Expression of specific matrix metalloproteinases in inflammatory ...

Brain (2001), 124, 341–351

Expression of specific matrix metalloproteinases in inflammatory myopathies Bernd C. Kieseier,1 Christiane Schneider,2 John M. Clements,3 Andrew J. H. Gearing,3 Ralf Gold,2 Klaus V. Toyka2 and Hans-Peter Hartung1 1Department

of Neurology, Karl Franzens University, Graz, Austria, 2Department of Neurology, Julius Maximilians University, Wu¨rzburg, Germany, and 3British Biotech Pharmaceuticals Limited, Oxford, UK

Correspondence to: Bernd C. Kieseier, MD, Department of Neurology, Karl-Franzens-Universita¨t, Auenbruggerplatz 22, 8036 Graz, Austria E-mail: [email protected]

Summary The family of matrix metalloproteinases (MMPs) comprises endopeptidases that are capable of degrading all extracellular matrix components. Given these actions, it is conceivable that MMPs may play a pathogenic role in inflammatory myopathies. These immune-mediated disorders are characterized by the invasion of mononuclear phagocytes and T lymphocytes and the loss of muscle fibres. We examined whether specific MMPs and their natural inhibitors (tissue inhibitors of metalloproteinases; TIMPs) are expressed in muscle during acute inflammatory attacks by studying muscle biopsies obtained from patients diagnosed as having polymyositis, dermatomyositis, sporadic inclusion body myositis and, for comparison, from cases of various muscular dystrophies. Quantitative polymerase chain reaction analysis revealed significantly elevated mRNA

expression of interstitial collagenase (MMP-1) and gelatinase B (MMP-9) in polymyositis and dermatomyositis and to a lesser extent in inclusion body myositis, whereas the level of expression of TIMPs remained unchanged in comparison with controls. Increased mRNA levels were associated with enhanced enzyme expression, as determined by immunoblotting, gelatin zymography and in situ zymography. Immunohistochemically, MMP-1 could be localized around the sarcolemma of diseased muscle fibres and to cells resembling fibroblasts, whereas MMP-9 seemed to be expressed primarily by invading T lymphocytes. Raised levels of MMPs could not be detected in the sera of affected patients, emphasizing the crucial action of MMPs in the inflamed muscle. Our results imply a pathogenic role for specific MMPs in the genesis of inflammatory myopathies, and open new strategies for therapeutic intervention.

Keywords: matrix metalloproteinases; polymyositis; dermatomyositis; inclusion body myositis; muscular dystrophy Abbreviations: ECM ⫽ extracellular matrix; GAPDH ⫽ glyceraldehyde-3-phosphate dehydrogenase; MMP ⫽ matrix metalloproteinases; PCR ⫽ polymerase chain reaction; SDS–PAGE ⫽ sodium dodecyl sulphate–polyacrylamide gel electrophoresis; TIMP ⫽ tissue inhibitor of metalloproteinases; TNF-α ⫽ tumour necrosis factor α

Introduction The inflammatory myopathies, as a group, are the commonest of the acquired muscle disorders, comprising three major and distinct subsets: polymyositis, dermatomyositis and inclusion body myositis. Although each subset has characteristic clinical, immunopathological and morphological features, their common histopathology is characterized by inflammation, loss of muscle fibres and fibrosis (Dalakas, 1991; Engel et al., 1994; Karpati and Durrie, 1994). Their association with other autoimmune diseases, the evidence for T-cell-mediated cytotoxicity or complement-mediated microangiopathy and the presence of autoantibodies support the notion of an autoimmune process being involved in the pathogenesis of these inflammatory muscle disorders (Dalakas and Sivakumar, 1996; Hohlfeld et al., 1997; Dalakas, 1998). © Oxford University Press 2001

Infiltrating lymphocytes are a histopathological hallmark of these myopathies, and trafficking of T-cell subsets through the extracellular matrix (ECM) towards the muscle membrane and beyond into the muscle fibre has been traced in polymyositis (Dalakas, 1995). A key process in the infiltration of mononuclear cells and tissue destruction in inflammatory disorders of the muscle may be the proteolytic disruption of the ECM. Several families of ECM-degrading enzymes have been identified, the largest of which are the matrix metalloproteinases (MMPs). The MMPs are members of a family of at least 23 zinc-dependent endopeptidases that operate extracellularly at neutral pH. They all have structural domains in common but differ in cellular source, substrate specificity and inducibility

