CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs.
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Tumor necrosis factor receptor-associated factors (TRAFs) associate with the CD40 cytoplasmic domain and initiate signaling after CD40 receptor multimerization by its ligand. We used saturating peptide-based mutational analyses of the TRAF1/TRAF2/TRAF3 and TRAF6 binding sequences in CD40 to finely map residues involved in CD40-TRAF interactions. The core binding site for TRAF1, TRAF2, and TRAF3 in CD40 could be minimally substituted. The TRAF6 binding site demonstrated more amino acid sequence flexibility and could be optimized. Point mutations that eliminated or enhanced binding of TRAFs to one or both sites were made in CD40 and tested in quantitative CD40-TRAF binding assays. Sequences flanking the core TRAF binding sites were found to modulate TRAF binding, and the two TRAF binding sites were not independent. Cloned stable transfectants of human embryonic kidney 293 cells that expressed wild type CD40 or individual CD40 mutations were used to demonstrate that both TRAF binding sites were required for optimal NF-kappaB and c-Jun N-terminal kinase activation. In contrast, p38 mitogen-activated protein kinase activation was primarily dependent upon TRAF6 binding. These studies suggest a role in CD40 signaling for competitive TRAF binding and imply that CD40 responses reflect an integration of signals from individual TRAFs. (+info)
Elevated levels and functional capacity of soluble CD40 ligand in systemic lupus erythematosus sera.
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OBJECTIVE: To measure soluble CD40 ligand (sCD40L) in sera from patients with systemic lupus erythematosus (SLE) and to study the functional capacity of sCD40L in mediating B cell activation. METHODS: A 2-site enzyme-linked immunosorbent assay (ELISA) was used to measure sCD40L in the sera of 66 SLE patients, 30 disease control patients, and 23 healthy subjects. Induction of B cell activation antigen expression was used to assess the functional capacity of sCD40L in SLE sera. RESULTS: The mean concentration of sCD40L was statistically significantly higher (P < 0.0001) in SLE patients than in disease controls or healthy subjects, and segregation of SLE patients by severe, moderate, or mild extent of disease showed a relationship between disease severity and sCD40L concentration. Western blot analysis demonstrated the presence of the 18-kd band of sCD40L in SLE sera, and the results of a 1-site ELISA protocol suggested that some of the product in SLE sera was present in dimer or trimer form. Functional studies showed that 10 ng/ml of recombinant CD40L, a level present in some SLE sera, induced increased expression of CD95 on B cells. Several SLE sera also induced CD95 or CD86 on Ramos B cells, a result that was inhibited by anti-CD40L monoclonal antibodies. CONCLUSION: The soluble form of CD40L is present in the sera of most patients with SLE and may have the capacity to mediate B cell activation. Aberrant expression of CD40L might be predicted to result in activation of bystander B cells, including those that have encountered self antigens, and to contribute to autoantibody secretion. (+info)
CD40- and HLA-DR-mediated cell death pathways share a lot of similarities but differ in their use of ADP-ribosyltransferase activities.
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CD40 and HLA-DR molecules are two major components of the immune system, and their engagement on several cell types leads to various cellular events that modulate cell function. In this study, we demonstrate that signaling via these molecules leads to a rapid B cell death. CD40-mediated cell death was mainly observed in Epstein-Barr virus (EBV)-transformed B cell lines, whereas, HLA-DR-induced response can be triggered in normal activated B cells as well as in EBV-transformed B cell lines. Cell death induced via both molecules does not require de novo protein synthesis, but involves the integrity of the cytoskeleton. The sensitivity of CD40- and HLA-DR-mediated cell death to various inhibitors is very similar to that previously reported for tumor necrosis factor receptor (TNFR)- and Fas-triggered apoptosis; however, caspases leading to poly(ADP-ribose) polymerase cleavage are not implicated in this response. Both B cell death forms do not involve Fas-Fas ligand and TNF-TNFR systems, but require LFA-1-independent cell-cell interactions mediated by still undefined molecules. Although CD40- and HLA-DR-mediated cell death appears to follow a common pathway, inhibitors of poly- and mono-ADP-ribosyltransferase activity differentially affect these responses. Defining the molecules involved in CD40- and HLA-DR-mediated death will provide a possible interrelation between the different B cell death programs that can lead to a better comprehension of regulation of B cell functions. (+info)
Co-ligation of CD44 on naive human tonsillar B cells induces progression towards a germinal center phenotype.
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The precise signaling pathways to induce a germinal center (GC) phenotype and somatic mutations in human B cells are presently not understood. Major phenotypical hallmarks of a human GC B cell are up-regulated expression of CD10 and CD95 together with a heterogeneous expression of CD77. Activation of resting human tonsillar B cells using anti-CD40 and anti-IgM antibodies normally only induces up-regulation of CD38 and CD71 but has no effect on the typical GC markers. However, we show here that an additional co-ligation of the glycoprotein CD44 on such tonsillar B cells up-regulated the typical human GC markers CD10, CD38, CD77 and CD95, and down-regulated CD24 and CD39 as well as induced progression towards apoptosis in these cells; all characteristics of GC B cells. These data indicate a functional role of CD44 during activation of human naive B lymphocytes and in the generation of GC B cells. (+info)
In situ expression of B7 and CD40 costimulatory molecules by normal human lung macrophages and epithelioid cells in tuberculoid granulomas.
