Interleukin-1 receptor antagonist blocks interleukin-1– induced expression of cyclooxygenase-2 in endometrium

Abstract

Objective: Our purpose was to test the hypothesis that the interleukin-1 receptor antagonist can inhibit interleukin-1–induced prostaglandin production and de novo expression of the inducible cyclooxygenase-2 isoform in a human endometrial epithelial cell line. Study design: A continuous line of human endometrial epithelial cells was established from a hysterectomy specimen from a nonmalignant uterus. Cells were maintained as a monolayer culture in medium 199 supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin. Cultures were treated with cytokines (interleukin-1α or interleukin-1β, interleukin-1 receptor antagonist, or tumor necrosis factor-α), and media were collected for analysis of prostaglandin E 2 and prostaglandin F 2α) by radioimmunoassay, whereas cells were harvested for ribonucleic acid and protein extractions and subsequent Northern blot or Western blot analyses, respectively. Results: When endometrial cells were incubated with interleukin-1α or interleukin-1β, each cytokine was shown to stimulate the production of prostaglandin E 2 and prostaglandin F 2α in a time- and dose-dependent fashion, with interleukin-1α being far more potent than interleukin-1β. Interleukin-1 receptor antagonist inhibited interleukin-1α– and interleukin-1β–induced prostaglandin formation, with 50% inhibitory concentration values of 30 ng/ml for prostaglandin E 2 and 90 ng/ml for prostaglandin F 2α. When Northern blots of interleukin-1α–treated cells were probed with a complementary deoxyribonucleic acid fragment specific for either cyclooxygenase-1 or cyclooxygenase-2, rapid de novo induction of cyclooxygenase-2 messenger ribonucleic acid was observed; however, cyclooxygenase-1 expression was constant regardless of interleukin-1α concentration or incubation time. Coincubation of cells with interleukin-1α (10 ng/ml) and cycloheximide caused superinduction of cyclooxygenase-2 messenger ribonucleic acid but had no effect on the expression of cyclooxygenase-1 messenger ribonucleic acid. Actinomycin D completely abolished interleukin-1α–induced cyclooxygenase-2 messenger ribonucleic acid expression, suggesting that the cytokine caused transcriptional activation of the cyclooxygenase-2 gene. Experiments were conducted to examine whether interleukin-1 receptor antagonist could suppress interleukin-1–induced cyclooxygenase-2 expression. Cells were preincubated for 30 minutes with interleukin-1 receptor antagonist and then challenged with interleukin-1α. Northern and Western analyses revealed that interleukin-1 receptor antagonist blocked interleukin-1α–induced expression of cyclooxygenase-2 messenger ribonucleic acid transcripts and the subsequent appearance of cyclooxygenase-2 protein. Interleukin-1 receptor antagonist had no effect on the constitutive expression of cyclooxygenase-1 messenger ribonucleic acid and protein. Interleukin-1 receptor antagonist failed to alter prostaglandin E 2 formation in response to tumor necrosis factor-α, indicating that the antagonist is specific for interleukin-1 family cytokines. Finally, interleukin-1 receptor antagonist acted as a partial agonist in some experiments in that relatively high concentrations (>100 ng/ml) caused a modest increase in prostaglandin E 2 and F 2α production. CONCLUSIONS: These data indicate that interleukin-1 receptor antagonist is a potent inhibitor of interleukin-1-induced arachidonic acid metabolism and could possibly serve as an endogenous or exogenous modulator of interleukin-1 action in the endometrial epithelium.