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(Goetz et al., 1996; Cawston, 1998). A group of natural tissue inhibitors of metalloproteinases (TIMPs) has been identified which allow the finely tuned regulation of the proteolytic activity (Goetz et al., 1996; Cawston, 1998). Knowledge about the involvement of MMPs in physiological as well as pathological conditions remains incomplete, but they appear indispensable for ECM degradation (Woessner and Nagase, 2000). Any imbalance in favour of inhibitors can lead to fibrotic processes, whereas any increase in enzymatic activity will result in tissue destruction or cell invasion (Birkedal-Hansen, 1995). Overexpression of gelatinases A and B in inflammatory myopathies has been reported recently (Choi and Dalakas, 2000), pointing to the potential involvement of MMPmediated pathomechanisms in this group of immune-mediated diseases. The present study was designed to investigate systematically the expression of MMPs and TIMPs in muscle biopsies and sera from patients with polymyositis, dermatomyositis, inclusion body myositis and muscular dystrophies. Such information could increase our understanding of the immunological mechanisms involved in the pathogenesis of these myopathies and open novel therapeutic avenues.

Material and methods Patients The study was approved by the local ethics committee at the Universities of Wu¨ rzburg and Graz and written consent was obtained from all participants. Three groups of patients were studied. The first group (n ⫽ 6) comprised patients with myositis, among whom three patients were diagnosed with polymyositis and three with dermatomyositis according to clinical and histological criteria (Dalakas, 1992; Engel et al., 1994). The second group of patients (n ⫽ 8) had sporadic inclusion body myositis (Engel et al., 1994; Griggs et al., 1995). The third group (n ⫽ 8) served as controls and comprised patients with various non-inflammatory forms of muscular dystrophy (Table 1). Nineteen of the 22 patients included in the study had not received immunomodulatory therapy before muscle biopsy and blood sampling.

Material processing All muscle biopsies were snap-frozen within 5 min of surgical intervention and stored at –80°C until analysis. Serum samples were collected and frozen immediately in aliquots and maintained at –80°C. All sera were processed in the same way and were analysed simultaneously.

Antibodies The following antibodies were used for immunohistochemistry: murine monoclonal anti-MMP-1 (ICN, Costa Mesa, Calif., USA), murine monoclonal anti-MMP-9 (R&D

Systems, Oxford, UK), murine monoclonal anti-CD3 (Serotec, Oxford, UK), murine monoclonal anti-CD68 (Dako, Hamburg, Germany), murine monoclonal anti-human fibroblast (Dako), and rabbit anti-mouse IgG (Dako).

Competitive polymerase chain reaction For quantitation of the expression of human MMP mRNA, polymerase chain reaction (PCR) assays, using a synthetic multicompetitor standard DNA containing tandem arrays of 5⬘ and 3⬘ priming sites for cDNAs of different MMPs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed. Primer pairs for interstitial collagenase (MMP-1), matrilysin, gelatinase A, gelatinase B (MMP-9), stromelysin-1, -2 and -3, TIMP-1, -2 and -3 and GAPDH were used (Table 2). Total cellular RNA was extracted from frozen muscles according to published methods (Chomczynski and Sacchi, 1987) and used as a template for cDNA synthesis using AMV (avian myeloblastosis virus) reverse transcriptase (Gibco, Karlsruhe, Germany). Threefold serial dilutions of competitive standard DNA were combined with a fixed amount of sample cDNA, and PCR was performed in 50 µl reaction/fluid containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, pH 9.0, and 0.1% Triton X-100 in the presence of 200 µM dNTP (Pharmacia, Freiburg, Germany), 50 pmol sense and antisense MMP primers, 1 U Taq DNA polymerase (Perkin Elmer, Branchburg, NJ, USA) and 1 µCi [32P]dCTP (Amersham, Braunschweig, Germany). Amplification was carried out using 35 cycles (95°C, 30 s/57°C, 30 s/72°C, 120 s) in a Hybaid Omnigene thermal cycler (MWG Biotech, Ebersberg, Germany). Ten microlitres of the reaction products was electrophoresed on 6% polyacrylamide gel. A Cyclon phosphor imaging system (Canberra Packard, Meriden, Conn., USA) was used to quantify bands on the gels. MMP mRNA levels were determined by plotting the ratio of sample cDNA to standard DNA against the standard dilution using a double logarithmic scale.

