Monoclonal Antibody Targeting the CD154 Cleavage Site Inhibits CD40-Dependent and -Independent Cleavage of CD154 from the Cell Surface.

In addition to the membrane-bound molecule, soluble CD154 (sCD154) is also detected at high levels in the medium of activated T cells and platelets and in the serum of patients suffering from different inflammatory diseases. This sCD154 is the result of cleavage of the full-length molecule between the glutamic acid residue at position 112 (E112) and methionine at position 113 (M113) and can be derived from the intracellular milieu and from cleavage of cell surface molecules. We have recently reported that substitution of both E112 and M113 by alanine inhibits intracellular and CD40-induced membrane cleavage of CD154 and procures to CD154 an increased biological function as compared with cleavable CD154. Thus, in this study, and in the aim of developing tools inhibiting cleavage of CD154 from the cell surface, we generated a panel of anti-human CD154 mAbs. One of the derived mAbs that did not alter the binding of sCD154 to CD40, named in this study Clone 8 mAb, totally lost its binding activity against cells expressing CD154 mutated at its E112 and M113 residues. Treatment with Clone 8 mAb was shown to completely abolish CD40-dependent and -independent cleavage of CD154 from the cell surface. Our study is highlighting the development and characterization of an innovative therapeutic tool capable of inhibiting the release/cleavage of CD154 from cells and thus maintaining its availability on the cell surface and the high probably of increasing its potency as an activator of CD40-induced responses.

CD154 Resistant to Cleavage from Intracellular Milieu and Cell Surface Induces More Potent CD40-Mediated Responses.

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.

Ochratoxin A mediates MAPK activation, modulates IL-2 and TNF-α mRNA expression and induces apoptosis by mitochondria-dependent and mitochondria-independent pathways in human H9 T cells.

Ochratoxin A (OTA) is a natural fungal secondary metabolite that contaminates food and animal feed. Human exposure and involvement of this mycotoxin in several pathologies have been demonstrated worldwide. We investigated OTA immunotoxicity on H9 cells, a human cutaneous CD4+ T lymphoma cell line. Cells were treated with 0, 1, 5, 10, and 20 µM OTA for up to 24 hr. Western blotting revealed increased phosphorylation of all three major mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun amino-terminal kinase, p38). OTA triggered mitochondrial transmembrane potential loss and caspase-3 activation. The 24-hr OTA treatment caused marked changes in cell morphology and DNA fragmentation, suggesting the occurrence of apoptotic events that involved a mitochondria-dependent pathway. Moreover, OTA triggered significant modulation of survivin, interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α): mRNA expression of survivin and IL-2 were decreased, while TNF-α was increased. OTA also caused caspase-8 activation in a time-dependent manner, which evokes the death receptor pathway activation; we suspect that this occurred via the autocrine pro-apoptotic effect of TNF-α on H9 cells.

Paraphenylene diamine exacerbates platelet aggregation and thrombus formation in response to a low dose of collagen.

Paraphenylene daimine (PPD) is an aromatic amine that is widely used in several industrial products; however, its toxicity has been reported in several cases of cardiac arrests. As platelets play a key role in cardiovascular diseases, we aimed to determine the impact of PPD in vitro and in vivo on platelet function. Our findings demonstrated that platelet activation and aggregation were strongly enhanced by PPD. Treatment with PPD primed human platelets that became more reactive in response to low doses of collagen. Furthermore, PPD exacerbated thrombus formation in rats in comparison with those untreated. Our results suggest that PPD is an important platelet primer predisposing platelets to promote thrombus formation in response to vascular injury. This should prompt the authorities to consider controlling the marketing of this product.

The Contribution of CD40/CD40L Axis in Inflammatory Bowel Disease: An Update.

Inflammatory bowel disease (IBD) is a chronic and multifactorial disease of the gastrointestinal tract. The exact etiology of IBD remains complex and unclear involving an inadequately defined relationship between microbial insult, genetic predisposition, altered intestinal barrier permeability, oxidative stress components and abnormal immune responses. The role of the co-stimulatory system made up of cluster of differentiation 40 protein (CD40) and its ligand (CD40L) in the response of the immune system to pathogens is now widely accepted. The implication of CD40/CD40L axis in immune system disorders due to its important role as signal transduction pathway among immune cells is well documented. Several studies have suggested that CD40/CD40L interactions regulate oxidative stress; this can affect various signaling pathways leading to IBD development. Hence, CD40/CD40L signaling pathway may become a new target for IBD treatment. This review will cover the general contribution of the CD40/CD40L dyad in the development of IBD in order to facilitate future approaches aiming to elucidate the immunological mechanisms that control gut inflammation.

Interaction of CD154 with different receptors and its role in bidirectional signals.

In addition to its classical receptor, CD40, it is now well established that CD154 also binds αIIbß3, α5ß1, and αMß2 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 α5ß1 and αMß2 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 α5ß1 are N151 and Q166, whereas those involved in αMß2 binding are common to residues required for CD40, namely Y145 and R203. Soluble CD40/CD154 or αMß2/CD154 complexes do not interfere with the binding of CD154 to α5ß1-positive cells, but inhibit the binding of CD154 to CD40- or αMß2-positive cells, respectively. Ligation of CD154 on CD154-positive cells with soluble CD40, αIIbß3, α5ß1, or αMß2 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.