Human endothelial cell activation and mediator release in response to Listeria monocytogenes virulence factors

The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umb ilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presenta tion of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purifi ed LLO. Studies of conditions lacking extracellular Ca2+ suggested LLO-elic ited Ca2+ flux as the underlying mechanism. In addition, HUVEC incubation w ith EGD turned out to be a potent stimulus for sustained (>12-h) upregulati on of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholi pase C (EGD plcA mutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible fur the cytokine response. Endothelial cells respon ded with diacylglycerole and ceramide generation as well as nuclear translo cation of NF-kappaB to the stimulation with the LLO-producing strains EGD a nd Listeria innocua. The endothelial PC-phospholipase C inhibitor tricyclod ecan-9-yl-xanthogenate as well as two independent inhibitors of NF-kappaB a ctivation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, sup pressed both the NF-kappaB translocation and the upregulation of cytokine s ynthesis. We conclude that L. monocytogenes is a potent stimulus of NO rele ase and sustained upregulation of proinflammatory cytokine synthesis in hum an endothelial cells, both events being largely attributable to LLO present ation. LLO-induced transmembrane Ca2+ flux as well as a sequence of endothe lial phospholipase activation and the appearance of diacylglycerole, cerami de, and NF-kappaB are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathog enic sequelae in severe listerial infection and sepsis.