Cytokines and growth factors inhibit tumor necrosis factor alpha-induced up-regulation of fibronectin binding on bovine endometrial cells.

Objective: To design a high-throughput cell assay to identify molecules modulating adhesion induced by tumor necrosis factor alpha (TNF-alpha) of endometrial cells to mesothelium.

Design: Prospective study.

Setting: Biotech company.

Patient(s): Bovine endometrial (BEND) and human mesothelial cells.

Intervention(s): Endometrial cells were treated with TNF-alpha and different proteins.

Main Outcome Measure(s): TNF-alpha increased binding of fibronectin-coated fluorescein isothiocyanate (FITC) beads. The ability of various proteins to inhibit TNF-alpha-induced fibronectin-bead binding was measured.

Result(s): Treatment of BEND cells with TNF-alpha increased binding of fibronectin-coated beads. Addition of TNF-alpha-binding protein abrogated the effect of TNF-alpha in a dose-dependent manner. The initial screen of 1014 proteins identified interferon-alpha2 (IFN-alpha2), inteleukin-17 (IL-17), transforming growth factor beta (TGF-beta), and platelet-derived growth factor (PDGF) as inhibiting TNF-alpha-induced bead binding. Interferon-alpha2, IL-17, and TGF-beta inhibited bead-binding with an IC50 (ng/mL, mean +/- SD) of 0.15 +/- 0.11, 0.098 +/- 0.008, and 5.91 +/- 0.72, respectively. All three isoforms of PDGF (AA, AB, and BB) were also found to inhibit TNF-alpha-induced bead-binding, with IC50s (ng/mL) of 1.8 +/- 0.45, 10.0 +/- 1.49, and 1.72 +/- 0.73, respectively.

Conclusion(s): We describe a novel high-throughput cell-based assay for endometrial cell binding to fibronectin. We show that IFN-alpha, IL-17, TGF-beta, and PDGF have inhibitory actions on adhesion of endometrial cells to fibronectin.