Disruption of the CD40–CD40 ligand (CD40L) co-stimulatory system, pivotal for cell-cell communication both in innate and adaptive immunity, may prevent or retard atherosclerotic plaque formation due to inflammation of the vessel wall.

We could previously demonstrate that cyclic stretch induces nitration of CD40 at one or both of its tyrosine residues followed by subsequent rapid degradation via the 20S proteasome in human primary cultured endothelial cells (HUVEC). Further investigations now revealed that CD40L-induced interleukin-12 subunit p40 (IL12-p40) mRNA expression in these cells is significantly decreased after brief exposure to exogenous peroxynitrite. Preincubation of the cells with the peroxynitrite scavenger urate reversed this effect. Moreover, scavenging by urate of intracellular peroxynitrite in stretched cells restored CD40L-dependent IL-12p40 mRNA and protein expression. Additional Western blot analyses showed a significant decrease in the localization of CD40 protein to the microsomal and plasma membrane fraction in cultured HUVEC and THP-1 monocyte/macrophages following exposure to exogenous peroxynitrite. Immunofluorescence studies with THP-1 cells demonstrated a peroxynitrite-dependent redistribution of cytokine-stimulated CD40 protein from the cell membrane to the cytoplasm. Depletion of membrane cholesterol with methyl-ß-cyclodextrin prevented this peroxynitrite-induced internalization of membrane-bound CD40.

These data corroborate our hypothesis of a stretch-induced proteasomal degradation of CD40 in human endothelial cells triggered by the peroxynitrite-dependent nitration of one or both of its tyrosine residues. Through this posttranslational oxidative modification of CD40 endothelial cells may endogenously limit their capacity to interact with pro-inflammatory CD40L-positive T cells or platelets, thus lowering the risk of atherosclerosis initiation and/or progression.