoid muscles) [1–3]. This muscle group contains type 2M fibers, a unique fiber type that is not present in limb muscles. While type 2M fibers have histochemical staining properties similar to those defined for type 2C fibers (i.e., acid stable following preincubation in acid media at pH 4.5 and 4.3), these fibers posses a unique myosin isoform, heavy chain and light chains [4]. Although type 2M fibers are also found in the masticatory muscles of other carnivores [2,3], an inflamma*
Corresponding author. Tel.: +39 0812536466; 0812536186. E-mail address: [email protected] (O. Paciello).
fax:
+39
0960-8966/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2007.01.012
tory myopathy selectively affecting this muscle group has only been identified and fully characterized in the dog [2]. CMMM can affect all breeds of dogs with no age or sex predilection [1,2]. Clinical presentations include an acute painful form with swelling of the masticatory muscles, jaw pain, trismus, and ocular signs (such as exophthalmus, conjunctivitis, and blindness), and a chronic form with progressive muscle atrophy (Fig. 1A and B) with or without restricted jaw mobility. Both forms are characterized histologically by myositis with cellular infiltration and myonecrosis [1,2] (Fig. 1B and C). Although the trigger(s) for CMMM have not yet been established, it is well known that this is an immune-mediated disease. CMMM is very responsive to corticosteroid therapy and circulating autoantibodies directed against type 2M fibers are found in most cases [3,5]. Although the clinical presentations are distinctly
314
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
Fig. 1. Clinical and histological features of canine masticatory muscle myositis (CMMM). (A) Marked atrophy of the temporalis muscles with an evident occipital crest (arrows) is a common clinical presentation of CMMM (case #1). (B) In addition to atrophy of this muscle group, there may be inability to open the jaw (case #9). (C) Diffuse infiltration of mixed mononuclear cells into masticatory muscles, with invasion of non-necrotic fibers and myonecrosis, is typical of acute CMMM (case #1). (D) In chronic cases, perimysial (asterisk) and endomysial fibrosis (not shown), and variability in myofiber size may be found with no detectable inflammatory cells (case #9). (Temporalis muscle, H&E stain, Original magnification 20· for both C and D.)
different, histologic and immunophenotypic similarities in muscle biopsy specimens between human dermatomyositis (DM) and CMMM have been described [6–8]. Although an angiopathy has not yet been identified in CMMM, perivascular accumulations of inflammatory cells are common [6]. In human IMs, expression of MHC class I and class II molecules have been of interest because they may be associated with a drastically changed potential to participate in immunological reactions related to T cell activation and anergization [7–9]. Muscle fibers do not normally express MHC class I and class II antigens, but in human IM, widespread over-expression of MHC class I and class II has been found even in areas remote from the inflammation [9,10]. These findings suggest that muscle cells may play an active role in the immune response [7] and are of critical importance in pathogenesis. In addition, localization of MHC class I and class II molecules may be important in the diagnosis of inflammatory myopathies (IM) as cellular infiltrates may be missed on individual biopsy specimens [9–11]. Considering the potentially important roles of MHC class I and class II expression on muscle cells in the pathogenesis of disease [7–9] and their possible use in the diagnosis of IMs in both animals and humans, we investigated MHC class I and class II expression in canine MMM using confocal
