Walid Mourad

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.

Essential role of PKCδ in platelet signaling, αIIbβ3 activation, and thromboxane A2release1

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCδ, but not PKCα or PKCβ, is required for collagen-induced phospholipase C-dependent signaling, activation of αIIbβ3, and platelet aggregation. Analysis of PKCδ phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by αIIbβ3 outside-in signaling. Moreover, PKCδ triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A2, which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCδ signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A2.

Enhanced Levels of Soluble CD40 Ligand Exacerbate Platelet Aggregation and Thrombus Formation Through a CD40-Dependent Tumor Necrosis Factor Receptor–Associated Factor-2/Rac1/p38 Mitogen-Activated Protein Kinase Signaling Pathway

Objective—CD40 ligand is a thromboinflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40 ligand (sCD40L), which has been shown to influence platelet activation, although its exact functional impact on platelets and the underlying mechanisms remain undefined. We aimed to determine the impact and the signaling mechanisms of sCD40L on platelets. Methods and Results—sCD40L strongly enhances platelet activation and aggregation. Human platelets treated with a mutated form of sCD40L that does not bind CD40, and CD40−/− mouse platelets failed to elicit such responses. Furthermore, sCD40L stimulation induces the association of the tumor necrosis factor receptor–associated factor-2 with platelet CD40. Notably, sCD40L primes platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, which leads to platelet shape change and actin polymerization. Moreover, sCD40L exacerbates thrombus formation and leukocyte infiltration in wild-type mice but not in CD40−/− mice. Conclusion—sCD40L enhances agonist-induced platelet activation and aggregation through a CD40-dependent tumor necrosis factor receptor–associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. This may explain the link between circulating levels of sCD40L and cardiovascular diseases.

CD154: an immunoinflammatory mediator in systemic lupus erythematosus and rheumatoid arthritis.

Systemic lupus erythematosus and rheumatoid arthritis are two major chronic inflammatory autoimmune diseases with significant prevalence rates among the population. Although the etiology of these diseases remains unresolved, several evidences support the key role of CD154/CD40 interactions in initiating and/or propagating these diseases. The discovery of new receptors (αIIbβ3, α5β1, and αMβ2) for CD154 has expanded our understanding about the precise role of this critical immune mediator in the physiopathology of chronic inflammatory autoimmune diseases in general, and in systemic lupus erythematosus and rheumatoid arthritis in particular. This paper presents an overview of the interaction of CD154 with its various receptors and outlines its role in the pathogenesis of systemic lupus erythematosus and rheumatoid arthritis. Moreover, the potential usefulness of various CD154-interfering agents in the treatment and prevention of these diseases is also discussed.

Ligation of MHC class II molecules differentially upregulates TNFβ gene expression in B cell lines of different MHC class II haplotypes

Although the production of selected cytokines by B cells is important for their regulation, little is known about MHC class II-induced cytokine expression in these cells. We designed the present studies to investigate MHC class II-mediated TNF-beta gene expression in 19 EBV-transformed homozygote B cell lines at similar stage of differentiation but presenting different MHC class II haplotypes. Our results demonstrate that in contrast to PMA, engagement of MHC class II with staphylococcal enterotoxin A (SEA), a natural ligand, or with anti-HLA-DR mAb L243, stimulates TNF-beta gene expression in some but not all B cell lines. The differential stimulation of TNF-beta gene expression via MHC class II was not due to the cells MHC class II expression level, nor to their capacity to bind the ligands as evidenced by SEA binding affinity studies. Together these results demonstrate that ligation of MHC class II molecules can stimulate TNF-beta gene expression in a B cell line-dependent manner. The differential cytokine gene expression might be due to an influence of MHC class II haplotype either by a linkage disequilibrium with TNF-beta gene or by a differential association with effector or cell surface molecules.

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.

Soluble CD40 ligand impairs the anti-platelet function of peripheral blood angiogenic outgrowth cells via increased production of reactive oxygen species

Adult peripheral blood angiogenic early outgrowth cells (EOCs), also known as early endothelial progenitor cells, interact with other blood and vascular cells and may regulate atherothrombosis. We have previously shown that endothelial progenitor cells inhibit platelet function and thrombus formation. The CD40L/CD40 axis is a thrombo- inflammatory mediator that affects platelet and endothelial functions. It has been shown that EOCs express CD40, whereas platelets represent the major source of its soluble ligand (sCD40L), which impairs EOC function.We aimed to test the hypothesis that the sCD40L/CD40 axis affects the anti-platelet function of EOCs. Human peripheral blood mononuclear cell-derived EOCs in culture inhibited platelet aggregation. Pre-treatment of EOCs with sCD40L reduced their inhibitory effect on platelet aggregation in a CD40-dependent manner. EOCs viability and release of the anti-aggregating agents, prostacyclin and nitric oxide, were not affected by sCD40L. However, production of reactive oxygen species (ROS) was increased in sCD40L-treated EOCs. Blockade of ROS reversed the effects of sCD40L-treated EOCs on platelet aggregation. This study reveals that the sCD40L/CD40 axis impairs the anti-platelet properties of EOCs through increased production of ROS. These data may explain the link between elevated levels of sCD40L, impaired activity of EOCs and enhanced platelet reactivity, and consequently the occurrence of atherothrombotic disease.

CD40 Ligand and Its Receptors in Atherothrombosis

Atherothrombosis is the main underlying determinant of cardiovascular diseases, which remain the leading cause of death in developed countries. Multiple lines of evidence now support the concept of atherothrombosis as a chronic inflammatory disease of the arterial wall.1, 2 This process involves a complex interplay between modified lipids and cells of the immune and vascular system, which usually evolves into the formation of atherosclerotic lesions yielding a stable necrotic plaque. If left untreated, plaque rupture and thrombosis may ensue, leading to important clinical manifestations, such as acute coronary syndromes and sudden death.3