Gelatin zymography Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) zymography was performed to determine gelatinase activity as described before (Kieseier et al., 1998b). Briefly, 20 ⫻ 10 µm frozen sections of muscle were incubated with 50 µl of Tris/glycine SDS sample buffer (Novex, San Diego, Calif., USA), and further homogenized by vortexing for 1 h at 4°C. The samples were applied to a 10% (w/v) polyacrylamide resolving gel containing 0.1% SDS and 0.1% gelatin type A from porcine skin (Sigma, St Louis, Mo., USA). Stacking gels were 5% (w/v) polyacrylamide. After electrophoresis, gels were washed in renaturing buffer (Novex) containing Triton X-100 to remove any SDS, and incubated in developing buffer (Novex) for 18 h at 37°C. Gels were stained for 6 h in 30% methanol/10% acetic acid containing 0.5% (w/v) Coomassie Brilliant Blue G-250 and

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Table 1 Patient characteristics Patient

Age (years)/ sex

Disease duration (months)

Diagnosis

CK† (U/l)

Treatment‡

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

55/M 65/F 30/F 69/F 46/F 41/F 72/M 75/M 73/M 59/M 35/M 65/M 61/M 57/M 37/M 41/M 25/M 49/M 6/M 36/F 26/F 36/F

30 36 30 2 2 3 120 48 24 72 6 36 96 3 180 24 84 84 30 288 120 156

PM PM PM DM DM DM 1BM IBM IBM IBM IBM IBM IBM IBM Becker-type MD Becker-type MD Becker-type MD Becker-type MD Duchenne MD Miyoshi myopathy Limb girdle MD Limb girdle MD

1600 142 1500 3660 3550 813 186 76 86 82 251 191 223 1030 550 1180 ⬍1000 600 ⬎4000 ⬎3000 890 1398

– – – – CST – CST – – CST – – CST – – – – – – – – –

M ⫽ male; F ⫽ female; PM ⫽ polymyositis; DM ⫽ dermatomyositis; IBM ⫽ inclusion body myositis; MD ⫽ muscular dystrophy; CST ⫽ corticosteroids. *Duration of disease until biopsy based on subjective assessment by patient; †creatine kinase (CK) measured before muscle biopsy; ‡drugs that had been used before muscle biopsy.

Table 2 Primers used for quantitative PCR MMP

Forward (5⬘–3⬘)

Reverse (5⬘–3⬘)

Interstitial collagenase Matrilysin Gelatinase A Gelatinase B Stromelysin-1 Stromelysin-2 Stromelysin-3 TIMP-1 TIMP-2 TIMP-3 GADPH

AAT GTG CTA CAC GGA TAC CC TTT GAT GGG CCA GGA AAC AC CCC CCT TTA ACT GGA GCA AA GAA GAT GCT GCT GTT CAG CG GAG GAA AAT CGA TGC AGC CA TGC AGC TGT TTC TGA CAA GG AGA TCT ACT TCT TCC GAG GC CGG GGC TTC ACC AAG ACC GAA GAA GAG CCT GAA CCA CA TCG GTA TCA CCT GGG TTG TA TGC CGT CTA GAA AAA CCT GC

CTT TGT GGC CAA TTC CAG GA GGG GAT CTC CAT TTC CAT AG TTT GGT TCT CCA GCT TCA GG ACT TGG TCC ACC TGG TTC AA CTC CAA CTG TGA AGA TCC AGG TGG CAT TGG GGT CAA ACT CA TTC CAG AGC CTT CAC CTT CA TCA GGC TAT CTG GGA CCG C GTC CTC GAT GTC GAG AAA CT GTC TGT GGC ATT GAT GAT GC ACC CTG TTG CTG TAG CCA AA

destained in the same buffer without dye. Gelatinase activity was detected as unstained bands on a blue background, representing areas of gelatin digestion.

with a murine anti-MMP-1 antibody overnight at room temperature. Murine anti-mouse IgG conjugated to horseradish peroxidase (Dako) was used as secondary antibody, and the reaction was developed using diaminobenzene substrate.