laser scanning microscopy in parallel with conventional immunohistology. 2. Materials and methods 2.1. Animal groups Biopsies (n = 12) from the temporalis and masseter muscles of dogs with a clinical diagnosis of CMMM were examined. Dogs were further divided into three groups according to the clinical presentation and on the basis of the presence or absence of cellular infiltrates. The clinical data is shown in Table 1. Group 1 (dogs 1–4): Acute onset of clinical signs with swollen and painful masticatory muscles and detectable infiltration of inflammatory cells in the muscle biopsy. The serum CK concentration was mildly elevated in 3 dogs (dogs 1–3) and normal in dog 4. Group 2 (dogs 5–7): Chronic disease with atrophy and muscle pain, and detectable clusters of infiltrating cells in the muscle biopsy. The serum CK concentrations were mildly elevated in all 3 dogs. These dogs may represent relapse of previously active disease, or most likely, a chronic slowly progressive course. Group 3 (dogs 8–12): Chronic stage of disease with severe masticatory muscle atrophy, no detectable masticatory muscle or jaw pain, an absence of inflammatory
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
315
Table 1 Clinical data on dogs with masticatory muscle myositis Dog #
Breed
1 2 3 4 5 6 7 8 9 10 11 12
Dalmatian German Shepherd Rottweiler Mixed German Shepherd German Shepherd Poodle Mixed Rottweiler Mixed German Shepherd Mixed
dog
dog dog
dog
Sex
Age (yr)
CK (<200 IU/L)
Clinical signs
Presence of inflammatory cells in the biopsy
M F F F M M M FS M FS M FS
3 4 8 8 3 6 9 12 7 6 8 5
300 380 260 <200 500 350 307 160 <200 <200 178 <200
Jaw and masticatory muscle pain Masticatory muscle swelling and pain Masticatory muscle swelling and pain Jaw and muscle pain Masticatory muscle atrophy, jaw and muscle pain Masticatory muscle atrophy, jaw and muscle pain Masticatory muscle atrophy, jaw and muscle pain Severe masticatory muscle atrophy and entropion Severe masticatory muscle atrophy and trismus Severe masticatory muscle atrophy Severe masticatory muscle atrophy and trismus Severe masticatory muscle atrophy and trismus
Yes Yes Yes Yes Yes Yes Yes No No No No No
M, male; F, female; FS, female spayed; CK, creatine kinase.
cells in the muscle biopsy and endomysial and perimysial fibrosis. The serum CK concentrations were normal in all dogs in this group. Group 4 (Normal and neuromuscular disease controls). Normal control (n = 3): Temporalis muscles obtained at necropsy from dogs without any known neuromuscular disease were used as controls. Neuromuscular disease controls (n = 6): Archived muscle biopsy specimens stored at 80 C from the temporalis and biceps femoris muscles of dogs with other histologically confirmed neuromuscular diseases were used as controls. Cases were selected based on the presence of generalized muscle disease affecting both the masticatory and limb muscles, and with clinical presentations including muscle atrophy, weakness, and myalgia. Cases included immune-mediated polymyositis (CPM) (2), dystrophin deficient muscular dystrophy (1), hypothyroid myopathy (1), and peripheral neuropathy with muscle atrophy in a neurogenic pattern with fiber type grouping consistent with chronic denervation (2).
2.2. Muscle biopsies Muscle biopsy specimens were frozen in isopentane pre-cooled in liquid nitrogen and stored at 80 C until further processed. Sections (8 lm) were stained by the following histological and histochemical techniques: haematoxylin and eosin (HE), Engel trichrome [12], and the myofibrillar ATPase reactions at pH 9.4 and 4.3. Biopsy samples were sectioned at several levels and then stained by the ATPase reaction to verify the selective involvement of type 2M fibers.
