Synergistic stimulation of amnion cell prostaglandin E2 synthesis by interleukin-1, tumor necrosis factor and products from activated human granulocytes

We examined the interactions between supernatant from FMLP-activated human granulocytes, recombinant interleukin-1 (IL-1) and recombinant tumor necrosis factor (TNF) in the stimulation of prostaglandin E2 (PGE2) production by human amnion cells. Amnion cells from elective term cesarian sections were cultured in monolayer culture. Human granulocytes were activated with FMLP and centrifuged to obtained cell-free supernatant. Amnion cells were treated with granulocyte supernatant, IL-1 alpha, IL-1 beta, TNF-alpha, TNF-beta, or different combinations of these. Each of the stimulators alone enhanced the PGE2 production 5- to 27-fold. Granulocyte supernatant was synergistic with each of the cytokines. The combinations of IL-1 alpha or IL-1 beta with either TNF-alpha or TNF-beta caused a synergistic stimulation of amnion cell PGE2 production as well, whereas the combinations of IL-1 alpha with IL-1 beta or of TNF-alpha with TNF-beta were not synergistic. Furthermore, granulocyte supernatant was synergistic with the combination of IL-1 and TNF, resulting in a more than 150-fold stimulation of PGE2 production. Indomethacin completely suppressed these effects. We propose that granulocyte products acting together with IL-1 and TNF enhance PGE2 synthesis during inflammation, and serve as signals for the initiation of preterm labor in the setting of intra-amniotic infection.     

Prostaglandin synthesis regulation in human amnion tissue: involvement of protein kinase C and dependence on ribonucleic acid and protein synthesis.

The role of protein kinase C (PKC) in the control of prostaglandin production by the human amnion was studied. Amnion membranes delivered spontaneously at term were minced and treated with phorbol esters, protein kinase inhibitors, cycloheximide, and actinomycin D; prostaglandin E2 (PGE2) output then was determined. Untreated tissue produced 3.97 +/- 1.13 ng PGE2/micrograms DNA/14 h (mean +/- SEM, n = 19). Phorbol dibutyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated PGE2 output up to 20-fold in a concentration-dependent manner with potencies corresponding to their efficacy as PKC activators. Four-beta-phorbol and 4-methoxy-TPA, which do not stimulate PKC, did not affect PGE2 output. Stimulation by TPA was blocked by staurosporine (IC50 = 57 nM) and H7; however, these PKC inhibitors did not decrease basal prostaglandin production. Cycloheximide inhibited basal and TPA-promoted PGE2 production and amino acid incorporation. Actinomycin D abolished TPA stimulation without decreasing unstimulated prostaglandin synthesis. These results show that amnion PGE2 production after labor is not maintained by PKC action, but PKC activation in this tissue causes a protein synthesis-dependent and RNA synthesis-dependent increase of PGE2 output. However, basal PGE2 production is dependent upon protein synthesis which, presumably, utilizes pre-existing mRNAs.     

The induction of cyclooxygenase-2 (COX-2) in intact human amnion tissue by interleukin-4

Infection is a major cause of preterm labor. Amniotic fluid from women in preterm labor associated with intrauterine infection contains increased concentrations of cytokines. The mechanism underlying this association may be a cytokine-mediated stimulation of amnion cell prostaglandin production. The biosynthesis of prostaglandins from arachidonic acid is regulated by the enzyme cyclooxygenase which exists in two forms; the constitutive form (COX-1) and the other mitogen inducible (COX-2). The purpose of this study was to evaluate the effect of the cytokine interleukin-4 (IL-4) on cyclooxygenase activity and PGE2 production in amnion. Amnion tissue was taken at caesarean section from term women not in labor and immediately incubated for 2 hours in media containing concentrations of IL-4 ranging from 1 to 100 ng/ml. An increase in both COX-2 enzyme and prostaglandin E₂ (PGE₂) production was observed for all concentrations of IL-4 greater than 25 ng/ml (P < 0.05, n = 8). No change in COX-1 was observed. Our data suggest that the cytokine IL-4 may be involved in the pathogenesis of premature labor by inducing COX-2 in amnion tissue resulting in increased production of PGE₂ and subsequent myometrial activity.     

Characterization of the major phosphoinositide-specific phospholipase C of human amnion

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1 alpha and IL-1 beta were incubated with amnion cells in culture, and prostaglandin E2 (PGE2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p greater than 0.05). Indomethacin blocked the effect of IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.     

Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.     