Immunoblotting Sections from muscle biopsy material and human sera were subjected to electrophoresis on SDS–PAGE under nonreducing conditions as described above, except that gelatin was not included in the gels. Samples were then electroblotted onto nitrocellulose (Schleicher and Schuell, Darmstadt, Germany) at 100 mA constant power using a semi-dry blotter (Bio-Rad, Hercules, Calif., USA). Thereafter, nitrocellulose was saturated with 2.5% dried milk, washed, and incubated

In situ zymography In situ zymography was performed as described by Oh and colleagues (Oh et al., 1999). Briefly, 10 µm cryosections of muscle biopsies were air-dried and overlaid with a solution containing 50 mM Tris–HCl, 5 mM CaCl, 0.2 mM NaN3 and 50 µg/ml fluorescence-labelled DQ gelatin and DQ collagen type I, respectively (Molecular Probes, Eugene, Oreg., USA). Sections were incubated for 18 h at 37°C and

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Fig. 1 Expression of MMP and TIMP mRNA in muscle biopsies from patients diagnosed with polymyositis (PM), dermatomyositis (DM), sporadic inclusion body myositis (IBM) and various forms of muscular dystrophies (MD). The polymyositis and dermatomyositis group comprised six cases (three of polymyositis and three of dermatomyositis), the inclusion body myositis group eight cases and the muscular dystrophy group eight cases. There were no statistically significant differences in mRNA levels between polymyositis and dermatomyositis biopsies. mRNA levels are expressed as the fraction of mRNA for GAPDH and are given as mean ⫾ standard error. *P ⬍ 0.05 versus muscular dystrophy cases; †P ⬍ 0.05 versus inclusion body myositis cases.

examined by fluorescence microscopy. Proteolytic activity cleaves the quenched substrate, resulting in fluorescent breakdown products that allow cellular proteinases within the section to be localized. Omission of the substrates served as a negative control.

substrate. Endogenous peroxidase activity was suppressed by incubating the sections with 3% H2O2 in methanol for 20 min prior to the secondary antibody. Sections were counterstained with haematoxylin, dehydrated, and mounted in Eukitt (Kindler, Freiburg, Germany).

Immunohistochemistry Serial cryosections (10 µm) of muscle biopsies were airdried and fixed for 10 min in 4% formalin and 50%/100%/ 50% acetone (2 min each) at room temperature. Sections were blocked with 10% BSA (Roth, Karlsruhe, Germany) in phosphate-buffered saline for 30 min at room temperature and incubated overnight with primary antibodies at 4°C. Thereafter, a biotinylated secondary antibody against mouse IgG and an avidin–biotinylated peroxidase complex (ABC; Dako) were used with 3,3⬘-diaminobenzidine as peroxidase

Enzyme immunoassay The concentrations of gelatinase B and TIMP-1 in serum were determined by dual-antibody solid-phase ELISA (enzymelinked immunosorbent assay) according to the instructions of the manufacturer (Amersham Buchler, Braunschweig, Germany) using an automated ELISA reader (Canberra Packard). The interassay variability was ⬍10% and the lower detection limits were 0.75 ng/ml for gelatinase B and 1 ng/ml for TIMP-1.


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Statistical analysis One-way analysis of variance and Student–Newman–Keuls multiple comparisons were used as the principal statistical tests. A P value of ⬍0.05 was considered significant. Data are given as mean ⫾ standard error.

Results MMP mRNA expression Using quantitative competitive PCR, we determined mRNA levels of seven MMPs and three of their natural inhibitors. In patients diagnosed with polymyositis and dermatomyositis, significant upregulation of mRNAs for interstitial collagenase and gelatinase B was found in the biopsy material and to a lesser extent in inclusion body myositis cases when compared with patients with muscular dystrophy (Fig. 1). In contrast, mRNAs for gelatinase A, matrilysin, stromelysin-1, -2 and -3 and TIMP-1, -2 and -3 were expressed at approximately the same level in all patient groups investigated. In the polymyositis and dermatomyositis groups, mRNA for gelatinase B was expressed at slightly higher levels, although this did not reach significance (P ⬎ 0.05), whereas mRNAs for interstitial collagenase were detected in equivalent amounts in polymyositis and dermatomyositis biopsies (data not shown).

