Biochemical and Biophysical Research Communications
Pro-inflammatory effect of TWEAK/Fn14 interaction on human umbilical vein endothelial cells☆
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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.