CD154 (CD40L) Induces Human Endothelial Cell Chemokine Production and Migration of Leukocyte Subsets

Abstract

CD154–CD40 interactions play key roles in humoral and cellular immune responses. With regard to the latter, ligation of CD40 on endothelial cells upregulates important intercellular adhesion molecules. Activated endothelial cells also regulate leukocyte trafficking into inflammatory sites by secreting chemokines. In this study we asked whether CD154 mediated signals induce human umbilical vein endothelial cells (HUVEC) to secrete neutrophil or peripheral blood mononuclear cell (PBMC) chemoattractants. HUVEC were cocultured with CD154+ Jurkat D1.1 cells in the presence or in the absence of anti-CD154 mAb or control mAb. Additionally, HUVEC were cocultured with control CD154− Jurkat B2.7 cells. Supernatants were harvested after 24 h and chemotaxis assays performed. Supernatants derived from Jurkat cells did not induce either neutrophil or PBMC chemotaxis. Resting endothelial cells produce at baseline some neutrophil and PBMC chemoattractants. However, there was significantly enhanced neutrophil and PBMC chemoattractant activity in supernatants derived from CD154 stimulated HUVEC. The enhanced leukocyte migration was specifically inhibited by anti-CD154 mAb. Anti-chemokine mAbs were used to identify specific chemokines mediating the enhanced leukocyte chemotaxis activity in CD154 stimulated HUVEC supernatants. There was complete or near complete inhibition of enhanced neutrophil and PBMC migration by anti-IL-8 and anti-monocyte chemoattractant protein-1 (MCP-1) mAbs, respectively. Anti-RANTES mAb partially blocked the enhanced PBMC migration, whereas anti-macrophage inflammatory protein-1α (MIP-1α) mAb had no effect. Utilizing specific ELISAs, we confirmed that CD40 ligation induces HUVEC to secrete IL-8, MCP-1, and RANTES, but not MIP-1α. Finally, we present evidence that the effects of CD154–CD40 interactions on HUVEC chemokine production are independent of IL-1β production. These findings demonstrate that CD154–CD40 interactions induce endothelial cells to produce specific neutrophil and mononuclear cell chemoattractants.