To address the question whether increased MMP expression at the mRNA level is associated with higher protein levels, immunoblotting was performed for interstitial collagenase. In agreement with the PCR results, a positive signal at the molecular size of 55 kDa was detected in biopsies with inflammatory myopathies, mirroring protein expression of the latent MMP-1 pro-form (Fig. 2A). Gelatin zymography was used to detect gelatinase B at the protein level. A pronounced increase in proteolytic activity at 92 kDa was visible in the myositis samples, representing increased gelatinase B activity; this is in line with the data obtained by quantitative PCR (Fig. 2B). Gelatinase A, which was detectable at 72 kDa in the same experiment, did not show any difference in proteolytic activity between the biopsies of the different groups investigated.

Fig. 2 Examples of protein expression of interstitial collagenase and gelatinase B in the inflamed muscle in patients with polymyositis (PM), dermatomyositis (DM), inclusion body myositis (IBM) and Becker-type muscular dystrophy (MD). Frozen sections from muscle biopsies obtained from patients were homogenized and subjected to electrophoresis on SDS–PAGE. (A) Immunoblotting with a monoclonal anti-interstitial collagenase antibody revealed protein expression at the molecular size of 55 kDa in the polymyositis, dermatomyositis and inclusion body myositis cases but not in the muscular dystrophy case, corresponding in molecular size to the pro-form of interstitial collagenase. (B) Using gelatin zymography, proteolytic activity was detectable at 92 and 72 kDa, corresponding in molecular size to gelatinases B and A, respectively. The proteolytic activity of gelatinase B appeared to be increased in the polymyositis and dermatomyositis cases; there was only weak activity in the inclusion body myositis case and no activity was detected in the muscular dystrophy muscle biopsy studied. Proteolytic activity of a molecule larger than 92 kDa is suggestive of covalent gelatinase B–neutrophil gelatinase B-associated lipocalin complexes (Rudd et al., 1999).

Detection of interstitial collagenase

with serial sections using a monoclonal antibody against human fibroblasts (Fig. 3).

Protein expression of interstitial collagenase and gelatinase B in the inflamed muscle

Immunohistological examination of muscle biopsies was carried out to determine the cellular source of interstitial collagenase. In biopsy material obtained from patients with polymyositis, dermatomyositis and inclusion body myositis, inflammation was active, as indicated by the invasion of non-necrotic muscle fibres by mononuclear cells. Positive immunoreactivity for interstitial collagenase could be visualized along the sarcolemma and localized to fusiform cellular nuclei, suggestive of fibroblasts, which correlated

Localization of gelatinase B Strong immunoreactivity for gelatinase B (MMP-9) could be localized to small cells with round nuclei within the inflammatory infiltrates in biopsies of patients diagnosed with polymyositis, dermatomyositis or inclusion body myositis. The amount and intensity of the positive signal was strong in polymyositis and dermatomyositis cases but sparse in

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Fig. 3 Immunohistochemistry for interstitial collagenase (MMP-1) in muscle biopsies from patients diagnosed with polymyositis (PM), sporadic inclusion body myositis (IBM) and muscular dystrophy (MD). In polymyositis cases, immunoreactivity was localized around the sarcolemma and cellular nuclei within the endomysium. Insert a: Immunohistochemistry for human fibroblasts on serial sections revealed that these nuclei belonged to fibroblasts. MMP-1 expression was detectable in inclusion body myositis biopsies, but at a lower level, whereas in muscular dystrophy cases no immunoreactivity was observed. Magnifications: ⫻200 and ⫻600 (insert a).

inclusion body myositis. To specify the cellular source of this MMP, serial sections were stained for common surface antigens on T cells (CD3) and macrophages (CD68). CD3⫹ and CD68⫹ cells were detected in the inflammatory infiltrates, and the staining pattern of CD3⫹ T cells correlated well with the immunoreactivity for gelatinase B. No positive signal for this MMP was recorded in the muscular dystrophy biopsies used as control (Fig. 4). Control sections after omission of the primary antibody showed only background staining.

body myositis and muscular dystrophy by immunoblotting and gelatin zymography. In all samples investigated, neither a positive signal for MMP-1 nor increased proteolytic activity of MMP-9 was detectable (data not shown). Furthermore, using ELISA, we determined the serum levels of gelatinase B and TIMP-1 in all patient groups and were again unable to find any significant difference in protein expression (Fig. 6).