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Normal alveolar macrophages (AM) are not efficient in inducing the proliferation of resting T lymphocytes, and, rather, tend to inhibit pulmonary immune responses. In contrast, epithelioid cells (EC), activated macrophages that play an essential role in the course of granulomatous responses, appear to stimulate T cell proliferation efficiently. The inability of macrophages to deliver potent costimulatory signals through the B7/CD28 and CD40/CD40L pathways could explain their weak accessory cell activity. Using MoAbs and immunohistochemical techniques, however, we found that essentially all AM in normal human lung tissue expressed B7-1, B7-2 and CD40 molecules, and most of these cells were strongly positive. Pulmonary macrophages in other compartments also expressed these costimulatory molecules; no differences in expression were observed comparing macrophages from smokers and non-smokers. Most AM recovered by bronchoalveolar lavage from normal lung segments also strongly expressed B7-1, B7-2 and CD40 molecules. In comparison, resting blood monocytes were B7-1- and only moderately positive for B7-2. Activation of monocytes with lipopolysaccharide (LPS) induced expression of these costimulatory molecules to levels similar to that of AM from the control subjects. EC in granulomatous lesions also expressed easily detectable levels of B7-1, B7-2 and CD40. T lymphocytes within and surrounding the granulomas expressed CD28, the counter-receptor for B7, and many of these T cells also expressed B7-1 and B7-2. These findings suggest that both AM and EC can deliver costimulatory signals through B7-1, B7-2 and CD40 molecules, and indicate that the impairment in accessory cell activity observed for normal AM cannot be attributed to the absence of expression of these costimulatory molecules. (+info)
Expression of mitogen activated protein kinases in labial salivary glands of patients with Sjogren's syndrome.
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OBJECTIVE: The expression of CD40 and CD40 ligand (CD40L) in mononuclear cells (MNCs) infiltrating the salivary glands of patients with Sjogren's syndrome (SS) has recently been reported. This study determined the expression of mitogen activated protein kinase (MAP kinase) superfamilies, which act as downstream effector molecules of CD40, in MNCs infiltrating labial salivary tissues in SS patients. METHODS: Six HTLV-I seronegative SS patients and 10 HTLV-I seropositive patients including five HTLV-I associated myelopathy (HAM) patients were examined. The expression of MAP kinase superfamilies in labial salivary glands was examined by immunohistochemistry containing the mirror section technique. RESULTS: Both active forms of c-Jun N-terminal kinase (JNK) and p38 were found in salivary infiltrating MNCs of SS patients. Only minimal expression of the active form of extracellular signal regulated kinase (ERK) was observed in these tissues, however, co-expression of active JNK and active p38 was confirmed by the mirror section technique. Furthermore, these protein kinases were co-expressed in CD40(+) MNCs. No difference in expression levels of active JNK and p38 was found in patients who were positive or negative for anti-HTLV-I antibody. CONCLUSION: These results indicate that JNK and p38, but not ERK, function as downstream effector molecules of CD40 in salivary infiltrating MNCs in SS patients, and suggest that these molecules may be involved in the pathological process of chronic sialadenitis in SS. (+info)
Leucocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization?
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OBJECTIVE: Microvessels in atherosclerotic plaques provide an alternative pathway for the recruitment of leucocytes in the lesions. The present study was designed to investigate the potential role of these microvessels in creating vulnerable sites in atherosclerotic plaques. METHODS: Thirty-four atherosclerotic plaques were obtained from 25 patients undergoing carotid endartherectomy (n = 16), femoral endartherectomy (n = 6) and aortic surgery (n = 12). Plaques were histologically classified as either lipid-rich (rupture prone, n = 21) or fibrous (stable, n = 13). Serial cryostat sections were immunohistochemically investigated using monoclonal antibodies against endothelial cells (ULEX-E and F-VIII), vascular endothelial growth factor (VEGF), endothelial adhesion molecules (ICAM-1, VCAM-1, E-Selectin, CD40) and inflammatory cells (macrophages (CD68) and T lymphocytes (CD3). RESULTS: The microvessel density in lipid-rich plaques was significantly increased as compared to fibrous plaques. Most of these vessels were located in the shoulder-region of the plaque and at the base of the atheroma. Microvessels in lipid-rich plaques also expressed increased levels of ICAM-1, VCAM-1, E-Selectin and CD40. Moreover, inflammation was most abundantly present in the proximity of microvessels. VEGF was only observed on vessels and mononuclear cells in lipid-rich plaques, suggesting that this factor may play a role in microvessels formation. CONCLUSIONS: Neovascularisation and expression of adhesion molecules by microvessels at sites of vulnerable lipid-rich plaques may sustain the influx of inflammatory cells and hence, could contribute to plaque destabilization. (+info)
Cutting edge: contrasting roles of TNF receptor-associated factor 2 (TRAF2) and TRAF3 in CD40-activated B lymphocyte differentiation.
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In B lymphocytes, CD40 signals contribute to the activation of Ab secretion, isotype switching, T cell costimulation, and immunological memory. TRAF proteins appear to be important components of the CD40 signal transduction complex, but their roles in the activation of B cell effector functions are poorly understood. We examined the contributions of TNF receptor-associated factor 2 (TRAF2) and TRAF3 to CD40-activated differentiation in mouse B cells transfected with inducible TRAF and dominant-negative TRAF cDNAs. We find that binding of TRAF2 and TRAF3 to CD40 is not required for the induction of Ab secretion, but that both TRAF molecules can regulate the activation process. We demonstrate a negative regulatory role for TRAF3 and that this activity is dependent on the availability of an intact TRAF3-binding site in the cytoplasmic domain of CD40. In contrast, TRAF2 appears to play a positive role in B cell differentiation, and this activity is apparent even when its binding site on CD40 is disrupted. (+info)