Cytokines and chemokines are both expressed by human myoblasts: possible relevance for the immune pathogenesis of muscle inflammation.

Marco De Rossi, Pia Bernasconi, Fulvio Baggi, Renè de Waal Malefyt1 and Renato Mantegazza

Laboratory of Neuroimmunology, Department of Neuromuscular Diseases, National Neurological Institute `Carlo Besta', 20133 Milan, Italy
1 Department of Immunobiology, DNAX Research Institute, Palo Alto, CA 94304-1104, USA

Correspondence to: M. De Rossi


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The idiopathic inflammatory myopathies are characterized by antibody- or cell-mediated immune response against unknown muscle tissue antigens. In these diseases a cellular infiltrate, composed of T and B lymphocytes, macrophages and NK cells, may invade muscle tissue with a gradient from the perivascular space to the endomysial compartment. Muscle cells may be actively involved in the processes of mononuclear cell recruitment and activation from the blood stream to the areas of inflammation. In order to verify this hypothesis, cultured human myoblasts were tested for their capacity to express different pro-inflammatory cytokines [IL-1{alpha}, IL-1ß, IL-6 and tumor necrosis factor (TNF)-{alpha}] and chemokines (IL-8, MCP-1 and RANTES) at the mRNA level and protein secretion, in the presence of the pro-inflammatory cytokines IFN-{gamma} and TNF-{alpha} alone or in combination. We confirmed that human myoblasts expressed IL-1{alpha} and IL-6 constitutively, while IL-1ß and TNF-{alpha} are detected only after treatment with pro-inflammatory cytokines; moreover, we observed that TNF-{alpha} was expressed on an autocrine fashion by myoblasts. IL-8 and RANTES were expressed constitutively while MCP-1 after proper induction. These molecular data were further confirmed by specific ELISA in the supernatant from cultured myoblasts. Our results underline the importance of human myoblasts in the recruitment of leukocytes from the blood stream and, most probably, in the cross-talk between infiltrating inflammatory cells and muscle cells, creating the conditions for a chronic inflammation. Moreover, the capacity of muscle cells to behave as cells of the immune system has to be kept in mind, also in view of i.m. vaccination and use of molecular engineered myoblasts as vehicles in gene therapy.

Keywords: chemokines, cytokines, human myoblasts, idiopathic inflammatory myopathies


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human muscle cells are involved as targets in different pathological conditions such as muscle infections (1), autoimmune muscle diseases (2) and myositis complicating graft versus host disease (3); these diseases are characterized by mononuclear cell infiltrates in the target muscle tissue (4). Moreover, i.m. vaccination may be a cause for local immune reactions eventually leading to systemic symptoms such as macrophagic myofascitiis (5,6).

Use of genetically modified myoblasts as vehicles in gene therapy (7) has increased the importance of understanding their capacity to act as facultative antigen-presenting cells (APC) and their interplay with infiltrating mononuclear cells, mediated by active transcription and release of cytokine and chemokine by both cell types (8,9).

A key factor for the initiation and sustainment of target tissue inflammation is the in situ cytokine expression by leukocytes, endothelial cells and mesenchymal cells (8). Human myoblasts were shown to produce IL-6, an important cofactor in T cell proliferation and cytotoxicity, and this secretion is greatly increased by tumor necrosis factor (TNF)-{alpha} (10). Other cytokines, and in particular IFN-{gamma}, are potent inducers of both MHC class I and II molecules as well as cell adhesion molecules in muscle cells (1114). IFN-{gamma}-treated myoblasts can present myelin basic protein to antigen-specific T cell clones in vitro, acting as non-professional APC able to deliver to primed cells the proper signal for proliferation (15). However, myoblasts cannot induce an in vitro primary alloreactive response, such as a mixed lymphocyte reaction, even if muscle cells were pre-treated with IFN-{gamma} or IL-4 plus granulocyte macrophage colony stimulating factor (16).

