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Inflammatory Lesions (CD80) and B7.2 (CD86) in Vitro and In Human Muscle Cells Express a Functional Costimulatory Molecule Distinct from B7.1 (CD80) and B7.2 (CD86) In Vitro and in Inflammatory Lesions This information is current as of September 26, 2021. Lüder Behrens, Martin Kerschensteiner, Thomas Misgeld, Norbert Goebels, Hartmut Wekerle and Reinhard Hohlfeld J Immunol 1998; 161:5943-5951; ; http://www.jimmunol.org/content/161/11/5943 Downloaded from References This article cites 49 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/161/11/5943.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Human Muscle Cells Express a Functional Costimulatory Molecule Distinct from B7.1 (CD80) and B7.2 (CD86) In Vitro and in Inflammatory Lesions1 Lu¨der Behrens,* Martin Kerschensteiner,* Thomas Misgeld,* Norbert Goebels,*† Hartmut Wekerle,* and Reinhard Hohlfeld2*† The B7 family of costimulatory molecules likely includes members distinct from B7.1 (CD80) and B7.2 (CD86). After stimulation with IFN-g or TNF-a, human myoblasts selectively express BB-1, but not B7.1 or B7.2. BB-1 is detected by anti-BB-1, a mAb cross-reacting with B7.1 (but not B7.2) and an as yet undefined costimulatory molecule. The absence of B7.1 and B7.2 in BB-1- positive myoblasts was confirmed by RT-PCR. The molecule detected by anti-BB-1 is functional, because anti-BB-1 mAb and CTLA4Ig (but not anti-B7.1- or anti-B7.2-specific mAbs) completely inhibit Ag presentation by cytokine-induced myoblasts to Downloaded from HLA-DR-matched Ag-specific CD41 T cell lines. Stimulation of myoblasts with IL-4 induces B7.1 and B7.2, as well as BB-1, but with different time kinetics. Stimulation of CD40-positive myoblasts with anti-CD40 mAb selectively induces BB-1, whereas stimulation with CD40L-transfected mouse L cells induces BB-1 and B7.1, with different kinetics. To assess whether BB-1 is expressed in muscle tissue, we investigated 23 muscle biopsy specimens from patients with polymyositis, dermatomyositis, inclu- sion body myositis, Duchenne muscular dystrophy, and nonmyopathic controls by immunohistochemistry and confocal laser microscopy. We found that, in all inflammatory myopathy cases, but not in normal muscle, many muscle fibers strongly react with http://www.jimmunol.org/ anti-BB-1. In contrast, muscle fibers did not react with B7.1- or B7.2-monospecific mAbs in any of the pathologic specimens or in normal muscle. Our results demonstrate that human muscle cells can be induced to selectively express BB-1, a functional co- stimulatory molecule distinct from B7.1 and B7.2. This molecule may play an important role in the immunobiology of muscle. The Journal of Immunology, 1998, 161: 5943–5951. uscle can be a site of desirable and undesirable im- nocytochemical and RT-PCR studies of cultured myoblasts. Fur- mune reactions (1). These are deliberately induced by thermore, we investigated human muscle biopsy specimens for ex- protein- or DNA-based vaccination (2, 3) or develop pression of the B7 family of costimulatory molecules, using mAbs M by guest on September 26, 2021 spontaneously during the course of autoimmune and infectious specific for B7.1 (12) or B7.2 (13), and anti-BB-1 (9), a mAb that muscle diseases (4, 5). Furthermore, local immune reactions can cross-reacts with B7.1 and a hitherto undefined member of the B7 pose a serious problem after intramuscular injection of vectors for family (10, 11). We observed that, whereas normal muscle fibers gene therapy (6, 7). do not express B7 molecules, there is abundant expression of It has been suggested that muscle cells can actively participate BB-1, but not B7.1 or B7.2, in different inflammatory myopathies. in local immune reactions (1). Notably, human myoblasts can Based on these observations, we propose that BB-1 plays an im- present Ags to CD41 T cells (8). However, little is known about portant role in immune reactions in muscle. the costimulatory molecules expressed by muscle cells in vivo and in vitro. Here, we report that human myoblasts can be induced to selectively express BB-1, a functional CTLA4Ig3-binding co- Materials and Methods stimulatory molecule that is distinct from B7.1 (CD80) and B7.2 Myoblast culture (CD86) (9–11). Human myoblasts were isolated from muscle obtained for diagnostic rea- Our evidence for the differential expression and regulation of the sons from patients with suspected myopathy. For in vitro experiments, different B7 molecules in muscle cells relies on functional, immu- myoblasts were obtained from nonmyopathic