Critical role of lipid rafts in CD154‐mediated T cell signaling

Abstract

Although signal pathways triggered via the CD40 molecule are well characterized, those induced via CD154 are less known. This study demonstrates that engagement of CD154 in Jurkat D1.1 cells with soluble CD40 leads to PKC a and d activation, calcium mobilization, and phosphorylation of the Map kinases ERK1/2 and p38. Such response is accompanied by significant recruitment of CD154 into lipid rafts. Disruption of lipid rafts integrity with nystatin or methyl b-cyclodextrin abrogated PKCa PKCd and p38 phosphorylation, but had no effect on ERK1/2 phosphorylation. Inhibition of PKC activation completely abolished p38 phosphorylation but had no effect on ERK1/2 phosphorylation, suggesting that localization of CD154 within lipid rafts is an absolute requirement for CD154-induced PKCaand PKCd-dependent p38 phosphorylation. Furthermore, CD154 acts as co-stimulator for the production of IL-2 in an APC-superantigen-T-cell activation model. The results obtained demonstrate for the first time, that lipid rafts are of immunological relevance for CD154-triggered signals, and reinforce the importance of CD154 in T-cell activation.Key words: CD154 . Lipid rafts . Signaling . T lymphocytes Introduction CD154 is a type II transmembrane protein, member of the TNF superfamily, originally detected on the surface of stimulated CD4 1 T cells. Since then, CD154 was found on the surface of activated CD8 1 T cells, gd T cells, B cells, basophils, mast cells, macrophages, dendritic cells, activated platelets, and nonhematopoietic cells such as endothelial cells and fibroblasts [1][2][3][4][5][6][7]. In addition to its transmembrane form, CD154 is also released as a soluble protein by cleavage of the extracellular domain [8,9]. CD154 acts as a ligand for its classical receptor CD40 that is expressed on various cell types of hematopoietic and nonhematopoietic origin such as B cells, macrophages, dendritic cells, platelets, and endothelial and epithelial cells [5]. Recently, three other functional receptors for CD154 have been described, namely the integrins aIIbb3 [6], a5b1 [10], and Mac-1 [11].CD154 is a crucial costimulatory molecule for the development of both humoral and cellular immune responses [5], and plays an important role in the development and/or maintenance of autoimmune and inflammatory diseases [12][13][14][15]. The absence of CD154 leads to hyper-IgM syndrome, abrogation of germinal center formation and failure of B cells to proliferate and differentiate [5,16].The downstream signals triggered via CD40 molecule are extensively studied [5]. However, the signaling pathways linked to CD154 remain poorly known. Previous studies indicate that CD154 accumulate at T cell-APC contact sites in the immunological synapse [17], and that ligation of CD154 on T cells with specific Ab triggers the activation of src kinases, PKC, and MAP kinases [18][19][20], leading to cytokines secretion [21,22] 770CD3-induced T-cell proliferation [23] and apoptosis [22]. However, the precise mechanism by which CD154-triggered signaling is accomplished r...

Requirement of Transmembrane Domain for CD154 Association to Lipid Rafts and Subsequent Biological Events

“…1C). Taken together, these results confirm our previously reported data demonstrating a translocation of CD154 into lipid rafts upon its stimulation [15], indicate that such translocation does not involve its cytoplasmic domain and does not therefore require CD154 intracellular signaling.…”

“…We have previously demonstrated that stimulating CD154 induced IL-2 production in T cells co-stimulated at their T cell receptor (TCR) [15]. Therefore, we aimed herein at investigating the role of lipid rafts in CD154-induced IL-2 production using T cells transfected with wild type or chimeric forms of CD154.…”

“…We have recently demonstrated that engaged CD154 translocates to lipid rafts to subsequently activate PKCα and PKCγ, and induces the phosphorylation of p38 MAPK. The importance of lipid rafts in the initiation of these CD154-mediated responses was demonstrated using cholesterol-chelating agents to biochemically disrupt lipid rafts [15]. The role of lipid rafts in CD154-mediated activation of PKC and p38 MAPK is not surprising given the importance of lipid rafts in the CD154 system as a whole.…”

CD154 Is Released from T-cells by a Disintegrin and Metalloproteinase Domain-containing Protein 10 (ADAM10) and ADAM17 in a CD40 Protein-dependent Manner

“…The anti-CD40 monoclonal antibody (clone G28.5) was purified from hybridoma cell lines as outlined previously (15). Human soluble CD40-Fc was generated in our laboratory as described previously (16). Mouse and human IgGs (isotype controls) were purchased from Santa Cruz Biotechnology.…”

Regulatory role of CD40 in obesity-induced insulin resistance

“…It was reported that CD154-mediated signaling in T cells acts as a co-stimulator to enhance T cell activation. 36 As the expression profiles of CD40 and CD154 are different, 17 and CD154 can function through distinct receptors additional to CD40, such as Mac-1/CD11b, 37 it is possible that the distinct phenotypes observed may result from overlapping but different physiologic alterations in mice lacking CD154 vs. CD40. More studies are required to delineate the specific roles of CD154 and CD40 in metabolic disease.…”