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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 15, 2003 as doi:10.1096/fj.03-0039fje. |
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* Department of Neurology, University of Tübingen, Medical School, Tübingen, Germany;
Department of Immunology, Mayo Clinic Rochester, Minnesota, USA ; and
Genzentrum, Friedrich-Baur Institute, and Department of Neurology, Ludwig Maximilians University, Munich, Germany
2 Correspondence: Department of Neurology, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany. E-mail: heinz.wiendl{at}uni-tuebingen.de
SPECIFIC AIMS
Muscle fibers and myoblasts can participate in immune cell interactions and may play an important role in initiating or perpetuating muscle-derived or directed immune responses. Up-regulation of major histocompatibility (MHC) class I and II antigens under pathological conditions is essential for the interaction of muscle with CD8 cytotoxic T cells and CD4 T helper cells, as observed in the idiopathic inflammatory myopathies. Human muscle cells can present antigens to CD4 T cells and are able to modulate T cell responses via accessory molecules. The expression of B7 costimulatory molecules distinct from B7.1/2 (CD80/CD86) has been postulated but lacks molecular definition and characterization of its functional significance.
PRINCIPAL FINDINGS
1. B7-H1 protein is expressed in TE671 rhabdomyosarcoma cells and cultured human myoblasts in the presence of IFN-
B7-H1 (PD-L1), is a type I transmembrane protein with 20% amino acid identity to B7.1 and 15% amino acid identity to B7.2. We investigated TE671 muscle rhabdomyosarcoma cells (TE671 wild-type cells, TE671-DR4 transfectants) and cultured human myoblasts for expression of this novel member of the B7 family of costimulatory molecules. TE671 did not express B7-H1 constitutively, but strongly up-regulated the cell surface expression of B7-H1 in response to IFN- (Fig. 1
A), with a maximum expression at 72 h. TNF-
had no effect on B7-H1 expression. Cultured myoblasts from five donors were examined for B7-H1 expression. Myoblasts constitutively express MHC-I, and MHC-II is induced by IFN- but not TNF- (Fig. 1B
). By analogy to TE671, unstimulated cultured myoblasts did not express B7-H1 in four of five myoblast preparations. IFN- strongly induced cell surface expression of B7-H1 in all myoblast lines but TNF- alone had no effect on B7-H1 protein expression (Fig. 1B
). No differences were observed between myoblasts derived from patients with inflammatory myopathies and nonmyopathic control subjects. When IFN- and TNF- were added together, a synergistic effect on up-regulation was observed (data not shown). Human myoblasts showed a similar time kinetic of up-regulation as TE671 cells (data not shown). In contrast to B7-H1, neither cultured myoblasts nor TE671 expressed B7.1 or B7.2 protein under any condition.
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Protein data were corroborated by analysis of RNA from purified cultured myoblasts and TE671 cells. As expected, human myoblasts did not express B7.1 or B7.2 mRNA. mRNA for B7-H1 was virtually absent in unstimulated myoblasts, whereas IFN- or IFN- plus TNF- induced B7-H1 (PD-L1) mRNA expression by a factor of 30.3 to 92.7. PD-L2 (also termed B7-DC) was recently identified as a second ligand for PD-1 predominantly expressed on dendritic cells. A slight up-regulation was measurable after treatment with IFN- or IFN- plus TNF- (factor 2.5 to 3.95).
2. Muscle-related B7-H1 inhibits activation of CD4 and CD8 T cells
Next we characterized the functional role of muscle-related B7-H1 expression for muscle immune cell interactions. Functional significance was assessed by experiments coculturing muscle cells with purified allogeneic CD4 and CD8 T cells in the presence of antigen. Relevance of B7-H1 for modulating T cell cytokine production and expression of cell surface markers of T cell activation was measured. Human myoblasts were preinduced to express B7-H1 and MHC-II by addition of IFN- and subsequently cocultured with CD4 or CD8 T cells and antigen (superantigen or glatirameracetate) in the presence of neutralizing anti-B7-H1 mAb 5H1 or respective isotype control. Release of IFN- and IL-2 into the supernatant was measured. 5H1 markedly augmented the production of IFN- and IL-2 by CD4 as well as CD8 T cells (factor 1.3 to 6.3; Fig. 2
). The inhibitory effect of B7-H1 was also demonstrated by assessing the expression pattern of T cell activation markers after coculture in the presence or absence of a neutralizing B7-H1 antibody. Neutralization of B7-H1 led to increased expression levels of CD25, CD69, and ICOS on CD4 and CD8 T cells, demonstrating the inhibitory role of muscle-related B7-H1 for T helper and cytotoxic T cell activation. Similar experiments were also carried out with TE671 cells and TE671-DR4 rhabdomyosarcoma cells corroborating the inhibitory effect of B7-H1 expression for T cell activation.
