CTLA-4 interacts with STAT5 and inhibits STAT5-mediated transcription

Cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152) is a member of the immunoglobulin gene superfamily with strong homology to the receptor CD28 with which it shares the ligands CD80 and CD86. Unlike CD28, a potent costimulator of T-cell responses, CTLA-4 is transiently expressed on the cell surface of activated T cells and appears to operate predominantly as a negative regulator of T-cell proliferation. Signal transduction mechanisms utilized by CTLA-4 remain unclear although several mechanisms have been implicated. In this study, we show that the cytoplasmic domain of CTLA-4, but not of CD28, binds to STAT5 in yeast two-hybrid assay and in coimmunoprecipitation assays. Mutations of Tyr165 and Tyr182 in CTLA-4 did not abrogate the interaction of STAT5 with CTLA-4. Finally, the overexpression of CTLA-4 in Jurkat T cells inhibits STAT-mediated activation of STAT5 responsive elements. These results suggest that CTLA-4 and STAT5 interact in T cells and that this interaction is important for CTLA-4 signalling.     

Modulation of anti-Candida activity of human alveolar macrophages by interferon-gamma or interleukin-1-alpha

The fungicidal and bactericidal activities of human alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 18 healthy volunteers were evaluated. The results showed that AM were able to phagocytize and kill Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. However, killing of the bacteria was already complete in 2 h, whereas killing of Candida required 4 to 6 h despite an early phagocytosis of yeast cells. The fungicidal activity of freshly collected AM and PBM was also tested after effector cell exposure to interferon-gamma (IFN-gamma), interleukin-1-alpha (IL-1 alpha), endotoxin lipopolysaccharide (LPS), or interleukin 2 (IL-2). It was found that treatment with IFN-gamma, IL-1 alpha, or LPS significantly augmented macrophage and PBM candidacidal activity, whereas the addition of IL-2 was ineffective. We also evaluated killing of C. albicans by AM cultured in vitro for different times. While phagocytosis was apparently unaffected, the candidacidal activity progressively decreased over the in vitro culture period, an effect that was largely reversed by cell exposure to IFN-gamma, IL-1 alpha, or LPS. In an experimental model in which mice infected with an agerminative C. albicans strain (PCA-2) resisted lethal microbial challenge, freshly harvested AM showed increased cytotoxic activity to Aspergillus fumigatus in vitro as well as enhanced IL-1 production. In conclusion, present data confirm the crucial role of AM in the surveillance of bacterial and fungal infections and indicate that treatment of these cells with IFN-gamma or IL-1 alpha is able to enhance their antimicrobial capability.     

The A20 protein interacts with the Epstein-Barr virus latent membrane protein 1 (LMP1) and alters the LMP1/TRAF1/TRADD complex.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) interacts with the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) molecules, which are important for LMP1-mediated signaling. Two domains of LMP1 can independently activate NF-kB, carboxyl-terminal activating region 1 (CTAR1) and CTAR2. The activation of NF-kB by CTAR1 occurs through direct interaction of LMP1 with the TRAF molecules, whereas CTAR2 interacts with the TNFR-associated death domain protein (TRADD) to activate NF-kB and the c-Jun N-terminal kinase (JNK). A20, which is induced by LMP1 through NF-kB, can block NF-kB activation from both domains of LMP1 and inhibit JNK activation from CTAR2. A20 also has been shown to associate with TRAF1 and TRAF2. In this study, an interaction between LMP1 and A20 was detected that was increased by TRAF2 overexpression. A20 did not affect the association of TRAF1 with TRAF2 but did displace TRAF1 from the LMP1 complex. The interaction of LMP1 and TRADD was decreased in the presence of A20, and the LMP1-A20 association was decreased by TRADD, suggesting that A20 and TRADD both interact with LMP1 and may compete for binding. These data indicate that A20 alters the interactions between LMP1 and the TRAF molecules and TRADD, affecting the activation of NF-kB, JNK, and perhaps other TRAF-mediated signaling events.     

The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-like receptors

The physiological function of the adaptor protein TRADD remains unclear because of the unavailability of a TRADD-deficient animal model. By generating TRADD-deficient mice, we found here that TRADD serves an important function in tumor necrosis factor receptor 1 (TNFR1) signaling by orchestrating the formation of TNFR1 signaling complexes. TRADD was essential for TNFR1 signaling in mouse embryonic fibroblasts but was partially dispensable in macrophages; abundant expression of the adaptor RIP in macrophages may have allowed some transmission of TNFR1 signals in the absence of TRADD. Although morphologically normal, TRADD-deficient mice were resistant to toxicity induced by TNF, lipopolysaccharide and polyinosinic-polycytidylic acid. TRADD was also required for TRIF-dependent Toll-like receptor signaling in mouse embryonic fibroblasts but not macrophages. Our findings definitively establish the biological function of TRADD in TNF signaling.     