Amnion cell prostaglandin E2 stimulatory activity in chorion laeve-conditioned medium

In this study we demonstrate the presence of a stimulant(s) to amnion cell prostaglandin (PG) E2 production in chorion-conditioned medium (CCM). The CCM induced a dose-dependent increase in amnion cell PGE2 production. This stimulatory activity was eliminated by heat and protease treatment. Maximal stimulation of amnion PGE2 by CCM did not occur until after 2 h of incubation, and treatment with cycloheximide (1 microgram/ml) effectively eliminated the ability of the amnion cells to respond to CCM. Additionally, CCM and arachidonic acid (2-40 microM) were synergistic in their stimulatory actions on amnion PGE2 production. CCM-treated amnion cells recover more quickly from acetylsalicylic acid pretreatment as compared to control. It is concluded that CCM contains a heat-labile protein which stimulates amnion cell PGE2 production by induction of prostaglandin endoperoxide synthase activity.     

Increased and intermittent prostaglandin release from amnion detected by a new superfusion technique for full thickness fetal membrane

Prostaglandin E2 (PGE) and F2 alpha (PGF) release by the intact fetal membranes is described using a novel superfusion technique allowing for the independent assessment of prostaglandin release from the amnion and chorio-decidua whilst maintaining the anatomical integrity of the fetal membranes. The effect of labour on prostaglandin release is described. Using this system it was confirmed that the amnion is a major site of prostaglandin release and possibly production. Labour resulted in a significant increase of both PGE and PGF release from the amnion side only (Pre-labour: PGE 918 pg/cm2/3h, PGF 370 pg/cm2/3h; Labour: PGE 2993 pg/cm2/3h, PGF 662 pg/cm2/3h). No change in either PGE or PGF release from the chorio-decidual side was observed in relation to labour. In addition a change in the pattern of prostaglandin release from the amnion was observed in tissues obtained after the onset of labour. In 6 of 8 samples obtained after spontaneous labour an intermittent or pulsatile release of both PGE and PGF was observed from the amnion side as compared to the steady state of prostaglandin release from all 10 samples obtained before labour.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Isolation of multiple substances in amniotic fluid that regulate amnion prostaglandin E2 production: The effects of gestational age and labor

The present study was undertaken to evaluate the effects of whole amniotic fluid (AF) and fractions of AF on amnion cell prostaglandin E2 (PGE2) production. Amnion cells were grown to confluence and then incubated in the presence of AF, or fractions thereof, obtained at 17-19 weeks gestation (MID), at term prior to the onset of labor (NIL), and at term after spontaneous onset of labor (LABOR). All whole AFs were stimulatory to amnion cell PGE2 production (p less than 0.001) but the stimulation by NIL and LABOR AFs was significantly greater (p less than 0.001) than stimulation by the MID AF. Fractionation of the AFs from the three groups (n = 9-10 per group) revealed multiple discernable peaks of stimulatory activity in each group. The majority of peaks had retention times that were similar among the three groups, and peak stimulatory activities were greater in NIL and LABOR samples than in MID samples.     

Interleukin-4 differentially regulates prostaglandin production in amnion-derived WISH cells stimulated with pro-inflammatory cytokines and epidermal growth factor.

Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.     

Progesterone represses interleukin-8 and cyclo-oxygenase-2 in human lower segment fibroblast cells and amnion epithelial cells

Labor is preceded by cervical ripening through upregulation of interleukin (IL)-1beta, IL-8, and increased prostaglandin synthesis via inducible type 2 cyclooxygenase (COX-2). Progesterone maintains myometrial quiescence during pregnancy. In this study, we examined the effects of IL-1beta and progesterone on IL-8 and prostaglandin E2 (PGE2) synthesis and IL-8 and COX-2 mRNA and promoter activity in amnion cells and lower segment fibroblast (LSF) cells. In both cell types, progesterone had no effect on basal IL-8 or PGE2 synthesis. In LSF cells, IL-1beta significantly increased IL-8 and PGE2 synthesis and COX-2 and IL-8 mRNA expression, but progesterone significantly attenuated these effects. In prelabor amnion cells, IL-1beta also increased IL-8 and PGE2 synthesis and both COX-2 and IL-8 mRNA and promoter expression; however, progesterone significantly attenuated these effects on IL-8 and PGE2 synthesis and COX-2 expression. In postlabor amnion cells, IL-1beta increased IL-8 and PGE2 synthesis and COX-2 expression, but progesterone did not attenuate the effect of IL-1beta upon IL-8 synthesis. Progesterone repression of IL-8 and COX-2 in LSF cells suggests that IL-8 and COX-2 have similar regulatory mechanisms in LSF cells and that progesterone may play a role in maintenance of cervical competence. The lack of effect of progesterone on IL-8 in postlabor cells may be the result of downregulation of the progesterone receptor during labor.     