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3. B7-H1 is expressed in inflamed muscle tissue in vivo
To further address the pathophysiological relevance of B7-H1, we examined muscle biopsy specimens from patients with idiopathic inflammatory myopathies [polymyositis (n=6), inclusion body myositis (n=5), dermatomyositis (n=6)] and from nonmyopathic and noninflammatory controls (n=6) for B7-H1 expression by immunohistochemistry. MHC class I expression and T cell markers (anti-CD8) were assessed in parallel. No B7-H1 expression was detected on muscle biopsy specimens from nonmyopathic controls or noninflammatory myopathies (6 of 6). In contrast, B7-H1 expression was detectable in inflammatory myopathies (PM: 6 of 6; IBM: 5 of 6; DM: 5 of 6). Muscle-related expression of B7-H1 was observed on the surface and to some extent in the cytoplasm of muscle fibers. It was localized in areas where inflammatory cells lay in close apposition to damaged or non-necrotic muscle fibers. B7-H1-positive muscle fibers were in direct contact with mononuclear cells, including T cells, thus corroborating the assumption of the in vivo relevance of B7-H1 interactions in inflammatory muscle disorders. Some regenerating or degenerating muscle fibers also showed B7-H1 staining. Mononuclear cells within the inflammatory tissue sections reflecting B cells, monocytes, or T cells stained positive for B7-H1, in accordance with the described lymphoid expression patterns of B7-H1. Some muscle capillaries in proximity to inflammatory infiltrates, especially in dermatomyositis, exhibited B7-H1 reactivity.
CONCLUSIONS AND SIGNIFICANCE
Insight into the immune regulatory properties of muscle is important for a better understanding of the desirable and undesirable immune reactions occurring in this tissue and necessary in order to control novel therapeutic strategies. Here we demonstrate that muscle cells express B7-H1, a novel member of the B7 family of costimulatory molecules. TE671 muscle rhabdomyosarcoma cells and cultured human myoblasts show high levels of B7-H1 mRNA and protein after stimulation with the inflammatory cytokine IFN- (Fig. 1)
. In biopsy specimens of inflammatory myopathies, B7-H1 expression was detected on muscle fibers in areas characterized by the presence of inflammatory cells and muscle fiber damage. B7-H1 expressed on muscle cells was a potent inhibitor of CD4 and CD8 T cell function, as it reduced cytokine production and expression of T cell activation markers in cocultures of muscle cells with lymphocytes in the presence of antigen (Fig. 2)
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B7.1/2(CD80/86) is believed to be a prerequisite for antigen-presenting cells to initiate immune responses according to the two-signal model of T cell activation. However, muscle fibers and cultured myoblasts do not express detectable levels of B7.1 or B7.2 protein under normal or inflammatory conditions, but nonetheless are able to modulate antigen-specific T cell responses. Therefore, other costimulatory molecules have been postulated, including BB-1, a putative B7-related costimulatory protein that interacts with CD28/CTLA-4 and has costimulatory function. The demonstration of muscle-related B7-H1 expression together with its functional consequences for muscle–immune cell interactions advances the understanding of immune regulatory mechanisms promoted by muscle cells. Costimulatory signals play a key role in regulating T cell activation and are decisive in inciting and perpetuating cellular effector mechanisms of various inflammatory conditions, including autoimmune diseases. Whereas B7.1/2/CD28 interactions have been studied extensively in various animal models and in human disease, the immunological role of novel members of the B7 family such as B7-H1 is far from being completely understood. The important advance of this work is the characterization of a molecularly defined B7 costimulatory molecule detectable on muscle cells in vitro and in vivo together with data suggesting an immunobiological role in autoimmune myopathies.
What might be the role of B7-H1 in muscle? In nonprofessional antigen-presenting cells such as muscle, B7-H1 expression might be a protective mechanism activated in response to immune-mediated damage of MHC-expressing target cells and provoked by inflammatory cytokines like IFN- (Fig. 3
). The expression patterns of B7-H1 observed in inflammatory myopathies and our functional experiments support this view. Our data thus identify B7-H1 as a novel negative immunoregulatory principle exerted by muscle cells and induced upon inflammatory stimuli. In autoimmune inflammatory myopathies this might be aimed at protecting muscle fibers from immune aggression. The observation that muscle-related B7-H1 directly inhibits cytotoxic CD8 T cell and CD4 T helper functions is of particular importance: B7-H1 on muscle fibers could directly interfere with cytotoxic T cell effector functions and inhibit the production of effector cytokines by interaction with CD4 effector/memory in vivo. Thus, B7-H1 expression might serve as an immunoprotective mechanism aimed at counterbalancing immune-mediated damage.
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The observation of muscle-related expression of B7-H1 has broad implications for the immunobiology of muscle, as it could play an important role in many different immune reactions that occur in this tissue. As hypothesized, B7-H1 could protect muscle fibers from cell-mediated injury in autoimmune muscle disorders (polymyositis, dermatomyositis, or inclusion body myositis) or in various muscle infections. B7-H1 could modulate the immune responses after protein- or DNA-based vaccinations and efficiently reduce muscle-directed antigen-specific and non-antigen-specific immune responses in either condition. Our data provide evidence for the strong capability of muscle to promote immunoprotective or immunosuppressive mechanisms and support a concept proposing that the expression of B7-H1 in the periphery may regulate local levels of immune inflammation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0039fje; doi: 10.1096/fj.03-0039fje 
3 These authors contributed equally.
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