Fibronectin fragments stimulate tumor necrosis factor secretion by human monocytes

Abstract: Fibronectin (Fn) is a high molecular-weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis fac-tor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gra-dient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatin-Sepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were as-sayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose-dependent manner. Intact Fn-induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R-G-D-containing peptide GRGDSP. Limited pro-teolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. High-performance liquid chromatographic (HPLC) purifica-tion of the fragments demonstrated that at least two frag-ments were capable of stimulating TNF secretion. Fur-ther purification by affinity chromatography and HPLC localized the stimulatory activity to the 120-kd cell-binding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially impor-tant molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.J. Leukoc. Biol. 51