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.


METHODS
Prospective study.


METHODS
Biotech company.


METHODS
Bovine endometrial (BEND) and human mesothelial cells.


METHODS
Endometrial cells were treated with TNF-alpha and different proteins.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Nataraja
Selvaraj
S
EMD Serono Research Institute, Rockland, Massachusetts 02370, USA. selva.nataraja@emdserono.com
Kagan
David
D
Clark
Ann
A
Palmer
Stephen
S
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D016207:Cytokines / Q000494:pharmacology*; D004717:Endometrium / Q000187:drug effects / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D019151:Peptide Library; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D016207:Cytokines; D005353:Fibronectins; D005456:Fluorescent Dyes; D036341:Intercellular Signaling Peptides and Proteins; D019151:Peptide Library; D014409:Tumor Necrosis Factor-alpha