2.3. Immunohistochemical examination Frozen muscle specimens were sectioned (6 lm), fixed in acetone at 4 C for 5 min, then blocked for endogenous peroxidase in 0.3% H2O2 in methanol for 20 min. Muscle sections were then incubated with two carefully selected and well characterized mouse monoclonal antibodies against canine MHC class I (1:100, clone H58A, VMRD, Inc., USA) [13] and canine MCH class II (1:50, clone H34A, VMRD, Inc., USA) [13] for 2 h at room temperature. Slides were washed three times with PBS, then incubated with biotinylated anti-mouse and labeled streptavidin biotin (LSAB kit; DakoCytomation, Denmark) for 30 min, followed by incubation with streptavidin conjugated to horseradish peroxidase (LSAB Kit; DakoCytomation, Denmark). Color development was obtained following 5–20 min of diaminobenzidine (DakoCytomation, Denmark) treatment. Sections were counterstained with Mayer’s haematoxylin. In the corresponding negative control section, the primary antibody was either omitted or replaced with normal mouse serum in place of the monoclonal antibody. 2.4. Immunofluorescence and confocal laser scanning microscopy Muscle sections were air dried, blocked with normal mouse serum diluted 1:10, then rinsed in 0.01 M PBS (pH 7.4) containing 0.2% Triton X-100 and 0.1% bovine serum albumin. Sections were then incubated for 24 h at 4 C with mouse primary monoclonal antibodies against the carboxy-terminus of dystrophin, against spectrin as a control for membrane integrity, and against desmin to identify regenerating fibers. All primary antibodies were obtained from Novocastra Laboratories Ltd, and diluted 1:50 in 1:5 normal goat serum. The sections were then washed in PBS followed by incubation with goat anti-mouse IgG-Fab fragment conjugated to TRITC
316
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
primary antibody against canine MHC class I and II (diluted 1:50 in 1:5 normal rabbit serum, antibody details are above) for 24 h at 4 C. After rinsing in PBS, the sections were treated with affinity-purified rabbit anti- mouse IgG-Fab conjugated to FITC fluorochrome (1:50; Jackson Laboratories), for 1 h at room temperature. Finally, the sections were rinsed with PBS and mounted with glycerin diluted with PBS 1:1. Specificity of the immunoreactivity in double immunolabeling using monoclonal antibodies raised in the same species (Fab fragment method) was tested by omitting the primary antibody in the second staining and using testing procedures recommended by Negoescu et al. [15]. 2.5. Scanning and photography Fig. 2. Temporalis muscle biopsy from a healthy control dog demonstrating that MHC reactivity is present only in endothelial cells (arrows). (Avidin-biotin-peroxidase complex method with Mayer’s hematoxylin counterstain, Original magnification 20·).
fluorochrome (1:50, Jackson Laboratories) for 1 h at room temperature. To avoid antibody cross-reaction in double immunofluorescence staining utilizing two primary mouse antibodies, fluorochrome conjugated Fab fragments were used (Jackson Laboratories) according to the method of Wessel and McClay [14]. The sections were again rinsed in PBS and incubated with the second
For scanning and photography, a confocal laser scanning microscope LSM-510 (Zeiss, Go¨ttingen, Germany) was used. FITC was irradiated at 488 nm and detected via a 505–560 nm band pass filter. TRITC was irradiated at 543 nm and detected with a 560 long pass filter. Two-channel frame-by-frame multi-tracking was used to avoid cross-reacting signals. The different frames were scanned separately, with appropriate installation of the optical path for excitation and emission of each scan according to the manufacturer’s instructions.
Fig. 3. Expression of both MHC class I and MHC class II was present on muscle fibers in CMMM in the presence or absence of cellular infiltration. In biopsies with obvious cellular infiltrations (case #5), there was strong immunoreactivity of MHC class I (A) and MHC class II (B) on muscle fibers (arrows), inflammatory cells (arrow heads), and endothelial cells. In biopsies without obvious cellular infiltration (case #12), many muscle fibers showed less intense but still obvious immunoreactivity for MHC class I (arrows, C) and MHC class II (arrows, D). (Avidin-biotin-peroxidase complex method with Mayer’s hematoxylin counterstain, Original magnifications 20· for all figures.)
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
2.6. Scoring of immunoreactivity Expression of MHC class I and class II was assessed as follows: 0, negative staining of muscle fibers; 1+, positive staining of endothelial cells and 1–25% positively stained fibers; 2+, positive staining of endothelial cells and 26–50% positively stained fibers; 3+, positive staining of endothelial cells and 51–70% positively stained fibers; 4+, positive staining of endothelial cells and 71–100% positively stained fibers.
