Background: CD154 is released from T-cells in a manner that remains elusive. Results: CD154 is released from Jurkat E6.1 T-cells by ADAM10 and ADAM17 upon its interaction with CD40 exclusively. Conclusion: The cleavage of CD154 is mediated through specific signaling events that bypass many other CD154 signaling events. Significance: These data may lead to the generation of novel targets for the treatment of CD154-associated disorders.
In addition to its classical receptor, CD40, it is now well established that CD154 also binds αIIbβ3, α5β1, and αMβ2 integrins. Although these integrins are all members of the same family, they bind CD154 differently. The current investigation aims to analyze the interaction of CD154 with α5β1 and αMβ2 and investigate its role in bidirectional signals in various human cell lines. Results obtained herein indicate that the CD154 residues involved in the interaction with α5β1 are N151 and Q166, whereas those involved in αMβ2 binding are common to residues required for CD40, namely Y145 and R203. Soluble CD40/CD154 or αMβ2/CD154 complexes do not interfere with the binding of CD154 to α5β1-positive cells, but inhibit the binding of CD154 to CD40-or αMβ2-positive cells, respectively. Ligation of CD154 on CD154-positive cells with soluble CD40, αIIbβ3, α5β1, or αMβ2 stimulates intracellular signaling, including MAPK phosphorylation. Given that CD154 exists as a trimer, our data strongly suggest that CD154 may bind concomitantly to two receptors of the same or different family, and biologically activate cells expressing both receptors. The characterization of CD154/receptor interactions helps the identification of new therapeutic targets for the prevention and/or treatment of CD154-associated autoimmune and inflammatory diseases.Keywords: Activation r Binding r CD154 r CD40 r Integrins Introduction CD154, also known as CD40 ligand (CD40L) or gp39, is a member of the tumor necrosis factor (TNF) superfamily. It was initially thought to be only expressed on activated CD4 + T cells; however, a wider distribution of CD154 is now established, including a variety of hematopoietic and nonhematopoietic cells [1,2]. Soluble CD154 (sCD154, encompassing residues M113-261), is released from activated T cells and platelets by an MMP-dependent Correspondence: Dr. Walid Mourad e-mail: mw.mourad@umontreal.ca cleavage [3,4]. Like other members of the TNF superfamily, both membrane and sCD154 form a noncovalently linked homotrimer [5,6], a structural requirement for CD154 biological activity [3,7].CD40, a type I transmembrane protein belonging to the TNF receptor superfamily, is the classical receptor for CD154. CD40 was initially considered a pan-B-cell antigen, but was subsequently shown to be expressed on a variety of cell types, including B lymphocytes, monocytes/macrophages, DCs, platelets, epithelial * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 592-602 Leukocyte signaling 593 cells, as well as endothelial cells [2,[8][9][10][11]. CD154/CD40 interactions were shown to be implicated in many biological responses, from inducing proliferation, differentiation, and Ig switching in resting B cells [12] to enhancing surface expression of costimulatory and adhesion molecules and stimulating cytokine production in immune and nonimmune cells [13,14]. It was considered that the CD154-mediated biological function involves only an interaction wi...
In addition to the membrane-bound form, CD154 also exists as a soluble molecule originating from an intracellular and membrane cleavage. We have previously shown that CD154 cleavage from T cell surface is mediated by CD40 and involves the action of ADAM10/ADAM17 enzymes. In the aim of defining the importance of CD154 maintained on cell surface, we generated a CD154 mutated at the cleavage site. Our data show that the double mutation of E112 and M113 residues of CD154 abolishes its spontaneous release and the CD40-mediated cleavage from cell surface but does not affect its binding to CD40. We also demonstrated that both the release of CD154 from the intracellular milieu and its CD40-mediated cleavage from cell surface are highly dependent on ADAM10/ADAM17 enzymes. The CD154-EM mutant was shown capable of inducing a more prominent apoptotic response in susceptible B cell lines than the wild-type (WT) form of the molecule. In addition, human B cells cultured in the presence of the CD154-EM mutant exhibited upregulated proliferative responses compared with the CD154-WT. The CD154-EM mutant was also shown to trigger differentiation of human B cells, reflected by an increased Ig production, more significantly than CD154-WT. Thus, our data strongly suggest that cleavage-resistant CD154 is a more prominent stimulant than the cleavable form of the molecule. Therefore, a maintained expression of CD154 on cell membrane and a disturbed cleavage of the molecule could be a mechanism by which CD154 is involved in some pathological conditions and should be revisited.
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