Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells

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Abstract

TWEAK, a member of the TNF family, induces cell death in some tumor cell lines, but also induces proliferation of endothelial cells and angiogenesis. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which may be responsible for the proliferation of endothelial cells and angiogenesis. In this study, we investigated the pro-inflammatory effect of TWEAK on human umbilical vein endothelial cells (HUVEC). We demonstrated that TWEAK could not only induce the proliferation and migration but also upregulate the cell surface expression of adhesion molecules such as ICAM-1 and E-selectin, and induce the secretion of chemokines such as IL-8 and MCP-1 in HUVEC. Moreover, by using an anti-Fn14 mAb that blocks the TWEAK/Fn14 interaction, we demonstrated that Fn14 was constitutively expressed on HUVEC and totally mediated the biological effects of TWEAK on HUVEC. These results indicated that TWEAK could induce pro-inflammatory reactions via Fn14 on HUVEC.

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Materials and methods

Cells. HUVEC were purchased from Clonetics (San Diego, CA) and routinely maintained in a specialized medium for human endothelial cells called ‘Endothelial Growth Medium’ (EGM-2, Clonetics) on collagen-coated plastic flasks (Asahi Techno Glass, Japan) according to the manufacturer’s instructions. Mouse T lymphoma L5178Y was obtained from American Type Culture Collection (ATCC) and cultured in RPMI 1640 containing 10% FCS, 100 μg/ml streptomycin and penicillin, and 2 mM glutamine (culture medium).

Expression and TWEAK binding of Fn14 on HUVEC

To examine the expression and function of Fn14 on HUVEC, we generated an anti-human Fn14 mAb (ITEM-2). ITEM-2 specifically reacted with hFn14/L5178Y (Fig. 1A) and hFn14/P815 (not shown), but not with L5178Y (Fig. 1A) or P815 (not shown). ITEM-2 also strongly reacted with HUVEC (Fig. 1A), indicating that HUVEC express Fn14 constitutively. ITEM-2 completely blocked CD8-TWEAK binding to HUVEC as well as hFn14/L5178Y (Fig. 1B), indicating that Fn14 is the predominant TWEAK receptor on HUVEC.

TWEAK-induced proliferation and migration of HUVEC is mediated by Fn14

It has

Discussion

In this study, we demonstrated that TWEAK could induce proliferation, migration, upregulation of ICAM-1 and E-selectin, and secretion of IL-8 and MCP-1 by HUVEC. These results are consistent with recent observations by others indicating TWEAK-induced proliferation and migration of human endothelial cells [6], ICAM-1 upregulation and IL-8 secretion by human astrocytes [24], and IL-8 secretion by human dermal fibroblasts and synoviocytes [25]. Moreover, by utilizing a newly generated blocking mAb

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    Abbreviations: TNF, tumor necrosis factor; FasL, Fas ligand; TRAIL, TNF-related apoptosis-inducing ligand; TWEAK, TNF-like weak inducer of apoptosis; DR, death receptor; Fn14, fibroblast growth factor-inducible 14; IL, interleukin; MCP-1, monocyte chemotactic protein-1; NF-κB, nuclear factor-κB; HUVEC, human umbilical vein endothelial cells; CD40L, CD40 ligand; ICAM-1, intercellular adhesion molecule-1; FITC, fluorescein isothiocyanate; PE, phycoerythrin; ELISA, enzyme-linked immunosorbent assay; TMB, 3,3,5,5-tetramethylbenzidine dihydrochloride; LLnL, N-acetyl-Leu–Leu-norleucinal; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt; TRAF, TNF receptor associated factor.

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