3. Results 3.1. Normal control tissue The temporalis muscle specimens were histologically normal without obvious cellular infiltrations in all healthy control dogs. MHC class I and class II expression was restricted to endothelial cells of arterioles, venules, and capillaries. No positivity was observed on muscle fibers (Fig. 2). 3.2. MHC class I and class II expression in CMMM and controls Group 1 (dogs 1–4): Acute onset of clinical signs with swollen and painful masticatory muscles and detectable infiltration of inflammatory cells in the muscle biopsy. Mononuclear cell infiltrates having an endomysial, perimysial, and perivascular distribution were present within muscle biopsies from all 4 dogs as previously described (6). Type 2M fibers constituted approximately 80% of the total fiber number. The expression of both MHC class I (Fig. 3a, score 4+) and MHC class II (Fig. 3b, score 3+ to 4+) was evident on the sarcolemma of the majority of muscle fibers (Fig. 3). Immunoreactivity was sometimes noted in the cytoplasm. In addition, inflammatory cells and endothelial cells strongly expressed both MHC class I and MHC class II. No difference in staining intensity between MHC class I and MHC class II was observed. Group 2 (dogs 5–7):Chronic disease with atrophy and muscle pain, and only small detectable clusters of infiltrating cells in the muscle biopsy. In this group, muscle biopsies showed only rare, small groups of lymphocytes. Type 2M fibers constituted approximately 40– 50% of the muscle fiber population. Sarcolemmal immunostaining for MHC class I (Fig. 3c, score 1+ to 3+) and MHC class II (Fig. 3d, score 1+ to 3+) was present not only on the sarcolemma of fibers close to areas of cellular infiltration, but also on the sarco-
317
lemma of fibers distant from the cellular infiltrates. Staining for MHC class I and MHC class II was also present in endothelial cells of capillaries, arterioles, and venules in all biopsies. Group 3 (dogs 8–12): Chronic stage of disease with severe masticatory muscle atrophy, no detectable masticatory muscle or jaw pain, and an absence of inflammatory cells in the muscle biopsy. In this group, type 2M fibers constituted 20–30% of the muscle fiber population. Sarcolemmal immunostaining for MHC class I (Fig. 4A, B and C, score 1+ to 3+) and class II (Fig. 4D, E and F; score 1+ to 3+) was present, but was limited to a lesser number of muscle fibers than in dogs in Groups 1 and 2. Furthermore, sarcolemmal staining was less intense than in CMM with obvious cellular infiltrates. Endothelial and interstitial cells were always positive. Group 4 (Neuromuscular disease controls): Muscle biopsies from 3 healthy dogs and from 6 dogs with other histologically confirmed neuromuscular diseases including polymyositis, dystrophin deficient muscular dystrophy, hypothyroid myopathy, and peripheral neuropathies were similarly tested. The staining pattern for MHC class I and class II antigens on muscle fibers from dogs with dystrophin deficient muscular dystrophy (not shown), hypothyroid myopathy (Fig. 5A and B), and peripheral neuropathy (not shown) was similar to control biopsies from healthy dogs, with no expression of either MHC class I or class II on the muscle fibers. The staining pattern for MHC class I and MHC class II in polymyositis was similar to the expression in CMMM, with both antigens expressed on muscle fiber membranes. MHC class I and MHC class II reactivity was also occasionally noted in the cytoplasm. Both normal and morphologically changed fibers expressed MHC class I and MHC class II antigens independently of the relation to foci of cellular infiltrates. Inflammatory cells and endothelial cells also expressed MHC class I and MHC class II antigens. No difference in staining pattern between PM and CMMM was recorded (Fig. 5C and D, score 4+). 3.3. Confocal laser scanning microscopy Immunofluorescence labeling gave a staining pattern superimposable to that of the immunohistochemical stainings in all groups (not shown). All muscle fiber membranes were positive using a monoclonal antibody against the carboxy-terminus of dystrophin, a membrane protein. Double-immunofluorescence staining analyzed by confocal laser scanning microscopy demonstrated a patchy, uneven distribution of co-localization of MHC class I and dystrophin, and MHC class II and dystrophin (Fig. 4). Double stained fibers were present in areas with or without inflammatory infiltrates. The pattern of co-localization of MHC class I
318
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
Fig. 4. Double immunofluorescence staining analyzed by confocal laser scanning microscopy showed co-localization of both MHC class I and class II with dystrophin. (A and D) Dystrophin localization to the muscle fiber membrane (red fluorescence). (B and E) Localization of MHC class I (B) and MHC class II (E) to the muscle fiber membrane (green fluorescence). Overlapping images demonstrated patchy areas of co-localization of MHC class I and dystrophin (C, yellow color), and MHC class II and dystrophin (F, yellow color). (Original magnifications 100· for all figures.)