Discussion Proteolytic activity in the inflamed muscle To support our findings on protein levels, in situ zymography using collagen and gelatin as substrates was performed. Strong collagenolytic activity was detected in polymyositis and dermatomyositis cases along the sarcolemma (Fig. 5A). On serial sections a similar distribution pattern was found for MMP-1 by immunohistochemistry (Fig. 5C). When DQ gelatin was used as the substrate, areas with strong, primarily round areas of gelatinolytic activity could be discerned in the biopsies from cases of polymyositis and dermatomyositis, and from some cases of inclusion body myositis (Fig. 5B). On serial sections, inflammatory infiltrates with positive immunoreactivity for MMP-9 were detectable in these areas (Fig. 5D). Control sections after omission of the quenched substrates revealed only background staining. These findings supplement and support observations made by immunoblotting, SDS–PAGE zymography and immunohistochemistry. All qualitative findings on the protein expression of MMP-1 and MMP-9 are summarized in Table 3.

MMPs in sera of patients with myositis To examine whether the increased expression of interstitial collagenase and gelatinase B in myositis is mirrored in the peripheral venous blood of affected patients, we studied the protein expression of these two MMPs in sera from patients diagnosed with polymyositis, dermatomyositis, inclusion

The results of the present study indicate that MMPs and TIMPs are present at low levels in muscle and appear to be differentially regulated in response to inflammatory stimuli. Raised levels of mRNA for interstitial collagenase were associated with increased protein expression, as shown by immunoblotting. Like most of the MMPs, interstitial collagenase is secreted by cells as a zymogen that requires activation before it is able to act on its substrates. This activation is achieved by dissociation of the propeptide from the catalytic domain (Van Wart and Birkedal-Hansen, 1990) through autoactivation and by the action of other proteinases (Kleiner and Stetler-Stevenson, 1993). The resulting active molecule has a size of 45 kDa (Wilhelm et al., 1984). In the present study we only found evidence for the presence of the pro-form of MMP-1. It can be assumed that the pro-form detected in the muscle biopsies is activated to the form with proteolytic potential. Whether lack of specificity of the antibody used or technical issues account for our inability to identify the active form of this MMP by immunoblotting remains unclear. Immunohistochemically positive signals for interstitial collagenase were noted along the sarcolemma of diseased muscle fibres and were associated with fusiform cellular nuclei, suggestive of fibroblasts, located primarily in the endomysium. These findings suggest a predominantly extracellular localization of the enzyme, which is potentially secreted by fibroblasts. This assumption is supported by previous observations in which interstitial collagenase was


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Fig. 4 Expression of gelatinase B (MMP-9) in muscle biopsies from patients with polymyositis (PM), inclusion body myositis (IBM) and muscular dystrophy (MD). In polymyositis biopsies, numerous MMP-9-immunoreactive cells were present within the inflammatory infiltrates. CD3-positive T cells exhibited morphology and a distribution pattern similar to those of gelatinase B-immunoreactive cells, whereas immunohistochemistry for CD68⫹ macrophages revealed a different staining pattern, suggesting that CD3⫹ T cells are the primary cellular source of MMP-9 in the inflamed muscle. In inclusion body myositis cases there was a similar expression pattern, but with lower intensity. In contrast, no immunoreactivity for MMP-9 and CD68 and few CD3-immunoreactive cells were found in muscular dystrophy cases. Serial 10 µm sections. Magnification: ⫻200.

found to be synthesized by fibroblasts (Woessner, 1994; Nagase and Okada, 1997). Interstitial collagenase is distinguished from other MMPs by its ability to degrade triple helical regions of interstitial collagens I, II and III at a specific site, generating two characteristic three-quarter and one-quarter size fragments (Fields et al., 1990). The localization of this MMP in the endomysium, as described in the present study, suggests a role of interstitial collagenase in endomysial fibrosis, which can be demonstrated in inflammatory myopathies, particularly in more advanced cases. In human muscle, the endomysium and perimysium contain both type I and type III collagen. The pathogenic relevance of increased amounts of endomysial collagen, as seen in myopathies, remains unclear. In the present investigation, positive staining for interstitial collagenase was also observed along the sarcolemma of primarily hypotrophic

muscle fibres. This localization suggests that this particular MMP is involved in muscle tissue remodelling. In situ zymography mirrored this observation by detecting collagenolytic activity in the endomysium and along fusiform cellular nuclei, suggestive of fibroblasts. However, it must be stated that in situ zymography is not specific for MMP-1 and that the observed collagenolytic activity might have been due to any enzyme capable of digesting collagen type I. Moreover, the distribution pattern found in the present study does not clarify the extent to which MMP-1 actively contributes to muscle cell destruction or necrosis. Further studies are needed to elucidate the precise role of interstitial collagenase in inflammatory myopathies. The second MMP found to be upregulated in the present investigation was gelatinase B. Significantly elevated mRNA expression, immunoreactive protein and enzyme activity were