Chemokines are directly involved in the process of leukocyte recruitment and activation in the site of inflammation of normal and neoplastic tissue (9). Chemokines are a novel class of small cytokines characterized by the presence of four conserved cysteines (Cys) linked by disulfide bonds, a short N-terminal domain and a long C-terminal region. Four families of chemokines have been identified: {alpha}-chemokines (Cys–X–Cys), ß-chemokines (Cys–Cys), {gamma}-chemokines (Cys) and {delta}-chemokines (Cys–X3–Cys) (9). IL-8 is the most studied member of the Cys-X-Cys chemokine family, activates leukocytes and has the properties of a potent neutrophil chemoattractant (17); monocytes, basophils, eosinophils, and T lymphocytes are weaker responders to IL-8 (18). MCP-1 and RANTES are members of the ß-chemokine group (Cys–Cys). MCP-1 attracts monocytes, memory T lymphocytes and NK cells in vitro, and its expression is often correlated with inflammatory diseases (19,20). RANTES acts through receptors expressed on eosinophils, monocytes and CD45RO+ T lymphocytes; it is also responsible for the recruitment of these cells from the blood stream to the inflamed area (9,21).

In the present paper we report on the transcription and production of different pro-inflammatory cytokines and chemokines by cultured human myoblasts, either constitutively or when stimulated by pro-inflammatory cytokines, to better understand the role of muscle cells in the inflammatory processes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell cultures
Human myoblasts were isolated from two muscle biopsies obtained for diagnostic reasons from patients with suspected myopathy and cultured in DMEM (Gibco, Gaithersburg, MD) containing 20% FCS (Hyclone, Logan, UT), 10 ng/ml epidermal growth factor (Gibco) and 10 µg/ml bovine insulin (Sigma, St Louis, MO) at 37°C/5% CO2 (22). Myoblast cultures were examined every day under an inverted phase-contrast microscope, and their purity was checked by FACS analysis using anti-CD56 mAb (NCAM/Leu19) (23), anti-CD11b mAb, anti-CD14 mAb and anti-VCAM mAb (all from Becton Dickinson). Only >95% pure cultures were used in all experiments.

Human fibroblasts were grown in DMEM supplemented with 10% FCS, penicillin–streptomycin, L-glutamine and ascorbic acid (all from Gibco/BRL, Eggenstein, Germany) and maintained in a 5% CO2 atmosphere at 37°C.

Peripheral blood mononuclear cells (PBMC) were separated from whole blood of healthy donors by Ficoll-Hypaque gradient (Sigma) centrifugation and then resuspended in RPMI 1640/10% FCS (Gibco/BRL) in the presence of 10 µg/ml of phytohemagglutinin (Sigma), 5 µg/ml concanavalin A (Sigma) and 10 µg/ml lipopolysaccharide (Difco, Detroit, MI) for 24 h.

Cytokine treatment of cultured cells
Human myoblasts and fibroblasts were incubated for 4 h in a 5% CO2 atmosphere at 37°C with 100 U/ml human recombinant IFN-{gamma} (Boehringer, Mannheim, Germany) and 500 U/ml human recombinant TNF-{alpha} (donated by Dr U. Traugott and Dr M. Müller-Neumann, BASF/Knoll, Ludwigshafen, Germany); these concentrations were reported to be effective in inducing expression of MHC class II and ICAM molecules by cultured myoblasts (12). Cytokine-stimulated cells were then lysed for RT-PCR or extensively washed and the medium, replaced with a fresh one, was collected after 48 h and stored at –20°C until the ELISA was performed.

