No abstract

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) o n the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFPl), from tomato. In contrast t o the few animal MAR DNA binding proteins thus far identified, MFPl contains a predicted N-terminal transmembrane domain and a long filament-like a-helical domain that is similar to diverse nuclear and cyto-plasmic filament proteins from animals and yeast. DNA binding assays established that MFPl can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFPl revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFPl is an in vitro substrate for casein kinase II, a nuctear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix local-ization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.

Inducible co-stimulator ligand (ICOSL), a member of the B7 family of co-stimulatory molecules related to B7.1/2, regulates CD4 as well as CD8 T-cell responses via interaction with its receptor ICOS on activated T cells. Here we examined the expression and the functional relevance of ICOSL in human muscle cells in vivo and in vitro. We investigated 25 muscle biopsy specimens from patients with polymyositis, dermatomyositis, inclusion body myositis, Duchenne muscular dystrophy and non-myopathic controls for ICOSL expression by immunohistochemistry. Normal muscle fibres constitutively express low levels of ICOSL. However, ICOSL expression is markedly increased in muscle fibres in inflammatory myopathies. Cell surface staining was most prominent in the contact areas between muscle fibres and inflammatory cells, which in turn show expression of ICOS as a marker of T-cell activation. Muscle endothelial cells show constitutive expression of ICOSL under normal and pathological conditions. We also detected mRNA and cell surface protein expression of ICOSL on myoblasts cultured from control subjects and patients as well as in TE671 muscle rhabdomyosarcoma cells. ICOSL expression was upregulated by tumour necrosis factor-alpha (TNF-alpha), whereas interferon-gamma (IFN-gamma) had no such effect. Co-culture experiments of major histocompatibility complex (MHC) class II-positive myoblasts with CD4 T cells together with superantigen demonstrated that the expression of muscle-related ICOSL has functional consequences: the production of Th1 (IFN-gamma) and Th2 cytokines [interleukin (IL)-4 and IL-10] by CD4 T cells was markedly reduced in the presence of a neutralizing anti-ICOSL monoclonal antibody (mAb HIL-131), thus showing the importance of ICOSL co-stimulation for T-cell activation. Taken together, our results demonstrate that human muscle cells express ICOSL, a functional co-stimulatory molecule distinct from B7.1 and B7.2. ICOSL-ICOS interactions may play an important role in inflammatory myopathies, providing further evidence for the antigen-presenting capacity of muscle cells.

B7-H1 is a novel B7 family protein attributed to costimulatory and immune regulatory functions. Here we report that human myoblasts cultured from control subjects and patients with inflammatory myopathies as well as TE671 muscle rhabdomyosarcoma cells express high levels of B7-H1 after stimulation with the inflammatory cytokine IFN-gamma. Coculture experiments of MHC class I/II-positive myoblasts with CD4 and CD8 T cells in the presence of antigen demonstrated the functional consequences of muscle-related B7-H1 expression: production of inflammatory cytokines, IFN-gamma and IL-2, by CD4 as well CD8 T cells was markedly enhanced in the presence of a neutralizing anti-B7-H1 antibody. This observation was paralleled by an augmented expression of the T cell activation markers CD25, ICOS, and CD69, thus showing B7-H1-mediated inhibition of T cell activation. Further, we investigated 23 muscle biopsy specimens from patients with polymyositis (PM), inclusion body myositis (IBM), dermatomyositis (DM), and nonmyopathic controls for B7-H1 expression by immunohistochemistry: B7-H1 was expressed in PM, IBM, and DM specimens but not in noninflammatory and nonmyopathic controls. Staining was predominantly localized to areas of strong inflammation and to muscle cells as well as mononuclear cells. These data highlight the immune regulatory properties of muscle cells and suggest that B7-H1 expression represents an inhibitory mechanism induced upon inflammatory stimuli and aimed at protecting muscle fibers from immune aggression.

Hyperosmotic stress is known to induce apoptotic cell death, an effect previously attributed to seemingly ligand-independent clustering of tumour necrosis factor alpha (TNF alpha) receptors. An alternative explanation for the clustering of TNF alpha receptors may be stimulation of TNF alpha production, with subsequent autocrine or paracrine stimulation of the receptors. The present study was performed to test for an effect of exposure to hyperosmotic extracellular fluid on cellular TNF alpha production. In both the macrophage cell line U937 and the B lymphocyte cell line LCL721, an increase of extracellular osmolarity to 500 mosmol/l indeed increased TNF alpha expression, an effect reversed by the p38 kinase inhibitor SB203580. In both cell types hyperosmotic stress triggered apoptosis, which in U937 cells was significantly inhibited by neutralizing antibodies against TNF alpha and by SB203580 and was similarly elicited by exogenous addition of TNF alpha. In contrast, osmotically induced apoptosis of LCL721 cells was only slightly blunted by anti-TNF alpha antibodies and rather increased by SB203580. In conclusion, through activation of p38 kinase hyperosmotic stress stimulates the expression of TNF alpha which at least in U937 macrophages may participate in the triggering of subsequent apoptotic cell death. However, the observations in LCL721 cells point to other, TNF alpha-independent, mechanisms mediating apoptotic cell death following an excessive increase of extracellular osmolarity.