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Fig. 5 Localization of proteolytic activity in the inflamed muscle by in situ zymography in a biopsy from a polymyositis case. (A) Collagenolytic activity is detectable along the sarcolemma (arrows), (B) primarily in round areas of gelatinolytic activity (arrows). Using immunohistochemistry on serial sections, the staining pattern for (C) MMP-1 (arrows) and (D) MMP-9 (arrows) was similar to the distribution of the proteolytic activity. Magnification: ⫻400.

Table 3 Protein expression of MMP-1 and MMP-9 Patient group

Polymyositis Dermatomyositis Inclusion body myositis Muscular dystrophy

Western blotting

Zymography

Immunohistochemistry

In situ zymography

MMP-1

MMP-9

MMP-1

MMP-9

MMP-1

MMP-9

3/3 3/3 4/8 0/8

3/3 3/3 5/8 1/8

3/3 3/3 4/8 0/8

3/3 3/3 5/8 0/8

3/3 2/3 1/8 0/8

3/3 2/3 2/8 0/8

Data are number of individual cases/total number of cases investigated in each group.

determined in polymyositis and dermatomyositis cases. The distribution pattern of positive immunoreactivity, with predominant localization to mononuclear infiltrates and perivascular spaces, suggests biological effects in stages of the disease process that are associated with inflammatory infiltration. This assumption finds support in the detection of

gelatinolytic activity localized to invading mononuclear cells, as demonstrated by in situ zymography. Invading macrophages and T lymphocytes are important effector cells in the pathogenesis of inflammatory myopathies. Staining for common surface antigens of macrophages and T cells on serial sections of the muscle biopsies revealed


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Fig. 6 Serum expression levels of gelatinase B (A) and TIMP-1 (B) pooled for all patients with myositis (polymyositis, dermatomyositis and inclusion body myositis) and control patients with various forms of muscular dystrophy (MD). Protein concentrations were measured by ELISA and the results are given as mean (ng/ml serum) ⫾ standard error of the mean.

that the predominant cellular source of MMP-9 in the present study is T cells. This finding is consistent with previous investigations in which T lymphocytes were found to secrete gelatinase B (Weeks et al., 1993). MMP-9 is known to digest gelatins and collagens of types III, IV and V, as well as elastin and aggregan. Moreover, as shown by previous in vitro studies, T-cell migration can be mediated by gelatinase B (Leppert et al., 1995), and an emerging body of evidence implicates MMP-9 in the disruption of vascular basement membranes (Gijbels et al., 1993; Rosenberg et al., 1996). In experimental studies investigating the role of MMP-9 in inflammatory demyelinating diseases of the central and peripheral nervous systems, MMP-9 was assigned a strategic role in mediating disruption of the blood–brain barrier (Clements et al., 1997; Kieseier et al., 1998b), the blood– nerve barrier (Hughes et al., 1998; Kieseier et al., 1998a) and the ECM (for review, see Cuzner and Opdenakker, 1999). The predominant localization of gelatinase B near T cells and around blood vessels in the present study suggests that MMP-9 plays a crucial role in the disruption of vascular basement membranes, thereby paving the way for infiltrating haematogenous cells. As the migration of inflammatory cells from blood into muscle is considered to be of paramount importance in the genesis of immune myopathies, such an action of MMPs would place these enzymes at the centre of this process. To support this notion, we investigated the expression of gelatinase B in an additional muscle biopsy obtained from a patient presenting the clinical picture of facio-scapulohumeral muscular dystrophy. In this type of muscular dystrophy, an inflammatory response is frequently seen. We were able to demonstrate MMP-9 expression in this biopsy, with a distribution pattern similar to that found in polymyositis, dermatomyositis and inclusion body myositis (data not shown). This finding supports the central role of MMP-9 in T-cell migration.