RT-PCR analysis
Total RNA was isolated from confluent cell cultures by the RNeasy Mini Kit (M-Medical SRL, Firenze, Italy). One microgram of total RNA was mixed with 2.5 µM random hexamers in 5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, pH 8.3, 1 mM each dNTP and 2.5 MMLV reverse transcriptase (Perkin-Elmer Cetus, Norwalk, CT) in a final volume of 20 µl. Reverse transcription was carried out at 42°C for 15 min. The resulting cDNA was then amplified by adding 2 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, pH 8.3, 2.5 U Taq DNA polymerase (Perkin-Elmer Cetus) and 0.15 µM each specific primer in a final volume of 100 µl. The PCR conditions were: initial denaturation 5 min, 94°C; denaturation 1 min, 95°C; annealing 1 min, 58°C; extension 1 min, 72°C for 30 cycles; followed by 10 min elongation step at 72°C. Twenty microliters of the reaction mixture was run on a 1.5% agarose gel stained with ethidium bromide. To exclude possible amplification of cytokine genomic sequence, PCR primers for individual cytokines were designed to amplify cDNA fragments that span at least one intron of the corresponding gene. In all cases, PCR-amplified bands of the appropriate size were generated with each primer pair. Primer sequences were: IL-1{alpha}, forward 5'-GTCTCTGAATCAGAAATCCTTCTATC-3', reverse 5'-CATGTCAAATTTCACTGCTTCATCC-3'; IL-1ß, forward 5'-ATGGCAGAAGTACCTGAGCTC-3', reverse 5'-GGAAGACACAAATTGCATGGT-3'; IL-6, forward 5'-ATGAACTCCTTCTCCACAAG-3', reverse 5'-ACATTTGCCGAAGAGCCCTCAG-3'; IL-8, forward 5'-CTTGGCAGCCTTCCTGATTT-3', reverse 5'-CTCAGCCCTCTTCAAAAACT-3'; TNF-{alpha}, forward 5'-TATACAAGTTATATCTTGGCT-3', reverse 5'-GGGCAATGATCCCAAAGTAGACC-3'; MCP-1, forward 5'-ACTGAAGCTCGTACTCTC-3', reverse 5'-CTTGGGTTGTGGAGTGAG-3'; RANTES, forward 5'-CGGCACGCCTCGCTGTCATC-3', reverse 5'-TGTACTCCCGAACCCATTT-3'; GAPDH, forward 5'-ACCACCTGGTGCTCAGTGTA-3', reverse 5'-ACCATCTTCCAGGAGCGAGA-3'.

Cytokine and chemokine ELISA
ELISA assay for measuring IL-1, IL-6 and IL-8 was performed as described by Abrams et al. (24). MCP-1, RANTES and TNF-{alpha} were measured using commercially available kits (Biosource, Camarillo, CA).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Different pattern of expression of pro-inflammatory cytokines by cultured human myoblasts
Cultured human myoblasts were evaluated for the expression of IL-1{alpha}, IL-1ß, IL-6 and TNF-{alpha} cytokine mRNAs (Fig. 1Go). In unstimulated myoblasts we observed a constitutive transcription only for IL-1{alpha} and IL-6, whereas IL-1ß and TNF-{alpha} were not found. Differences in mRNA levels were observed when cultured myoblasts were treated with IFN-{gamma}, TNF-{alpha} or both. Differences in cytokine mRNA expression were evaluated by densitometric analysis of band intensity on agarose gel stained with ethidium bromide and the relative amount was related to the housekeeping gene GAPDH (Fig. 2AGo). IL-1{alpha} and IL-1ß transcripts were slightly affected by IFN-{gamma} treatment, while a marked increase was observed when myoblasts were treated with TNF-{alpha} or with IFN-{gamma} plus TNF-{alpha}. Only a moderate effect was observed on IL-6 by the different treatments. Interestingly, TNF-{alpha} transcript was not observed in untreated myoblasts, but it was induced when cultured cells were treated by exogenous TNF-{alpha} either alone or in combination with IFN-{gamma}.



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Fig. 1. Expression of IL-1{alpha}, IL-1ß, IL-6, TNF-{alpha}, RANTES, MCP-1 and IL-8 mRNAs, by human fibroblasts (hF) and human myoblasts (hMYO) examined by RT-PCR. Cells were unstimulated (unst.) or stimulated with IFN-{gamma} (100 U/ml, 4 h), with TNF-{alpha} (500 U/ml, 4 h) or with IFN-{gamma} (100 U/ml, 4 h) + TNF-{alpha} (500U/ml, 4 h). Positive control (pos. ctrl): mRNA from mitogen-activated PBMC. GAPDH was used as housekeeping gene for densitometric analysis. Blank: no cDNA was added in the PCR reaction. MW: {Phi}X174 DNA cleaved with HaeIII and pBR322 DNA cleaved with HaeIII.

