Cytokines and lipopolysaccharide enhance basal and thrombin-stimulated production of PGI 2 by cultured human pulmonary artery smooth muscle cells

Abstract

We evaluated the thrombin-stimulated production of prostacyclin (PGI 2) by cultured human pulmonary artery smooth muscle cells (HPASMC) that were pretreated with cytokines (IL-1β, TNFα) and lipopolysaccharide (LPS). Cultured HPASMC, obtained from autopsied cases, were identified as smooth muscle cells by immune staining with mouse anti-human α-smooth muscle actin monoclonal IgG. A 3 hour incubation of HPASMC with LPS, IL-1β, or TNFα followed by a 10 min exposure to 2 U/ml thrombin was sufficient to generate a greater amount of PGI 2 than observed in control cells. The increase in PGI 2 production peaked after 8 h in the IL-1β- and TNFα-treated HPASMC, and continued to increase for 24 h in the LPS-treated HPASMC. We then investigated the effect of incubation time of thrombin on PGI 2 production in HPASMC pretreated with cytokines or LPS for 24 h. PGI 2 production by LPS- and cytokine-treated HPASMC peaked after a 15 min exposure to thrombin. In contrast, the exposure of LPS- or IL-1β-treated HPASMC to buffer seemed to increase the release of PGI 2 for up to 30 min of incubation. However, the PGI 2 released was less than that in the thrombin-stimulated HPASMC. After incubation with various concentrations of LPS or cytokines, the production of PGI 2 by thrombin-stimulated HPASMC showed significant, dose-dependent increases beginning at 0.1 μg/ml of LPS, 20 U/ml of IL-1β, and 50 U/ml of TNFα. We conclude that LPS, IL-1β, and TNFα enhanced both the basal and thrombin-stimulated production of PGI 2 by HPASMC. This enhanced production of PGI 2 might help in lowering the pulmonary vascular tone and modifying pulmonary hemodynamics in cytokine- or endotoxin-mediated inflammation and acute injury of the lung.