Recombinant non-structural 1 (NS1) protein of dengue-2 virus interacts with human STAT3beta protein

A combination of yeast two-hybrid library screening, co-immunoprecipitation and immunofluorescence microscopy demonstrated that dengue-2 virus non-structural 1 (NS1) protein can interact with an N-terminally truncated form of human STAT3beta (DeltaN40-STAT3beta) protein. The NS1 protein interacted with the activated STAT3beta protein in vesicle-like structures in the cell cytoplasm. In addition, transfection of dendritic cells with plasmid expressing NS1 protein also resulted in significant induction of tumor necrosis factor-alpha (TNFalpha) and interleukin-6 (IL-6). Since the STAT3beta protein is an acute-phase response factor, its interaction with NS1 protein may influence the pathological changes observed in dengue fever, dengue hemorrhagic fever and dengue shock syndrome.     

Human metapneumovirus inhibits IFN-alpha signaling through inhibition of STAT1 phosphorylation

The recently discovered human metapneumovirus (hMPV) is a major cause of lower and upper respiratory tract infections worldwide. Acute viral infection initiates the interferon response that is critical in mediating viral clearance, viral host defense, and development of adaptive immunity. Mouse models of infection suggest that hMPV can cause persistent lung infections, yet the mechanisms of evading host viral clearance are unknown. Here we report that hMPV can subvert host type I interferon signaling by a mechanism distinct from other paramyxoviruses. Two lung epithelial cell lines and primary normal human bronchial epithelial cells (NHBE) were permissive for hMPV, consistent with its tropism for the respiratory tract. Treatment of hMPV-infected cells with exogenous IFN-alpha failed to reduce viral replication. Moreover, in lung epithelial cells, hMPV infection prevented IFN-alpha-mediated transactivation of the interferon-stimulated response element (ISRE) and up-regulation of interferon-stimulated genes (ISGs). Further examination of the IFN-alpha signaling cascade showed that hMPV infection prevented IFN-alpha-induced phosphorylation and nuclear translocation of STAT1. The inhibitory effects of hMPV on STAT1 phosphorylation and translocation were abolished by ultraviolet inactivation. Regulation of STAT1 by hMPV was specific, as phosphorylation of STAT2, Tyk2, and Jak1 by IFN-alpha and the surface expression of the IFN-alpha receptor were unaltered by hMPV infection. These findings demonstrate that hMPV can inhibit the type I interferon response through regulation of STAT1 phosphorylation, and provide important insight into the viral pathogenesis of hMPV infection in the respiratory tract.     

Comparison of blocking effects of monoclonal antibodies anti-MO1-alpha and anti-LFA1-alpha on human neutrophil functions.

In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MO1) recognizing an epitope of the MO1-alpha chain and the other (25.31) an epitope of the LFA1-alpha chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean +/- 1 SD) on plastic nuclon plates; this figure decreased to 33 +/- 5% and 23 +/- 6% of control adherence when the neutrophils had been pretreated with anti-LFA1-alpha (anti-alpha L) and anti-MO1-alpha (anti-alpha M), respectively. On another support (plastic culture chambers), 84 +/- 6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-alpha L or anti-alpha M was 10% and 43% of the control figure, respectively. These results show that adherence of neutrophils is dependent upon the plastic used. Moreover, inhibition of adhesion by the two MAbs was also dependent upon the support used for the assay, suggesting that MO1 and LFA1 may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-alpha L inhibited (congruent to 50%) locomotion more than did anti-alpha M (congruent to 25%), without altering chemoattractant-induced shape changes. These results suggest that the two MAbs inhibit chemokinesis but not chemotaxis. Many other adherence-associated functions, such as ingestion of opsonized Klebsiella pneumoniae, and cytotoxicity towards K/562 cells were decreased more by anti-alpha L than by anti-alpha M. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-alpha M than by anti-alpha L. Degranulation induced by zymosan or opsonized zymosan was altered by anti-alpha M only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-alpha M than by anti-alpha L, while degranulation induced by phorbol myristate acetate was not altered by either of the two Mabs.     

Glucagon-like peptide 1 receptor signaling influences topography of islet cells in mice

Glucagon-like peptide 1 (GLP-1) amplifies glucose-induced insulin release in vivo and in vitro. Activation of GLP-1 receptor (GLP-1R) signaling leads to differentiation of exocrine cells towards a beta-cell phenotype in vitro and stimulation of islet cell proliferation in vitro and in vivo, suggesting a potential role for GLP-1 in the modulation of islet growth and differentiation. To determine whether basal levels of GLP-1R signaling are essential for islet development, we examined islet cell composition and topography in GLP-1R-/- mice. Total beta-cell volume and number are not altered, but the topography of beta cells is markedly different in GLP-1R-/- mice compared with GLP-1R+/+ controls. The distribution of beta cells is shifted from large to small and medium-sized islets in the absence of GLP-1R signaling (large islets: 50 +/- 3% in GLP-1R+/+ vs 28 +/- 4% in GLP- 1R-/-, P < 0.01 and medium islets: 32 +/- 2% in GLP- 1R+/+ vs 48 +/- 3% in GLP-1R-/-, P < 0.001). Furthermore, GLP-1R-/- islets exhibit abnormalities in cell topography, with two to threefold more centrally located alpha cells detected in GLP-1R-/- islets. These alterations in alpha- and beta-cell topography indicate that basal levels of GLP-1 signaling in the normal rodent are involved in the normal cellular organization of the endocrine pancreas.