tissue. Myoblasts were cul- tured as previously described (8) with some modifications. Briefly, muscle specimens were mechanically dissociated and passed through a steel sieve *Department of Neuroimmunology, Max-Planck Institute of Neurobiology, D-82152 (Tissue Grinder Kit, Sigma, Deisenhofen, Germany). The homogenate was Martinsried, Germany; and †Department of Neurology, Klinikum Grosshadern, Mu- digested in trypsin-EDTA-solution (Life Technologies, Eggenstein, Ger- nich, Germany many) for 30 min at 37°C. The resulting suspension was centrifuged (5 min, 1500 rpm) and washed with PBS. The cells were transferred into Received for publication April 23, 1998. Accepted for publication July 30, 1998. plastic tissue culture flasks (Falcon, Heidelberg, Germany) and incubated The costs of publication of this article were defrayed in part by the payment of page in skeletal muscle cell growth medium (modified MCDB 120 supple- charges. This article must therefore be hereby marked advertisement in accordance mented with 5% FCS, 10 ng/ml epidermal growth factor (EGF), 1 ng/ml with 18 U.S.C. Section 1734 solely to indicate this fact. basic fibroblast growth factor (bFGF), 0.5 mg/ml fetuin, 0.1 mg/ml insulin, 1 This study was supported by the Max-Planck Society, Deutsche Forschungsgemein- 0.4 mg/ml dexamethasone, 50 mg/ml gentamicin sulfate, 50 ng/ml ampho- schaft (SFB 217, Project C13), and a European Community Grant (Project BMH4- tericin B; PromoCell, Heidelberg, Germany). After 30 min, nonadherent CT96-0893). cells were removed and seeded in new tissue flasks coated with poly-L- 2 Address correspondence and reprint requests to Dr. Reinhard Hohlfeld, Department lysine (Sigma) and laminin (kindly provided by Dr. H. Neumann, MPI of of Neuroimmunology, Max-Planck Institute of Neurobiology, D-821521 Martinsried, Neurobiology, Martinsried, Germany) and cultured at 37°C at 5% CO2 in Germany. E-mail address: [email protected] skeletal muscle cell growth medium (see above). The myoblasts were re- 3 Abbreviations used in this paper: CTLA4Ig, CTL-associated protein 4 Ig; MBP, peatedly purified during the culture period and 1 wk before experiments, myelin basic protein; NCAM, neural cell adhesion molecule; TCL, T cell line. using a magnetic cell separation system (Dynal, Hamburg, Germany) and Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 5944 COSTIMULATORY MOLECULES IN HUMAN MUSCLE anti-CD56 mAb (8) (NCAM/Leu-19; Becton Dickinson, Heidelberg, Ger- myoblasts, the MBP-specific T cell lines SS014 and SS018 were used many). Purity was checked by FACS analysis. Only .95% pure cultures between days 13 and 15 after the last restimulation with peptide Ag. were used in all experiments. The cultured myoblasts do not contain pro- fessional APC (macrophages or dendritic cells), since they are are negative Coculture experiments of myoblasts and T cells for CD11b, CD14, and VCAM by flow cytometry and do not express B7.1 or B7.2 by RT-PCR. However, a small proportion (,5%) of NCAM-neg- Myoblasts were plated in 96-well flat-bottom plates (Costar, Bodenheim, 3 4 ative fibroblasts may be present. To exclude the possibility that fibroblasts Germany) at a density of 2 10 cells/well and induced for 72 h with the 3 4 also express BB-1, we isolated and expanded the NCAM-negative cell indicated cytokines. In previous experiments, a cell density of 1–2 10 population and treated them with IFN-g or TNF-a, alone or in combina- myoblasts per flat-bottom well was found optimal for T cell stimulation, tion. The fibroblasts remained negative for B7.1, B7.2, and BB-1 by flow because the myoblasts grow essentially confluent at this seeding density cytometry and did not transcribe B7.1 or B7.2 mRNA by RT-PCR. (8). After induction, myoblasts were irradiated with 50 Gy, washed in The HLA-DR type of the myoblasts was determined by Drs. E. Albert RPMI 1640 (Life Technologies) and cultured in the presence or absence of 3 5 and S. Scholz (Labor fu¨r Immungenetik, Ludwig-Maximilians-Universita¨t, Ag and 2 10 HLA-DR-matched or autologous Ag-specific T cells. Mu¨nchen, Germany), using PCR and sequence-specific oligonucleotide Irradiated PBMC, isolated from blood using standard density gradient cen- primers (Dynal). trifugation (Lymphoprep, Nycomed, Oslo, Norway), were used as profes- sional APC in control experiments. T cell proliferation was measured by Culture of CD40L-transfected mouse fibroblasts [3H]TdR incorporation in a gas scintillation counter (Matrix 96 Direct Beta Counter, Packard, Frankfurt, Germany).
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