The role of cyclooxygenase‐1 and cyclooxygenase‐2 in lipopolysaccharide and interleukin‐1 stimulated enterocyte prostanoid formation

Abstract

Lipopolysaccharide is an inflammatory agent and interleukin-1 is a cytokine. Their pro-inflammatory effects may be mediated by prostanoids produced by inducible cyclooxygenase-2. The aim of this study was to determine the prostanoids produced by lipopolysaccharide and interleukin-1 stimulated enterocytes through the cyclooxygenase-1 and 2 pathways. Cultured enterocytes were stimulated with lipopolysaccharide or interleukin-1beta with and without cyclooxygenase inhibitors. Low concentrations of indomethacin and valerylsalicylic acid (VSA) were evaluated as cyclooxygenase-1 inhibitors and their effects compared with the effects of a specific cyclooxygenase-2 inhibitor, SC-58125. Prostaglandin E2, 6-keto prostaglandin F1alpha, prostaglandin D2 and leukotriene B4 levels were determined by radioimmunoassay. Immunoblot analysis using isoform-specific antibodies showed that the inducible cyclooxygenase enzyme (COX-2) was expressed by 4 h in LPS and IL-1beta treated cells while the constitutive COX-1 remained unaltered in its expression. Interleukin-1beta and lipopolysaccharide stimulated the formation of all prostanoids compared with untreated cells, but failed to stimulate leukotriene B4. Indomethacin at 20 microM concentration, and VSA inhibited lipopolysaccharide and interleukin 1beta stimulated prostaglandin E2, but not 6-keto prostaglandin F1alpha formation. SC-58125 inhibited lipopolysaccharide and interleukin-1beta stimulated 6-keto prostaglandin F1alpha but not prostaglandin E2 release. The specific cyclooxygenase-2 inhibitor also inhibited lipopolysaccharide produced prostaglandin D2 but not interleukin-1beta stimulated prostaglandin D2. While SC-58125 inhibited basal 6-keto prostaglandin-F1alpha formation it significantly increased basal prostaglandin E2 and prostaglandin D2 formation. As SC-58125 inhibited lipopolysaccharide and interleukin-1beta induced 6-keto prostaglandin F1alpha production but not prostaglandin E2 production, it suggests that these agents stimulate prostacyclin production through a cyclooxygenase-2 mediated mechanism and prostaglandin E2 production occurs through a cyclooxygenase-1 mediated mechanism. Prostaglandin D2 production appeared to be variably produced by cyclooxygenase-1 or cyclooxygenase-2, depending on the stimulus.

Cyclooxygenase-2 Is Required for Tumor Necrosis Factor-α– and Angiotensin II–Mediated Proliferation of Vascular Smooth Muscle Cells

“…21 Moreover, in enterocytes, the COX-2-selective antagonist SC58125 was shown to inhibit basal and lipopolysaccharide-stimulated 6-keto-PGF 1␣ levels but not PGE 2 , suggesting that PGI 2 production occurred mainly by a COX-2-dependent mechanism, whereas PGE 2 formation occurred via a COX-1-mediated mechanism. 22 PGI 2 has several vasoprotective effects, including vasodilation, anti-platelet aggregation, and inhibition of smooth muscle cell proliferation. Thus, the increase in PGI 2 production in response to either Ang II or TNF-␣, in the context of an increase in proliferation, may be indicative of a compensatory mechanism that limits the mitogenic activity of these molecules.…”

Endotoxin Inhibits Intestinal Epithelial Restitution through Activation of Rho-GTPase and Increased Focal Adhesions

“…LPS is one of the most abundant proinflammatory stimuli in the gastrointestinal tract, and a break in the mucosal barrier leads to translocation of LPS and a sustained LPS challenge (19,20). This has direct local effects on the mucosal cells, including activation of the enterocytes (21)(22)(23)(24), as well as systemic effects with activation of immune cells (25,26). Sustained inhibition of epithelial restitution by LPS would lead to further bacterial translocation, potentiation of the inflammatory response, and further epithelial injury.…”

The Effect of Selective Cyclooxygenase Inhibitors on Intestinal Epithelial Cell Mitogenesis

“…The IEC-18 cell line was employed because of our experience with this cell line in previous studies evaluating COX inhibitors on cytokine-stimulated prostanoid formation [13]. The WB-2054 cell line was developed by Glenn Steele, M.D.…”

“…This cell line was employed because not only is it a suitable cancer cell line to employ in vitro but it is also able to selectively produce liver metastases without immunosuppression [19]. The culture, passage, and freezing techniques of these cells have been described previously [13,[17][18][19].…”

“…In previous experiments, utilizing an intestinal epithelial cell line, we found that a COX-1 inhibitor, valerylsalicyclic acid (VSA) inhibited cytokinestimulated PGE 2 production [13].…”