 


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Fig. 2. Levels of transcription in human myoblasts of the genes for cytokines (A) and chemokines (B) before and after treatment with IFN-{gamma}, TNF-{alpha} and their combination. Densitometric analysis was performed on ethidium bromide-stained agarose gel by software program Kodak Digital Science; the net intensity of each band was compared to that one of the housekeeping gene GAPDH and their ratio are reported.

 
Human myoblasts express transcripts for chemokines
The Cys–Cys chemokine MCP-1 was not found to be constitutively expressed by cultured myoblasts, but its transcription could be induced by treatment with pro-inflammatory cytokines either alone or in combination (Figs 1 and 2BGoGo). On the contrary, RANTES was constitutively expressed and its transcription level was affected by TNF-{alpha} (alone or in combination with IFN-{gamma}) and not by IFN-{gamma} (Figs 1 and 2BGoGo). IL-8, the prototype of the Cys–X–Cys chemokines, was constitutively expressed at the mRNA level; differences in its transcription were observed when myoblasts were cultured in the presence of inflammatory cytokines (Figs 1 and 2BGoGo).

Human fibroblasts express transcripts for pro-inflammatory cytokines and chemokines either constitutively or when treated with inflammatory cytokines
Cultured myoblasts were negative for the expression of CD11b, CD14 and VCAM, suggesting that professional APC such as B cells, macrophages or dendritic cells were not present in our cultures; moreover, the culture medium was not permissive for long-lasting survival of T and B cells. However, a small proportion (< 5%) of NCAM-negative fibroblasts may be present. For this reason we studied the expression of cytokines and chemokines on stimulated and unstimulated cultured human fibroblasts (Figs 1 and 3GoGo). These cells were found to be positive constitutively for the cytokines and chemokines studied, even if with different extent, but not for TNF-{alpha} and RANTES. An increase in their expression was observed when fibroblasts were treated with pro-inflammatory cytokines. TNF-{alpha} and RANTES were undetectable and at the lower sensitivity limit of our densitometric analysis system, and their expression was not affected by IFN-{gamma} treatment. However, the transcripts were measurable after cell treatment with exogenous TNF-{alpha} or IFN-{gamma} plus TNF-{alpha}.



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Fig. 3. Levels of transcription in human fibroblasts of the genes for cytokines (A) and chemokines (B) before and after treatment with IFN-{gamma} , TNF-{alpha} and their combination. Densitometric analysis was performed on ethidium-bromide stained agarose gel by software program Kodak Digital Science; the net intensity of each band was compared to that one of the housekeeping gene GAPDH and their ratio are reported.

 
Human myoblasts produce pro-inflammatory cytokine and chemokine proteins in vitro
Cytokine and chemokine release in culture supernatants by human myoblasts was analyzed by ELISA either under basal conditions and after treatment with IFN-{gamma} and TNF-{alpha}, alone or in combination (Fig. 4Go). IL-1 was detected in the culture medium of untreated myoblasts (13 pg/ml) with very little increase after treatment. IL-6 expression was constitutive in human myoblasts (3312 pg/ml) and was affected by IFN-{gamma} (5918 pg/ml) or by TNF-{alpha} (16811 pg/ml) treatment; however, the maximum increase was obtained with the combination of inflammatory cytokines (27,040 pg/ml). TNF-{alpha} was detected in supernatants of human myoblasts only when cells were treated with exogenous TNF-{alpha} (467 pg/ml) and further enhanced by IFN-{gamma} plus TNF-{alpha} treatment (701 pg/ml), but it was not influenced by IFN-{gamma} alone.



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Fig. 4. Cytokine and chemokine production by human myoblasts. Cells were cultured in the absence (basal) and in the presence of IFN-{gamma} (100 U/ml for 4 h), TNF-{alpha} (500 U/ml for 4 h) or a combination of both, and the supernatants were collected after 48 h and stored at –20°C until ELISA was performed. Values are expressed in pg/ml.

 
Among chemokines, RANTES was not found in the supernatants of cultured human myoblasts either in the basal condition or after induction with IFN-{gamma}; RANTES was induced by TNF-{alpha} (695 pg/ml) and by IFN-{gamma} plus TNF-{alpha} (6883 pg/ml) treatments. MCP-1 expression was not found in supernatants from untreated myoblasts but was clearly induced by treatment with IFN-{gamma} (2510 pg/ml), TNF-{alpha} (2915 pg/ml) or both (3670 pg/ml). Only IL-8 was shown to be constitutively expressed in untreated human myoblasts (114 pg/ml); again, it was inducible after IFN-{gamma} (865 pg/ml) or TNF-{alpha} (1153 pg/ml) or both (1532 pg/ml).

