Four different classes of inhibitors of receptor-mediated endocytosis decrease tumor necrosis factor-induced gene expression in human endothelial cells.

Abstract

We have investigated the relationship between receptor-mediated endocytosis of TNF and TNF-induced gene expression in cultured human endothelial cells. Exposure of cells to hypertonicity, to cytoplasmic acidification, to treatment with phenylarsine oxide, or to treatment with primary amines such as putrescine or dansylcadaverine each inhibited receptor-mediated endocytosis by 30 to 75%, as measured by uptake of acetylated Dil-low density lipoprotein or of 125I-TNF. All four treatments also inhibited TNF-induced surface expression of ELAM-1 by 50 to 100%. Among these four treatments, only hypertonicity inhibited pinocytosis, as measured by uptake of fluorescein-BSA, and only phenylarsine oxide irreversibly inhibited protein synthesis, as measured by [35S]methionine incorporation. Notably, acidification or treatment with primary amines selectively inhibited the response to TNF, compared with the response to PMA, a drug that induces ELAM-1 through a pathway that bypasses surface receptors. Primary amines, which can be used for sustained periods under physiologic culture conditions without causing toxicity, were investigated further. Pretreatment of endothelial cells with 10 mM putrescine or 100 microM dansylcadaverine also inhibited TNF induction of ICAM-1 expression and VCAM-1 expression. Primary amines also inhibited IL-1-induced increases in ELAM-1, ICAM-1, and VCAM-1 measured 4 to 6 h after treatment and inhibited IFN-beta- and IFN-gamma-mediated induction of class I MHC molecules and IFN-gamma-mediated induction of class II MHC molecules measured 72 h after treatment with cytokine. Levels of mRNA encoding cytokine-inducible molecules were also selectively reduced by primary amines. A constitutively expressed surface molecule, gp96, was not affected in the same cells. These data are consistent with a role for receptor-mediated endocytosis in TNF-mediated gene induction and suggest a new potential target for anti-inflammatory therapy.