Regulatory mechanisms appear to be of critical importance in controlling the digestion of the ECM in discrete areas, where circumscribed ECM degradation or vectorial cell movement should occur. Studying the mRNA expression of the natural MMP inhibitors (TIMPs) in the muscle biopsies, we could not detect any difference between the various dystrophy groups investigated. In particular, TIMP-1 is known to inhibit both MMP-1 and MMP-9. Selective upregulation of interstitial collagenase and gelatinase B in the absence of increased TIMPs indicates that increased proteolytic activity may develop within the inflamed muscle. Our observation of differential expression of MMP and TIMP is in accord with a recent in vitro study in which it was demonstrated that MMP-1, MMP-9 and TIMP-1 were differently regulated by cytokines, through prostaglandin-dependent and -independent mechanisms (Zhang et al., 1998). These cytokines include the proinflammatory tumour necrosis factor α (TNF-α), which is known to induce the synthesis of MMP-1 in fibroblasts and MMP-9 in T lymphocytes (Dayer et al., 1985; Ries and Petrides, 1995; Johnatty et al., 1997). On the other hand, MMPs are involved in the regulation of bioactive proteins, including cytokines. The release of TNF-α has been reported to depend on an MMP-like mechanism of action (Gearing et al., 1994; Mohler et al., 1994). However, the TNF-α converting enzyme (TACE) has been cloned by two independent groups (Black et al., 1997; Moss et al., 1997) and identified as an adamylysin belonging to the larger group of the metzincin metalloproteinases. In a recent study with a different design, Choi and Dalakas investigated various MMPs in relation to the expression of MHC (the major histocompatibility complex) on muscle fibres involved in antigen recognition, to autoinvasive T cells, and to the presence of amyloid in inclusion body myositis (Choi and Dalakas, 2000). Interestingly, they described overexpression of MMP-9 and MMP-2 in muscle biopsies obtained from patients diagnosed with polymyositis

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and inclusion body myositis, but not from patients with dermatomyositis or muscular dystrophy. In the same study, matrilysin and stromelysin-1 were not seen at the protein level. The presence of interstitial collagenase was not investigated. Using immunohistochemistry, this group was able to localize the gelatinases to muscle fibres, whereas MMP-9 was also found in invading CD8⫹ T lymphocytes. In our present study, primarily addressing the role of MMPs in cell invasion and in connective tissue, we confirm these results in part, but there were discrepancies between the two sets of results. Whereas the expression of gelatinase B and its colocalization to invading T cells were also demonstrated in the present study, in contrast to the results of Choi and Dalakas we observed increased levels of MMP-9 in dermatomyositis biopsies at both the mRNA and the protein level. Moreover, in our study no significant elevation of MMP-2 was detectable, at least at the mRNA level. It is obvious that both studies were designed to investigate MMP expression at distinct phases of disease pathogenesis. However, future studies are required in order to determine whether technical issues may have contributed to this discordance—different assays and antibodies were used—or whether there was unintentional selection bias in the choice of biopsies. In the present investigation we did not find any difference in the circulating levels of MMP-1 or MMP-9 in the serum, as measured by immunoblotting, gelatin zymography and ELISA. This finding underlines the crucial action of MMPs in inflamed muscular tissue in situ. In contrast, elevated levels of MMP-9 were reported in multiple sclerosis. In this disease, a strict correlation between MMP-9 level in the serum and disease activity in the target tissue, as determined by magnetic resonance tomography, has been reported (Yong et al., 1998; Lee et al., 1999). The elevated local production of interstitial collagenase and gelatinase B in polymyositis, dermatomyositis and inclusion body myositis and their distinct distribution pattern provide evidence that MMPs participate in the immunopathogenesis of inflammatory myopathies. It is obvious that a crosssectional study such as ours allows only a snapshot of the disease process at a given time point. It cannot provide a more comprehensive view of an underlying dynamic disease process that develops over months and years. Studies on animal models of inflammatory myopathies will be helpful in examining this issue and furthering our knowledge of the temporospatial expression patterns of selected MMPs during the course of the disease. It is hoped that such insight may help in the design of specific MMP inhibitors that could be administered at critical checkpoints in the evolution of immune-mediated damage to the muscle, and that such compounds may enlarge our still restricted therapeutic armamentarium.

Acknowledgements We thank Heidrun Pischel for excellent technical assistance. This study was supported in part by a grant from the

Gemeinnu¨ tzige Hertie Stiftung (to H.-P.H). Part of this study was presented at the 9th annual meeting of the European Neurological Society.

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Received January 31, 2000. Revised July 31, 2000. Accepted September 25, 2000