Human fibroblasts produce pro-inflammatory cytokine and chemokine protein in vitro
Cytokine and chemokine release by human fibroblasts was studied and results are illustrated in Fig. 5Go. Pro-inflammatory cytokines IL-1 (10 pg/ml), IL-6 (16,775 pg/ml) and TNF-{alpha} (171 pg/ml) were found in untreated fibroblasts. Higher amounts of IL-6 were observed in fibroblasts than in myoblasts (Fig. 4Go) and its secretion was only affected by IFN-{gamma} treatment (26,898 pg/ml). TNF-{alpha} was greatly increased only by exogenous TNF-{alpha} treatment (533 pg/ml). MCP-1 and IL-8 were found constitutively expressed (2596 and 1338 pg/ml respectively), while RANTES was not found in fibroblast supernatant. Fibroblast treatment with pro-inflammatory cytokines did not greatly affect MCP-1 level, while an increase in protein release was observed by IFN-{gamma} plus TNF-{alpha} treatment for IL-8 (3075 pg/ml) and RANTES (5721 pg/ml).



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Fig. 5. Cytokine and chemokine production by human fibroblasts. Cells were cultured in the absence (basal) and in the presence of IFN-{gamma} (100 U/ml for 4 h), TNF-{alpha} (500 U/ml for 4 h) or a combination of both, and the supernatants were collected after 48 h and stored at –20°C until ELISA was performed. Values are expressed in pg/ml.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present paper we report on the transcription and secretion by cultured human myoblasts of several cytokines and chemokines that might play a role in the inflammation processes within muscle tissues. We demonstrate by molecular and protein data that human myoblasts can spontaneously produce different cytokines and chemokines; moreover, their transcription and expression can be affected by treatment with specific pro-inflammatory cytokines. These results suggest that muscle cells may behave as immunologically active cells during inflammation. Indeed human myoblasts produce cytokines of the monocyte–macrophage lineage (IL-1{alpha}, IL-1ß, IL-6 and TNF-{alpha}) as well as Cys–X–Cys chemokines (IL-8) and Cys–Cys chemokines (MCP-1 and RANTES). IL-1, IL-6, IL-8 were expressed constitutively, whereas the other cytokines/chemokines are expressed only in the presence of specific pro-inflammatory stimuli. Lundberg et al. reported the presence of IL-1{alpha} and IL-1ß in muscle biopsies of idiopathic inflammatory myopathies (IIM) by immunohistochemistry (25); IL-1{alpha} was localized not only in the inflammatory cells but also in the endothelial cells of capillaries and venules. They suggested that this cytokine might play a role in the pathogenesis of IIM and that this molecule could be a possible target of therapeutic strategies (25). They reported also that IIM muscle cells were negative for IL-1{alpha} and this is different with our data on cultured myoblasts: the different techniques employed (RT-PCR versus immunohistochemistry) might partially account for the observed differences. RT-PCR is a very sensitive technique and the results must be biased by the presence of contaminating mRNA molecules from other cell types. In order to avoid misleading results, special precautions need to be considered, such as keeping PCR condition very stringent, not exceeding the amplification cycles (`saturating conditions'), assessment of cell purity and highly specific culture conditions. The state of muscle cell differentiation should also be considered, which was shown to be a critical point for the expression of molecules of the immune system such as MHC class II on myocytes (22).

