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. 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 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.

Insulinlike growth factors. Their regulation of glucose and amino acid transport in placental trophoblasts isolated from first-trimester chorionic villi.

The transport of glucose and amino acids from the maternal to fetal circulation through the placenta is critical to the delivery of fuel for normal fetal growth and development. Little information indicates that transplacental glucose or amino acid transport is influenced by hormones or polypeptide growth factors. We developed a continuous cell line of cytotrophoblastlike cells derived from first-trimester human chorionic villi as a model system to study the regulation of glucose and amino acid transport by insulinlike growth factors (IGFs). Using immunocytochemical and biochemical criteria, the cells were shown to manifest a trophoblastlike phenotype. The cells were maintained in serum-supplemented medium until confluent, at which time they were shifted to serum-free medium for one day. Experiments were initiated by transferring the cells to glucose-free assay buffer and incubating them with IGF-I, IGF-II or insulin. Glucose uptake was measured by the transport of 2-deoxy-D-[1,2-3H]glucose (2[3H]DG) in the presence or absence of cytochalasin B, which has been shown to competitively inhibit glucose uptake. IGF-I, IGF-II and insulin each enhanced 2[3H]DG transport in a dose-dependent fashion. Amino acid transport was measured by incubation of the cells with IGF-I for 60 minutes, followed by a 5-minute challenge with alpha-[methyl-3H]aminoisobutyric acid. IGF-I caused a dose-dependent increase in uptake of the amino acid analog. Radioreceptor assays using [125I]insulinlike growth factor I ([125I]IGF-I) demonstrated that the trophoblast-derived cells contained high-affinity, saturable receptors for IGF-I that also bound IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid hormones inhibit DNA synthesis in glial cells cultured in chemically defined medium.

Primary cultures of glial cells from 2-day-old rat cerebellum were used to examine the growth control properties of steroid hormones. Immunocytochemical staining with antiglial fibrillary acidic protein (anti-GFAP) demonstrated that the cultures were highly enriched for astrocytes (90%). In an effort to avoid the potential influence of serum-borne steroids, cultures were switched from serum-supplemented to serum-free, chemically defined medium prior to experimentation. Assays for DNA synthesis used [3H]thymidine incorporation with either liquid scintillation counting of TCA-insoluble material or light microscopic autoradiography. Glial cells grown in serum-free, chemically defined medium (F12 basal medium supplemented with putrescine, selenium, insulin, transferrin, and BSA) replicated their DNA to a limited extent even in the absence of serum mitogens. When the glial cells were shifted to defined medium supplemented with various steroid hormones (corticosterone, dexamethasone, hydrocortisone, 17 beta-estradiol, progesterone, or testosterone) at a concentration of 10(-7) M, it was found that the glucocorticoids, corticosterone and dexamethasone inhibited synthesis of DNA by 49.6 and 56.9%, respectively. Hydrocortisone, another glucocorticoid, caused only a small reduction in DNA synthesis. The growth-controlling activity of the glucocorticoids was dose-dependent with concentrations of 10(-7) -10(-6) M showing maximal effect on DNA synthesis. These results suggest that physiological concentrations of glucocorticoid hormones may exert negative control over DNA synthesis of glial cells in the developing or injured central nervous system.