Angiogenic Effects of Interleukin 8 (CXCL8) in Human Intestinal Microvascular Endothelial Cells Are Mediated by CXCR2

Mary F. Otterson,Michael B. Dwinell,Norbert Lügering,Wolfram Domschke,Parvaneh Rafiee,Henning R. Gockel,David G. Binion,Christian Maaser,Jan Heidemann,David M. Ota,Hitoshi Ogawa

doi:10.1074/jbc.m208231200

Abstract

Angiogenesis plays a critical role in metastasis and tumor growth. Human tumors, including colorectal adenocarcinoma, secrete angiogenic factors, inducing proliferation and chemotaxis of microvascular endothelial cells, eventually leading to tumor neovascularization. The chemokine interleukin 8 (IL-8; CXCL8) exerts potent angiogenic properties on endothelial cells through interaction with its cognate receptors CXCR1 and CXCR2. As CXCR1 and CXCR2 expression is differentially regulated in tissue-specific endothelial cells and effects of IL-8 on intestinal endothelial cells are not defined, we characterized the potential IL-8-induced angiogenic mechanisms in primary cultures of human intestinal microvascular endothelial cells (HIMEC) and IL-8 receptor expression in human intestinal microvessels. CXCR1 and CXCR2 expression on HIMEC were defined using reverse transcriptase-PCR, immunohistochemistry, flow cytometry, and Western blot analysis. IL-8-induced downstream signaling events were assessed using immunoblot analysis and immunofluorescence. The angiogenic effects of IL-8 on HIMEC were determined using proliferation and chemotaxis assays. HIMEC responded to IL-8 with rapid stress fiber assembly, chemotaxis, enhanced proliferation, and phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2). HIMEC express CXCR2, but not CXCR1. Neutralizing antibodies to CXCR2 diminished IL-8-induced chemotaxis and stress fiber assembly. Specific inhibitors of ERK 1/2 and phosphoinositide 3-kinase abrogated endothelial tube formation and IL-8-induced chemotaxis in HIMEC. IL-8 elicits angiogenic responses in microvascular endothelial cells isolated from human intestine by engaging CXCR2. We confirmed tissue expression of CXCR2 in human intestinal microvessels. Supported by the notion that malignant colonic epithelial cells overexpress IL-8, CXCR2 blockade may be a novel target for anti-angiogenic therapy in colorectal adenocarcinoma.

Highlights

Human tumors, including colorectal adenocarcinoma, secrete angiogenic factors, inducing proliferation and chemotaxis of microvascular endothelial cells, eventually leading to tumor neovascularization
Angiogenesis plays a critical role in metastasis and tumor growth
human intestinal microvascular endothelial cells (HIMEC) Constitutively Express CXCR2, but Not CXCR1 mRNA—To assess CXCR1 and CXCR2 expression in cultured HIMEC, we performed reverse transcriptase-PCR using receptor-specific primers (Fig. 1)

Summary

Introduction

Human tumors, including colorectal adenocarcinoma, secrete angiogenic factors, inducing proliferation and chemotaxis of microvascular endothelial cells, eventually leading to tumor neovascularization. Both colonic epithelial cells [17] and microvascular EC of human gut secrete IL-8 in a regulated fashion in vitro [18], 1 The abbreviations used are: EC, endothelial cell; ERK, extracellular signal-regulated kinase; HIMEC, human intestinal microvascular endothelial cells; IL-8, interleukin 8; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor; TNF-␣, tumor necrosis factor ␣; IFN, interferon; LPS, lipopolysaccharide; FBS, fetal bovine serum; BSA, bovine serum albumin; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; DARC, Duffy antigen receptor for chemokines.

Results

Conclusion