Claire Léveillé

CD40 Ligand Binds to α5β1 Integrin and Triggers Cell Signaling

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L–/– and CD40–/– mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an αIIbβ3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and αIIbβ3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-α5β1 monoclonal antibody P1D6, and soluble α5β1. The direct binding of sCD40L to purified α5β1 was confirmed in a solid phase binding assay. Binding of sCD40L to α5β1 was modulated by the form of α5β1 expressed on the cell surface as the activation of α5β1 by Mn2+ or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of α5β1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an α5β1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble α5β1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, α5β1, and αIIbβ3) for CD40L.

CD20 is physically and functionally coupled to MHC class II and CD40 on human B cell lines

Engagement of MHC class II and CD40 on B cell lines triggers intracellular signals that activates cell surface adhesion receptors, resulting in LFA‐1‐dependent and ‐independent cell‐cell adhesion. In this study, a murine monoclonal antibody (mAb R21) has been produced against a LFA‐1‐negative human B cell line and proven to completely block MHC class II‐and CD40‐induced LFA‐1‐independent homotypic adhesion. However, this mAb failed to prevent MHC class II‐ or CD40‐induced homotypic adhesion in LFA‐1‐positive Raji B cells, and alone, it triggered LFA‐1‐dependent cell‐cell adhesion. Biochemical characterization indicated that the CD20 molecule, a tetraspan phosphoprotein expressed on B cells that functions as a Ca2+ ‐conductive ion channel, is the target of mAb R21. Interestingly, further biochemical analysis demonstrated that CD20 is physically associated with MHC class II and CD40 molecules on the cell surface of LFA‐1‐negative and LFA‐1‐positive B cell lines. Although these three molecules are associated with each other, the complex formation between any two of them is not dependent on the simultaneous expression of the three molecules. Altogether, these results indicate that CD20 is physically and probably functionally coupled to the MHC class II and CD40 molecules; thereby it may have certain modulatory effects on their functions.

CD40 associates with the MHC class II molecules on human B cells

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell‐cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross‐linking of MHC class II or CD40 molecules leads to the association as well as the co‐association of both molecules to the NP‐49‐insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.

Requirement of Oxidation-dependent CD40 Homodimers for CD154/CD40 Bidirectional Signaling

It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys238 of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling.

Functional Interaction of CD154 Protein with α5β1 Integrin Is Totally Independent from Its Binding to αIIbβ3 Integrin and CD40 Molecules

Background: CD154, an immuno-inflammatory molecule, binds to four receptors. Results: CD154 differentially binds its various receptors and is capable of simultaneously interacting with multiple ones, inducing synergistic responses in monocytes. Conclusion: The simultaneous engagement of CD154 receptors can create a cross-talk between them. Significance: Concomitant binding of CD154 to multiple receptors is greatly significant in therapies of CD154-related diseases. In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ∼4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.

Implication of CD154/CD40 Interaction in Healthy and Autoimmune Responses

The discovery of new functions for CD154 and CD40 molecules has expanded our knowledge to claim that CD154 plays well beyond its postulated role in adaptive immunity. Active research in this area has outlined an important role of CD154 and its receptor CD40 in the physiopathology of autoimmunity. CD154/CD40 interactions have been shown to underlay inflammatory events characterizing autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosous, and multiple sclerosis. Besides CD40, three additional receptors were recently discovered for CD154, namely, � IIb� 3, � 5� 1 and Mac-1 integrins. This review gives an overview on CD154 and its receptors, and outlines the function of CD154/CD40 interactions in both normal and autoimmune states. Moreover, the potential usefulness of various CD154-interfering agents in treatment/prevention of autoimmune events is discussed.

A colorimetric assay for the enumeration of Candida albicans in biological samples after amplification in a selective medium

Abstract The MTT (3 (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) colorimetric assay was redesigned to enumerate C. albicans in biological samples. Major modifications include the total permeabilization of the Candida cells to the tetrazolium salt and the solubilization of the intracellular purple-colored formozan for spectrophotometric analysis. A preliminary amplification of the Candida population to be quantified is carried out using a low pH antibiotic-containing synthetic selective medium. Growth curves are logarithmic and similar for 11 strains of C. albicans . All strains grew in the blastopore phase whether inoculated as blatospores or hyphae. C. guillermondii and C. pseudotropicalis also grow in this selective medium and give similar standard curves, whereas C. tropicalis, krusei, parapsilosis and glabrata cannot be amplified due to the presence of cycloheximide. The assay was used to enumerate Candida in aqueous mouth washes and in mucosal tissues. The presence of indigenous bacteria, debris and host cells do not interfere with Candida growth and enumeration. The sensitivity and the adaptability of the test make it a powerful tool in epidemiological and diagnostic studies as well as in vitro assays of Candida growth or growth inhibition.

In situ detection of Candida albicans using oral mucosal imprints

Abstract Candida albicans is a common commensal organism of the human mouth and can cause superficial infections when Candida overgrowth occurs. A sampling technique of the oral mucosa was devised which permits the in situ detection of C. albicans . Technical optimization was carried out using an in vitro approach with infected keratinocyte monolayers, and an experimental model of oral candidosis. The oral population of Candida was then assessed in normal subjects and individuals at risk. Small pieces (1 cm 2 ) of polypropylene tape were pressed onto selected parts of the buccal, palatine or labial mucosae after drying the area with air. The pieces of tape were then processed for histology and immunohistology and mounted in Immu-mount. This sampling method allows the recovery of the superficial layers of intact keratinocytes or epithelial cells. C. albicans was easily detected as infectious foci or as a sparse population in these imprints by PAS staining or calcofluor white. Specific antibodies may be used to localize the microorganism, with minimal background. In our experimental model of oral candidosis, the extent of infectious foci (as observed in mucosal imprints) is correlated with the peak of colonization (as measured by colony-forming units) and epithelial invasion by hyphae elements (observed by conventional histology). Since these imprints are of good optical quality, they allow morphological analysis of host-parasite interactions and quantification of cells. It is anticipated that such mucosal microbiopsies could be used as a diagnostic tool for the detection of oral candidoses and possibly other oral infections.