Fig. 5. Immunofluorescence staining of MHC class I and class II in neuromuscular disease controls. MHC class I (A) and MHC class II (B) expression in a dog with hypothyroid myopathy was restricted to endothelial cells of arterioles, venules, and capillaries. Expression of MHC class I (C) and MHC class II (D) in a dog with polymyositis was similar to CMMM, with both antigens present on muscle fibers, inflammatory cells, and endothelial cells. (Original magnifications 40· for all figures.)
and spectrin, and MHC class II and spectrin was similar to that of dystrophin. Small numbers of regenerating muscle fibers were identified using the antibody against desmin. These fibers were positive for MHC class I but in all cases negative for MHC class II (not shown).
4. Discussion In this study we demonstrate that muscle fibers in the focal canine inflammatory myopathy CMMM and in the generalized inflammatory myopathy PM, express both MHC class I and MHC class II antigens in the
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
presence or absence of cellular infiltration. Neither healthy muscle or muscle from other non-inflammatory myopathies such as dystrophin deficient muscular dystrophy and hypothyroid myopathy, or in cases of peripheral neuropathy with neurogenic atrophy, showed obvious expression on muscle membranes. This supports the contention that these molecules are induced and not constitutively expressed on muscle fibers. This is also consistent with the findings that under inflammatory conditions with expression of pro-inflammatory cytokines, human muscle cells can express MHC class I as well as MHC class II molecules [16]. Although we did not specifically address the question of whether type 2M fibers specifically expressed MHC antigens in this study, there was an obvious trend of decreased expression of MHC antigens on muscle membranes with loss of type 2M fibers in the acute and chronic groups. In vivo, under physiologic conditions, myofibers do not normally express MHC class I and class II antigens on the cell surface [17], although both antigens are upregulated in various human inflammatory myopathies [17–19]. In dogs, expression of MHC class I has been previously described in a case of canine polymyositis [20], and in a case of myositis associated with neosporosis [21]. Expression of MHC class II has been demonstrated in both CMMM and in canine polymyositis [6]. Expression of these antigens indicates that the muscle fibers are immunologically activated, but the mechanism underlying their expression as well as the consequences are as yet unknown. In both human [16] and canine IMs [5], there is evidence for the presence of antigen processing and presentation within muscle. In a recent microarray study of IMs in dogs, several genes associated with macrophage and dendritic cell function were up-regulated in both CMMM and CPM [5]. These include genes important in activation, migration, and antigen processing, and antigen presentation including cathepsin precursors and cathepsin S proteases, MHC class II-associated invariant chain, MHC class I (DLA-A, beta 2 microglobulin), and MHC class II (DLA-DR, DQ). If DLA-DR and DLA-DQ are expressed on the surface of canine muscle fibers in vivo, the muscle fibers could theoretically present not only viral or bacterial antigens but also muscle autoantigens or alloantigens to CD4+ T cells. The expression of HLA-DR on the surface of muscle fibers in human inflammatory lesions has been inconsistent [9,19]. Whether the expression of MHC on the muscle fiber membranes switches the activation or the anergy in T lymphocytes could depend on the presence of other co-stimulatory molecules such as the B7 family [22,23]. The co-stimulatory molecules CD80 and CD86 were both identified in canine IMs using monoclonal antibodies [5]. MHC expression could be an earlier event than leukocyte infiltration in initiating or maintaining pathological condition in MMM.