We confirmed the expression and release of IL-6 by human myoblasts as already published (10); however, in contrast to this previous report we found a synergistic effect of IFN-{gamma} and TNF-{alpha}. Recently, TNF-{alpha} and its receptors have been immunolocalized in muscle biopsies of IIM patients; TNF-{alpha} was detected in invading macrophages, in myonuclei of regenerating muscle fibers and freely dispersed in endomysial or perimysial connective tissue. Many endothelial cells in dermatomyositis expressed TNF-{alpha} (26). TNF-{alpha} is a very active cytokine and possesses a variety of functions not only by stimulating the de novo production of various lymphokines and chemokines such as IL-1{alpha}, IL-1ß, MCP-1 and RANTES, but also by increasing the level of expression of IL-6 and IL-8. Interestingly, we showed that in myoblasts TNF-{alpha} can induce its own expression (see Figs 1 and 2GoGo). Furthermore, TNF-{alpha} is known to up-regulate on endothelial cell surface adhesion molecules like intercellular adhesion molecule-1, E-selectin and VCAM-1, which are molecules critical for mononuclear cell trafficking into target tissues (2730). On the basis of our data and those of De Bleecker et al. (26), we suggest that TNF-{alpha} may play a crucial role in inducing and maintaining inflammation in IIM.

We showed that IL-8 was expressed in human myoblasts on a constitutive basis, but its possible role in the pathophysiology of autoimmune muscle diseases remains undefined. In this regard, IL-8 is known to play an important role for neutrophil recruitment, whereas its effect on monocytes, basophils and eosinophils is only moderate(18). T lymphocytes express receptors for IL-8 which can be detected only by RT-PCR and not by Northern blotting, thus suggesting that their numbers are low on T cells (18,31). At the present time no information are available with regard to IL-8 immunolocalization in muscle tissue from IIM patients.

We reported on the expression by human myoblasts of two important Cys–Cys chemokines, i.e. MCP-1 and RANTES. MCP-1 has been implicated in many diseases such as atherosclerosis, rheumatoid arthritis, tumors and multiple sclerosis, which all present a hallmark of a monocyte-rich infiltrate (1921). This chemokine plays a central role not only in monocyte trafficking but even in their activation. Only recently, MCP-1 has been shown to have additional activities toward basophil and eosinophil granulocytes, NK cells and memory T lymphocytes (9,20,21). We have already demonstrated that MCP-1 is actively transcribed only in IIM muscle biopsies, and the protein localized in infiltrating inflammatory cells and extracellular matrix (32). These data together with the new results confirm the presence of this Cys–Cys chemokine in association with a state of inflamed muscle.

RANTES is another Cys–Cys chemokine which may have a pivotal role in the trafficking of peripheral blood leukocytes involved in immunoregulatory and inflammatory processes of IIM. RANTES is the ligand for the CCR5 receptor which is characteristic of Th1 lymphocytes (33), which are mainly involved in chronic inflammatory diseases. Moreover, its presence, at least at the transcriptional level, as has already been shown in IIM (34). The constitutive expression of RANTES observed by RT-PCR analysis was not confirmed when this chemokine was assayed by specific ELISA in culture supernatant; RANTES was detected at high level only after myoblast treatment with IFN-{gamma} plus TNF-{alpha}.

Our study was performed on myoblasts and fibroblasts cultured in vitro, and thus does not allow us to correlate the expression of studied molecules neither to the disease status of the patients nor to the inflammatory milieu of the muscle tissue. However, we hypothesize that human myoblasts, after acute or chronic inflammation caused by an unknown agent, might drive the formation of the inflammatory exudate, and sustain its persistence by means of the synthesis of cytokines and chemokines, key factors for the migration of leukocytes from the blood stream to the target tissue. This might be also true for fibroblasts present in muscle tissue, since these cells produce both pro-inflammatory cytokines and chemokines: human fibroblasts as well as human myoblasts could determine activation and recruitment of immune cells in the inflammatory exudate.

In conclusion, we have demonstrated that human myoblasts are not only the target of immune-mediated cytotoxic reactions, but that they may directly release cytokines/chemokines necessary to recruit immunocompetent cells in the site of inflammation characteristic of autoimmune muscle disease.


    Acknowledgments
 
We wish to thank Professor R. Hohlfeld for critical reading of the manuscript and fruitful suggestions that have significantly helped our work.


    Abbreviations
 
APC antigen-presenting cell
IIM idiopathic inflammatory myopathies
PBMC peripheral blood mononuclear cell
TNF tumor necrosis factor

    Notes
 
Transmitting editor: L. Steinman

Received 28 February 2000, accepted 23 May 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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