Endotoxin and Mast Cell Granule Proteases Synergistically Activate Human Coronary Artery Endothelial Cells to Generate Interleukin-6 and Interleukin-8

Abstract

Mast cells (MC) are strategically located along blood vessels and, on activation, exocytose granules that contain many vasoactive mediators. Endothelial cell (EC) activation, which includes the production of such cytokines as interleukin-6 (IL-6) and IL-8, is a key event in vascular inflammation. In this study, the effects of purified MC granules (MCG) on the production of IL-6 and IL-8 by human coronary artery EC (HCAEC) were examined. HCAEC were cocultured with MCG in the presence or absence of lipopolysaccharide (LPS), and IL-6 and IL-8 levels in the culture medium were assayed by ELISA. Unactivated HCAEC produced only low levels of IL-6 or IL-8, and the addition of MCG alone resulted in little or no increase in production of these cytokines. LPS-activated HCAEC produced significant amounts of IL-6 and IL-8 in a dose-dependent and time-dependent fashion, which was amplified 2–3-fold by MCG at EC/MC ratios of 16:1–2:1. Scanning electron microscopy revealed direct communication between MCG and HCAEC. The enhancement of IL-6 and IL-8 production by MCG was abrogated when MCG were pretreated with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). These results demonstrate that MCG interaction with HCAEC causes amplification of endotoxin-stimulated cytokine production via serine proteases present in MCG. The synergistic activation of EC by endotoxin and MCG proteases emphasizes the role of MC in amplifying vascular inflammation.