319
We also demonstrated that MHC expression can occur independently of the presence of inflammatory cell infiltration. This finding is of diagnostic importance, since cellular infiltrations in inflammatory myopathies can have a patchy or multifocal distribution and be missed on individual biopsy specimens, whereas MHC class I staining is diffuse throughout the sample. Several authors [10,11] suggested that localization of MHC class I is a valid test for the diagnosis of idiopathic inflammatory myopathies when inflammatory cells may be undetectable during short term use of immunosuppressive agents. The serum CK concentration was within the reference range in 6 of 12 dogs with CMMM, in which 5 dogs had the chronic form. Even in dogs with acute CMMM, it is not unusual to find the CK value within the reference range or only mildly elevated (2–3X normal). The amount of muscle mass involved in CMMM is small compared to the entire body mass, so only mild elevations would be expected even with acute disease. In dogs with chronic CMMM, muscle is replaced by fibrous tissue and CK concentrations are usually normal. Although the CK concentration is a sensitive indicator of muscle fiber injury, it is not elevated in all human patients with polymyositis and dermatomyositis [24]. In the two cases of CPM used as controls, the serum CK concentrations were elevated at 1800 and 1010 IU/L (5–10X normal), respectively. Multifocal areas of mixed mononuclear cell infiltrations were present involving multiple muscle groups, and both dogs showed clinical evidence of muscle weakness and stiffness in addition to generalized muscle atrophy. While not specifically addressed in this study, similar to human IMs, the serum CK concentration is not always elevated in cases of canine PM (Shelton, unpublished observations). A normal CK can occur in canine PM when the pathological process is concentrated in the perimysium and fiber necrosis is minimal. Although the data is conflicting, muscle cells, as well as invading lymphocytes and endothelial cells, secrete pro-inflammatory cytokines such as IL-1a, IL-1b, TNF-a, IL-6, IL-2 and IFN-c, but not TGF-b [23]. It is unlikely that induction of MCH expression in IMs occurs in response to local lymphocyte products in muscle without obvious cellular infiltration [25,26]. It is theoretically possible that muscle fibers create a local environment for chronic inflammation by secretion of pro-inflammatory cytokines. Reports have documented an increased expression of pro-inflammatory cytokines such as IL-1a in capillary endothelial cells in IM, and IL-1a has been found in muscle tissue of patients with clinical signs of myositis in the absence of detectable infiltration of inflammatory cells [27]. Pro-inflammatory cytokines have recently been shown to influence MHC expression on human myoblasts and myotubes in
320
O. Paciello et al. / Neuromuscular Disorders 17 (2007) 313–320
in vitro experiments [16]. It is theoretically possible that MHC class I and MHC class II are at least partially regulated in response to IL-1a [26]. In the recent microarray study of CMMM and CPM, there was increased expression of several pro-inflammatory cytokine genes in both CMMM and CPM [5]. In conclusion, this study documents the muscle membrane expression of both MCH class I and class II molecules in CMMM and CPM, in the presence or absence of cellular infiltrations. These findings suggest that these glycoproteins may play a crucial role in the pathogenesis of CMMM and PM, and possibly other idiopathic inflammatory myopathies. Further, using double-immunofluorescence staining and confocal laser microscopy, we demonstrate for the first time that MHC class I and class II molecules are expressed on the sarcolemma and co-localize with dystrophin in the presence or absence of cellular infiltrates. Thus, expression of MHC class I and class II molecules in CMMM and CPM is not an artifact caused by shedding of molecules from the contacting inflammatory cells. As suggested by other authors [6,8], this study provides further support for the role of canine IMs as an animal model for the study of many aspects of human IMs. References [1] Podell M. Inflammatory myopathies. Vet Clin North Am Small Anim Pract 2002;32:147–67. [2] Shelton GD, Cardinet G, Bandman E. Canine masticatory muscle disorders: a study of 29 cases. Muscle Nerve 1985;10:783–90. [3] Shelton GD, Cardinet G, Bandman E, Cuddon P. Fiber typespecific autoantibodies in a dog with eosinophilic myositis. Muscle Nerve 1985;8:783–90. [4] Shelton GD, Bandmann E, Cardinet III GH. Electrophoretic comparison of myosins from masticatory muscles and selected limb muscles in the dog. Am J Vet Res 1985;46:493–8. [5] Shelton GD, Hoffman EP, Ghimbovschi S, et al. Immunopathogenic pathways in canine inflammatory myopathies resemble human myositis. Vet Immunol Immunopathol 2006;113:200–14. [6] Pumarola M, Moore PF, Shelton GD. Canine inflammatory myopathy: analysis of cellular infiltrates. Muscle Nerve 2004;29:782–9. [7] Dalakas MC, Hohlfeld R. Polymyositis and dermatomyositis. Lancet 2003;362:1762–3. [8] Dalakas MC. From canine to man: on antibodies, macrophages, and dendritic cells in inflammatory myopathies. Muscle Nerve 2004;29:753–5. [9] Englund P, Lindroos E, Nennesmo I, Klareskog L, Lundberg IE. Skeletal muscle fibers express major histocompatibility complex class II antigens independently of inflammatory infiltrates in inflammatory myopathies. Am J Pathol 2001;159:1263–73. [10] Topaloglu H, Muntoni F, Dubowitz V, Sewry C. Expression of HLA class I antigens in skeletal muscle is a diagnostic marker in juvenile dermatomyositis. J Child Neurol 1997;12:60–3.
