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


METHODS
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 micrograms/ml gentamicin. Cultures were treated with cytokines (interleukin-1 alpha or interleukin-1 beta, interleukin-1 receptor antagonist, or tumor necrosis factor-alpha), and media were collected for analysis of prostaglandin E2 and prostaglandin F2 alpha) 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-1 alpha or interleukin-1 beta, each cytokine was shown to stimulate the production of prostaglandin E2 and prostaglandin F2 alpha in a time- and dose-dependent fashion, with interleukin-1 alpha being far more potent than interleukin-1 beta. Interleukin-1 receptor antagonist inhibited interleukin-1 alpha- and interleukin-1 beta-induced prostaglandin formation, with 50% inhibitory concentration values of 30 ng/ml for prostaglandin E2 and 90 ng/ml for prostaglandin F2 alpha. When Northern blots of interleukin-1 alpha-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-1 alpha concentration or incubation time. Coincubation of cells with interleukin-1 alpha (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-1 alpha-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-1 alpha. Northern and Western analyses revealed that interleukin-1 receptor antagonist blocked interleukin-1 alpha-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 E2 formation in response to tumor necrosis factor-alpha, 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 E2 and F2 alpha 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.
Kniss
D A
DA
Department of Obstetrics and Gynecology, Ohio State University, College of Medicine, Columbus, USA.
Zimmerman
P D
PD
Garver
C L
CL
Fertel
R H
RH
D016718:Arachidonic Acid / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D051546:Cyclooxygenase 2; D015237:Dinoprost / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004717:Endometrium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000494:pharmacology*; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D007536:Isomerism; D008565:Membrane Proteins; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011863:Radioimmunoassay; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D012795:Sialoglycoproteins / Q000494:pharmacology*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; D012795:Sialoglycoproteins; D014409:Tumor Necrosis Factor-alpha; D016718:Arachidonic Acid; D003513:Cycloheximide; D015237:Dinoprost; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone