Interleukin-1 beta independently stimulates production of prostaglandin E2 and cyclic AMP from human decidual cells.

Interleukin-1 beta (IL-1 beta) increased the production of cyclic AMP and prostaglandin E2 (PGE2) by cultured human decidual cells during 24 h of stimulation, but not over short incubation times (< 6 h). At concentrations of IL-1 beta ranging from 1 to 100 pg/ml, there were parallel changes in cyclic AMP and PGE2 levels, but 1000 pg of IL-1 beta/ml inhibited cyclic AMP production while still stimulating PGE2 synthesis. The possible link between cyclic AMP and PGE2 was therefore studied further. Inhibition of IL-1 beta-stimulated PGE2 synthesis by indomethacin and direct addition of PGE2 had no effect on cyclic AMP levels, indicating that PGE2 did not increase cyclic AMP production by human decidual cells and confirming the independent synthesis of cyclic AMP and PGE2. The increase in cyclic AMP production induced by IL-1 beta is dependent on protein synthesis, but it is not known which component of the adenylate cyclase is increased. A phosphodiesterase inhibitor potentiated the effects of IL-1 beta on cyclic AMP synthesis, indicating that the cytokine may increase cyclic AMP metabolism. We suggest that high concentrations of IL-1 beta activate phosphodiesterase activity more than adenylate cyclase, which gives rise to the low levels of cyclic AMP noted above. IL-1 beta also decreased forskolin-stimulated cyclic AMP production, which again indicates increased cyclic AMP metabolism. Since most concentrations of IL-1 beta alone increased cyclic AMP levels, this stimulation must out-weigh the increase in metabolism apparent in the presence of forskolin, phosphodiesterase inhibitor or high levels of interleukin. It is clear that IL-1 beta increased decidual PGE2 production independently of cyclic AMP, and that other second messenger must mediate the action of this cytokine.

Activation of oestrogen receptor complexes: evidence for the distinct regulation of ligand and oligonucleotide binding sites.

The activation of the rat uterine oestrogen receptor has been measured in vitro by its binding to oligodeoxythymidylate cellulose (oligo(dT] and was found to be sensitive to the time and temperature of prior incubation of cytosol with oestradiol. The presence of 20 mM dithiothreitol promoted receptor activation and was partially inhibited by 10 mM molybdate; molybdate also inhibited the time- and temperature-dependent activation of receptor. The nucleotides GTP, ATP, ADP, CTP and UTP all promoted receptor activation; the effect of GTP was significantly greater than that of ATP. It is unlikely that phosphate donation is involved in receptor activation as the effects of GTP could be reproduced by p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate), while PPi was also effective in activating receptor. The results provide evidence for the distinct regulation of the oligonucleotide- and ligand-binding domains, since manipulations which promoted binding to oligo(dT) did not affect either ligand binding capacity or the rate constant and composition the biphasic dissociation of the ligand receptor complex.

The regulation of progesterone and hCG production from placental cells by interleukin-1beta.

We have investigated the roles of interleukin-1beta as a regulator of progesterone and chorionic gonadotrophin production from human placental cells. In primary placental cells IL-1beta increased hCG synthesis through a cyclic AMP-independent pathway, and was without effect on progesterone or cyclic AMP production. Since dibutyryl cyclic AMP increased progesterone production, this suggests that there is no coupling between the IL-1beta receptor and the adenylate cyclase enzyme in these cells. Immortalised trophoblast cells responded to IL-1beta by increasing progesterone production through a cyclic AMP-dependent mechanism, but hCG production by these cells was unaffected by IL-1beta or dibutyryl cyclic AMP. Further studies are needed to identify the role of IL-1beta as a possible regulator of progesterone production in primary placental cells. While hCG production in first-trimester trophoblast was increased by dibutyryl cyclic AMP and IL-1beta, both these effects may involve other factors such as IL-6, and their second messenger systems.

Metabolism of prostaglandins E2 and F2 alpha by human fetal membranes.

Prostaglandin E2 (PGE2) is important in the early stages of human labour, leading particularly to cervical ripening and dilatation. The source of PGE2 is thought to be either the amnion or the decidua, but the chorion interposes between the amnion and the target tissues, namely the myometrium and cervix. In order to investigate the role of the chorion in modulating prostanoid production, [3H]PGE2 was added to the amnion side of fetal membranes, and the production of metabolites on both sides of the fetal membrane followed by HPLC. The major metabolite was 13,14-dihydro-15-oxo-PGE2 with smaller amounts of 13,14-dihydro-15-oxo-PGA2 and PGB2. The production of all metabolites of PGE2 was time dependent. [3H]PGF2 alpha, which is normally produced by the decidua, was also added to fetal membranes and found to be metabolised to 13,14-dihydro-15-oxo-PGF2 alpha and PGE2. These results suggest that the metabolic enzymes in the chorion may determine intra-uterine levels of prostaglandins, and may also determine the identity of the eicosanoids released by intact fetal membranes.

Formation and metabolism of 14,15-epoxyeicosatrienoic acid by human reproductive tissues.

Human granulosa-luteal cells cultured in the presence of arachidonic acid produced low levels of the epoxygenase metabolite 14,15-epoxy-5,8,11-(Z,Z,Z)-eicosatrienoic acid (14,15-EpETrE) as determined by HPLC analysis and gas chromatography mass spectrometry. When authentic 14,15-[3H]EpETrE was incubated with these cells in the absence of serum it was metabolised initially to the dihydroxy derivative (14,15-dihydroxy-5,8,11-eicosatrienoic acid, 14,15-DiHETrE) and subsequently to a number of more polar metabolites as determined by HPLC. Fetal calf serum protected 14,15-EpETrE from metabolism for at least 2 h. A similar pattern of metabolism was obtained when 14,15-[3H]EpETrE was incubated with a human choriocarcinoma cell line (BeWo). Microsomes from this cell line converted arachidonic acid to a large number of radioactive metabolites including 14,15-DiHETrE and 11,12-DiHETrE although there was no evidence for the parent epoxides. These results extend earlier findings that human reproductive tissues produce epoxygenase metabolites, and demonstrate the rapid metabolism of these compounds by intact cells in the absence of serum.

Detection of platelet desensitization in pregnancy-induced hypertension is dependent on the agonist used.

The aggregation of platelets from women with pregnancy-induced hypertension (P.I.H.), or with normal pregnancies, in response to arachidonic acid, ADP, collagen or platelet activating factor (PAF) was examined. No differences in platelet aggregation between the normotensive and hypertensive women were detected when arachidonic acid or collagen were used to stimulate in vitro platelet aggregation. Higher concentrations of ADP and PAF were required to aggregate platelets from women with P.I.H. compared with platelets from normotensive controls. Platelets from women with normotensive pregnancies (n = 80) aggregated maximally in response to 20 nM PAF without exception. Reversible aggregation by platelets from women with P.I.H. (n = 25) was observed at the same concentration of PAF; again, this was found in all subjects tested. These results indicate that PAF at a concentration of 20 nM can clearly demonstrate differences in aggregation of platelets from women with normotensive pregnancy and women with P.I.H.

The regulation of platelet aggregation in vitro by interleukin-1beta and tumor necrosis factor-alpha: changes in pregnancy and in pre-eclampsia.

Platelet activation occurs in early pregnancy in women at risk of developing pre-eclampsia. Cytokines have been implicated in the pathogenesis of pre-eclampsia, so we determined the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on the in vitro aggregation of human platelets. IL-1beta increased aggregation of platelets from non-pregnant and pre-eclamptic women, and inhibited the aggregation of platelets from normal pregnant women. This latter effect was linked to a diminished P-selectin expression on ADP-stimulated whole blood platelets in normal pregnant women (p = 0.011). Platelet aggregation in response to ADP was found to be inhibited after preincubation with TNF-alpha in non-pregnant (38%, p = 0.01) and in normal pregnant women (54%, p = 0.001) and not affected in pre-eclamptic women. The inhibitory effects of TNF-alpha were mediated through the P75 receptor for TNF-alpha.

Titration of antiplatelet treatment in pregnant women at risk of preeclampsia.

We recruited 111 patients who were considered to be at significantly increased risk of preeclampsia on the basis of previous obstetric history or preexisting medical disorders. All patients were treated with low dose aspirin (75 mg/day) from the first occasion the patient attended the antenatal clinic, regardless of gestational age. If the maternal mean platelet volume (MPV) increased significantly (by > 0.8 fl) from the baseline, antiplatelet treatment was increased. Five pregnancies were lost during the second trimester and 106 of the treated patients had live infants. The incidence of neonatal death (3/106 infants) was much lower than in the previous pregnancies in these patients (32/134 infants). Patients who were treated from the first trimester of pregnancy (group A, 89 patients) did substantially better than those treated from the second trimester (group B, 17 patients) as assessed by the incidence of pre-eclampsia or intrauterine growth restriction (IUGR), gestational age and birthweight at delivery. These data suggest that longitudinal monitoring of the MPV may identify the women who could benefit from increased antiplatelet treatment, and that antiplatelet treatment may be more effective when initiated in the first trimester rather than later in pregnancy.

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.

1. It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre-eclampsia (PE) and intra-uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal-term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG-nitro-L-[2,3,4,5(-3)H]-arginine ([3H]-L-NOARG) binding, quantitative in vitro autoradiography, [3H]-arginine to [3H]-citrulline conversion and Western blotting. 2. Specific, high affinity (KD = 38 nM) [3H]-L-NOARG binding was demonstrated in the villous trophoblast of normal-term placentae. Binding was calcium-independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L-NOARG > or = L-NMMA > or = 7-NI > L-NAME > L-Arg > or = L-NIO > ADMA). 3. [3H]-L-NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal-term placentae. 4. Western blotting, using an endothelial NOS peptide antiserum, demonstrated a approximately 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal-term placentae. 5. Specific [3H]-L-NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]-L-NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal-term controls. 6. The role of trophoblast-derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]-L-NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.

Differential localization of endothelin ETA and ETB binding sites in human placenta.

1. The localization and differential distribution of endothelin (ET) receptor subtypes (ETA and ETB) was investigated in sections of human placenta by use of quantitative in vitro autoradiography and receptor selective ligands. 2. Specific, high density [125I]-ET-1 binding sites were localized to the decidua and foetal membranes as well as to arteries and veins in the chorionic plate and throughout the villous tree. Moderate to low density binding was found in the extravillous and villous trophoblast respectively. 3. [125I]-ET-1 binding sites exhibited a rank order of inhibition by unlabelled peptide sequences (ET-1 > ET-3 > [Ala3,11,18Nle7]-ET-1 > BQ123 > or = sarafotoxin 6c). However, in contrast to the monophasic inhibition curve of ET-1, the other sequences produced a significantly better fit to a two component inhibition curve suggesting the presence of a heterogeneous population of ET binding sites. 4. ETA and ETB receptors were distinguished by competitive inhibition of [125]-ET-1 binding with increasing concentrations of unlabelled ET-3, [Ala3,11,18Nle7]-ET-1, sarafotoxin 6c and BQ123 and by incubating sections with the ETB agonist, [125I]-BQ3020. ET receptor subtypes exhibited a differential distribution in the placenta. ETA type binding sites predominated (approximately 80% of the total) on veins and arteries in the chorionic plate. Veins in stem villi, blood vessels in distal regions of the villous tree and decidual cells displayed a high density (approximately 60-70% of the total) of the ETB receptor subtype. 5. No difference was detected in either the relative density of [125I]-ET-1 binding sites or the proportion of ETA to ETB sites in placentae from pregnancies complicated by pre-eclampsia compared with normal term controls.6. ET may have a local autocrine or paracrine role in the placenta, acting via specific receptors to influence foetoplacental blood flow and other aspects of placental function.

A novel oestrogen-binding species in rat granulosa cells.

The dissociation constants (Kd) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (Kd 0.35 +/- 0.09 nmol/l) and cytosol (Kd 0.39 +/- 0.03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75-96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of Kd 45.8 +/- 6.9 nmol/l (capacity 16.7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of Kd 0.58 +/- 0.22 nmol/l (capacity 2.8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured. The second oestrogen-binding species displayed affinity for oestradiol-17 beta, oestriol, oestrone, testosterone, 5 alpha-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of the second oestrogen-binding species of the rat granulosa cell.

Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A-Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4 degrees C (1.72 +/- 0.27(S.E.M.) X 10(8) mol/h) and 25 degrees C (4.50 +/- 0.36 X 10(8) mol/h) was faster than with the oestrogen receptor (7.20 +/- 0.15 X 10(7) mol/h and 1.23 +/- 0.15 X 10(8) mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25 degrees C (rate constants k-1 = 0.30 +/- 0.07/min and k-2 = 3.73 +/- 0.57 X 10(-3)/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10.3S, Stokes radius greater than 5.2 nm) in low ionic strength buffers and as a small species (4.4S, Stokes radius 3.5 nm) in buffers containing 0.4 M-KCl. However, the SOB fractionated as 2-3S, Stokes radius 3.7-4.0 nm at low ionic strength and as 5.8S, Stokes radius 3.5 nm in 0.4 M-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4.6S, mol. wt 69-72000 on SDG.

Alterations in progesterone receptors in the rat uterus bearing an intra-uterine device during the oestrous cycle and early pregnancy.

The binding characteristics, content and intracellular distribution of cytosolic and nuclear progesterone receptors have been investigated, using [3H]progesterone as ligand, in the rat uterus bearing a unilateral intra-uterine device (IUD) during the oestrous cycle and from days 3 to 6 of pregnancy. The dissociation constants of nuclear and cytosolic progesterone-receptor complexes for IUD-containing and control uterine horns were similar. Cytosolic receptor concentrations in the IUD-containing uterus were always lower but changed in a manner similar to the control during the periods studied. Nuclear receptor concentrations in the control horn reflected changes in hormone levels during the oestrous cycle although concentrations measured were greater than previously reported. However, in IUD-containing uteri the pattern was completely reversed with minimal levels at pro-oestrus. Nuclear receptor concentrations were little different in both horns during early pregnancy. Total progesterone receptor synthesis determined between metoestrus and pro-oestrus in IUD-containing horns was significantly less than that of control horns. This correlated with the attenuated rise of nuclear oestrogen receptor levels previously observed between these times in IUD-containing uterine horns.

Oestrogen receptor in the granulosa cell during postnatal development of the rat ovary.

Numbers of granulosa cells obtained from follicles of immature rats increased from 1.6 X 10(5) cells/ovary on day 8 to 7.1 X 10(6) cells/ovary on day 40 of age, the day of vaginal opening and first pro-oestrus. Very high levels of cytosol oestrogen receptor were found on day 8 (175,000 sites/cell) but by day 19 20,000 sites/cell were found. Nuclear receptor concentrations were highest on day 12 (5400 +/- 1470 (S.D.)sites/cell) and again on day 21 (5400 +/- 2300 sites/cell). After day 21 both cytosol and nuclear oestrogen receptor concentrations fell and remained low until nuclear concentrations rose at day 40. Two consecutive daily injections of FSH/LH (5 i.u.) increased cell number over control in animals killed on day 22, gave no significant alteration in animals killed on day 26 or 28 but decreased numbers in animals aged 32 and 35 days. Only on day 22 was the increase in cell number associated with an increase in nuclear oestrogen receptor concentrations. Indeed on days 32 and 35 increased nuclear receptor concentrations were associated with a decreased cell number.

Effect of an intra-uterine device on intracellular relationships of the uterine oestrogen receptor, particularly during pregnancy.

The presence of an intra-uterine device in the rat results in a lower nuclear concentration of the oestrogen receptor in the treated horn at pro-oestrus when it is compared with the contralateral control horn. This effect was also seen after the administration of hyperphysiological doses of oestradiol and when the horn was exposed in vitro to high concentrations of oestradiol. The cyclic changes during the oestrous cycle in the activity of the oestrogen-induced enzyme peroxidase were similar in the treated and control horns. These observations have discounted the possibility that the relatively lower nuclear receptor content in the treated horn at pro-oestrus was due to a decreased exposure to oestrogen. A significantly lower nuclear content was also observed in the treated horn on days 4 and 5 of pregnancy. This was not associated with a deficiency in cytosol receptor content which increased concurrently with that of the control horn in the 6 days of pregnancy that were studied. The proportional content of the putative cytosol factor implicated in receptor translocation was similar in both horns, increasing on days 4 and 6 in concert with reported changes in 'induced protein' synthesis. There appeared to be reduced levels of nuclear receptor at a time when blastocyst implantation normally occurs.

A comparison of cytokine and hormone production by decidual cells and tissue explants.

Previous work has shown that enzymatic digestion of human placental tissue can induce the production of the cytokine interleukin-1 beta. Most studies of the feto-maternal interface of human pregnancy have used decidual cells prepared in a similar way, but the effects of tissue dissociation on the production of growth factors, cytokines, prostaglandins or hormones have not been investigated. Our studies show human decidual explants produce substantially lower levels of a range of factors than do human decidual cells cultured under the same conditions, indicating that induction may be a general process during the dissociation of tissues in vitro as the production of interleukins-1 beta, -6 and -8, granulocyte-macrophage colony-stimulating factor, transforming growth factor-beta 2, tissue necrosis factor-alpha, prostaglandins E2 and F2 alpha, and prolactin were all affected. The induction of cytokine production (expressed per mg tissue protein) ranged from 10- to 300-fold, indicating that isolated cells cultured in vitro may not reflect accurately the in vivo situation.

Regulation of human villous trophoblast by insulin-like growth factors and insulin-like growth factor-binding protein-1.

Many studies have implicated the insulin-like growth factors (IGFs) and insulin-like growth factor-binding protein-1 (IGFBP-1) in the control of the feto-maternal interface of human pregnancy, but many of the data are from cell lines derived from primary trophoblast or from extravillous trophoblast. We have obtained highly enriched villous cytotrophoblast (VCT) from first trimester and term human placentae, and investigated the effects of IGF-I, IGF-II and phosphoisoforms of IGFBP-1. First trimester villous trophoblast cells were regulated by all these factors. IGF-II increased cell numbers 3.5-fold after 96 h in culture, and IGF-I had less effect (1.5-fold increase) (both P<0.05). IGF-II also had a greater effect on the levels of matrix metalloproteinase (MMP)-2 and MMP-9. Phosphorylated and non-phosphorylated iso-forms of IGFBP-1 added alone increased cell numbers and MMP levels (P<0.05). IGFBP-1 did not modify the effects of IGF-II on cell numbers or on MMP production. Term VCT numbers and MMP production in vitro were unaffected by IGFs (P>0.05). Cell numbers were increased only by 100 nM IGFBP-1 isoforms (P<0.05), whereas MMP levels released from term cells were optimally increased by 1-10 nM IGFBP-1. Overall, our data show that IGFs regulate only first trimester, but not term, VCT. IGFBP-1 regulates VCT from both gestations, but the effects are concentration and end-point specific. In particular, first trimester cell numbers are more affected by low levels of IGFBP-1, whereas high levels of IGFBP-1 are needed to increase MMP and the converse applies to term VCT; low levels of IGFBP-1 have more effect on MMP levels.

A spontaneous induction of fetal membrane prostaglandin production precedes clinical labour.

Fetal membranes from term human pregnancies produce prostaglandins, and may respond to bacterial endotoxin or interleukin-1 beta (IL-1 beta) with increased prostaglandin E2 (PGE2) production. The effects of endotoxin persisted for up to 24 h, whereas those of IL-1 beta were maximal 4-8 h after addition. The maximum levels of PGE2 (200-350 pg/ml) were similar in all experiments, and were independent of the stimulus used. Not all tissues responded to these stimuli; those which did not had basal levels of PGE2 production of 200-350 pg/ml, which was not further increased by endotoxin or IL-1 beta. The basal production from these tissues was therefore similar to the maximal production from those tissues which responded to endotoxin or IL-1 beta. The high basal production of PGE2 was attributed to prior in vivo activation of the membranes such that PGE2 synthesis could not be further stimulated in vitro. Overnight pretreatment with aspirin decreased basal PGE2 production from these activated membranes to < 100 pg/ml/4 h during subsequent culture in aspirin-free medium. Both endotoxin and IL-1 beta increased PGE2 production from the activated aspirin-pretreated membranes during this culture time, but this was transient as after 12 h of culture basal PGE2 production rose to over 200 pg/ml despite aspirin pretreatment.

Regulation of prostaglandin production in intact fetal membranes by interleukin-1 and its receptor antagonist.

There is strong evidence for the involvement of inflammatory mediators such as interleukin (IL)-1 in the biochemical mechanisms of parturition. Therefore the effects of the IL-1 family (IL-1alpha (1 ng/ml), IL-1beta (1 ng/ml) and the IL-1 receptor antagonist (IL-1ra) (10 ng/ml)) on the regulation of prostaglandin synthesis in term human fetal membranes were investigated. It was found that, after 4 h of culture, IL-1beta increased prostaglandin E2 (PGE2) output approximately twofold. This was associated with both a significant increase in cyclo-oxygenase-2 (COX-2) mRNA levels (approximately fourfold compared with control) and translocation of cytoplasmic phospholipase A2 (cPLA2) from the cytosol to the membrane fraction. IL-1alpha was less effective than IL-1beta at stimulating PGE2 production through similar mechanisms. IL-1ra had no effect on PGE2 output. However, in combination treatments, IL-1ra did not inhibit IL-1alpha- or IL-1beta-stimulated PGE2 output, and increased PGE2 production further compared with IL-1beta alone. IL-1ra decreased IL-1beta-induced COX-2 mRNA expression by about half and significantly increased cPLA2 protein levels, as detected by immunoblotting, when used alone and together with IL-1beta. These results suggest that IL-1ra has partial agonist properties when used together with IL-1alpha and IL-1beta in fetal membranes by increasing cPLA2 protein levels, which leads to an increase in the production of prostaglandins.

Regulation by interleukin-1 beta of growth and collagenase production by choriocarcinoma cells.

Impaired trophoblastic invasion and proliferation have been implicated in the pathogenesis of eclampsia, pre-eclampsia, spontaneous abortions and intra-uterine growth retardation (IUGR). First trimester trophoblast cells (which do not grow in culture) and choriocarcinoma (BeWo) (which grow spontaneously, and are used as a model for proliferating trophoblast) were incubated with interleukin-1 beta (IL-1 beta). BeWo cell growth was decreased dose-dependently by exogenous IL-1 beta at concentrations of 100-1000 pg/ml. This effect was first detected after 24 h of incubation with IL-1 beta, and persisted for up to 96 h of culture. In contrast, trophoblast cells isolated from first trimester placental tissue showed no growth response when stimulated with IL-1 beta. The levels of active interstitial collagenase produced by BeWo cells were increased by IL-1 beta (100-1000 pg/ml), which paralleled the decrease in cell growth. First trimester trophoblast cells produced lower levels of collagenase and this was not affected by incubation of the cells by IL-1 beta. These results indicate that IL-1 beta may regulate placental development, but further development of culture systems for first trimester trophoblast will be needed before this result can be confirmed.