[11] Van der Pas J, Hengstman GJ, ter Laak HJ, Borm GF, van Engelen BG. Diagnostic value of MHC class I staining in idiopathic inflammatory myopathies. J Neurol Neurosurg Psychiatry 2004;75:136–9. [12] Engel WK, Cunningham GG. Rapid examination of muscle tissue. An improved trichrome method for fresh-frozen biopsy sections. Neurology 1963;13:919–23. [13] Davis WC, Marusic S, Lewin HA, et al. The development and analysis of species specific and cross reactive monoclonal antibodies to leukocyte differentiation antigens and antigens of the major histocompatibility complex for use in the study of the immune system in cattle and other species. Vet Immunol Immunopathol 1987;15:337–76. [14] Wessel GM, McClay DR. Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies. J Histochem Cytochem 1986;34:703–6. [15] Negoescu A, Labat-Moleur F, Lorimier P, et al. F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J Histochem Cytochem 1994;42:433–7. [16] Wiendl H, Hohlfeld R, Kieseier BC. Immunobiology of muscle: advances in understanding an immunological microenvironment. Trends Immunol 2005;26:373–80. [17] Karpati G, Pouliot Y, Carpenter S. Expression of immunoreactive major histocompatibility complex products in human skeletal muscles. Ann Neurol 1988;23:64–72. [18] Appleyard ST, Dunn MJ, Dubowitz V, Rose ML. Increased expression of HLA ABC class I antigens by muscle fibres in Duchenne muscular dystrophy, inflammatory myopathy, and other neuromuscular disorders. Lancet 1985;1:361–3. [19] Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibility complex class I antigen expression, immunolocalization of interferon subtypes, and T cell-mediated cytotoxicity in myopathies. Hum Pathol 1989;20:224–31. [20] Morita T, Shimada A, Yashiro S, et al. Myofiber expression of class I major histocompatibility complex accompanied by CD8+ T-cell-associated myofiber injury in a case of canine polymyositis. Vet Pathol 2002;39:512–5. [21] Paciello O, D’Orazi A, Borzacchiello G, Martano M, Restucci B, Maiolino P, Papparella S. Expression of Major Histocompatibility Complex Class I and II in a case of Neospora caninum myositis in a dog. Acta Myol 2004;23:151–3. [22] Figarella-Branger D, Civatte M, Bartoli C, Pellissier JF. Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies. Muscle Nerve 2003;28:659–82. [23] Marino M, Scuderi F, Mannella F, Bartoccioni E. TGF-b1 and IL-10 modulate IL-1b-induced membrane and soluble ICAM-1 in human myoblasts. J Neuroimmunol 2003;134:151–7. [24] Engel AG, Hohlfeld R. The polymyositis and dermatomyositis syndromes. In: Myology. McGraw-Hill; 2004. [25] Isenberg DA, Rowe D, Shearer M, Novick D, Beverley PC. Localization of interferons and interleukin 2 in polymyositis and muscular dystrophy. Clin Exp Immunol 1986;63:450–8. [26] Kalovidouris AE. The role of cytokines in polymyositis: interferon-gamma induces class II and enhances class I major histocompatibility complex antigen expression on cultured human muscle cells. J Lab Clin Med 1992;120:244–51. [27] Nyberg P, Wikman AL, Nennesmo I, Lundberg I. Increased expression of interleukin 1alpha and MHC class I in muscle tissue of patients with chronic, inactive polymyositis and dermatomyositis. J Rheumatol 2000;27:940–8.