Regulation of cyclooxygenase-2 expression by phosphatidate phosphohydrolase in human amnionic WISH cells.

Prostaglandins are known to play a key role in the initiation of labor in humans, but the mechanisms governing their synthesis in amnion are largely unknown. In this study, we have examined the regulatory pathways for prostaglandin E(2) (PGE(2)) production during protein kinase C-dependent activation of human WISH cells. In these cells, PGE(2) synthesis appears to be limited not by free arachidonic acid availability but by the expression levels of cyclooxygenase-2 (COX-2). Concomitant with the cells being able to synthesize and secrete PGE(2), we detected significant elevations of both COX-2 protein and mRNA levels. Specific inhibition of COX-2 by NS-398 totally ablated PGE(2) synthesis. All of these responses were found to be strikingly dependent on an active phosphatidate phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 activity by three different strategies (i.e. use of bromoenol lactone, propranolol, and ethanol) resulted in inhibition of COX-2 expression and hence of PGE(2) production. These data unveil a novel signaling mechanism for the regulation of PGE(2) production via regulation of COX-2 expression and implicate phosphatidate phosphohydrolase 1 as a key regulatory component of eicosanoid metabolic pathways in the amnion.     

Phorbol ester-induced stimulation of prostaglandin biosynthesis in human amnion cells

Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production by human amnion cells, with maximum stimulations of 10.8-fold and 5.9-fold, respectively. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester analogue, had little or no effect on PGE2 production by amnion cells. PMA and phorbol 12,13-dibutyrate (10(-7) M) induced a maximal increase in the rate of PGE2 biosynthesis within 15 min of treatment. These results suggest that there is an active protein kinase C present in amnion cells that is linked to arachidonic acid release and/or metabolism.     

Identification of calmodulin-like activity in term human amnion: effect of calmodulin inhibitors on prostaglandin biosynthesis

Human amnion prostaglandin E2 (PGE2) synthesis increases with the onset of labour, and this synthesis is Ca2+-dependent. To understand better the mechanism of Ca2+-stimulated PGE2 biosynthesis, studies were performed to identify the presence of the intracellular Ca2+-mediator, calmodulin, in human amnion and to examine its role in PGE2 synthesis. Calmodulin-like activity was identified by the ability of the microsomal and cytosolic fractions of the 105,000g centrifugation of amnion homogenate to stimulate cyclic AMP-dependent phosphodiesterase activity. Cytosolic fractions consistently stimulated phosphodiesterase activity more than microsomal fractions (P less than 0.001) in paired samples from term human amnions. This activity was calcium-dependent. The cytosolic and microsomal factors increased the Vmax but not the Km of phosphodiesterase. There were no differences in these parameters with the onset of labour. The distribution of calmodulin-like activity between microsomes and cytosol was similar to the distribution of calmodulin mass as determined by radioimmunoassay. Three structurally different inhibitors of calmodulin activity, calmidazolium, trifluoperazine and W7, were tested for their ability to inhibit cytosolic factor-stimulated phosphodiesterase activity and to inhibit PGE2 output from dispersed amnion cells obtained before the onset of labour at term (cesarean section cells) or after spontaneous labour and vaginal delivery (spontaneous labour cells). The 50% inhibitory concentrations of the calmodulin antagonists in the phosphodiesterase assay were: trifluoperazine (6.7 microM), calmidazolium (0.11 microM), and W7 (24 microM). Trifluoperazine inhibited both basal and calcium ionophore (A23187)-stimulated PGE2 output from cesarean section cells and spontaneous labour amnion cells. Calmidazolium inhibited basal PGE2 output in cesarean section cells and spontaneous labour cells, but had no effect on A23187-stimulated output. W7 inhibited only the ionophore-stimulated PGE2 output in cesarean section amnion cells. The rank order of inhibition of both phosphodiesterase activation and basal PGE2 output was: calmidazolium greater than trifluoperazine greater than W7. These results suggest that human amnion contains calmodulin and that its distribution, concentration and activity remain unchanged with the onset of labour. The data suggest, although not conclusively, that calmodulin may, in part, play a role in amnion cell PGE2 production. Further investigation of calmodulin effects upon specific enzymes in the PGE2 synthetic pathway will be necessary to elucidate a role for calmodulin in PGE2 production.     

The “interleukin 1 receptor antagonist” is a partial agonist of prostaglandin synthesis by human decidual cells

In many systems the interleukin-1 receptor antagonist opposes the effects of interleukin-1β. We considered that it might block interleukin-1β-stimulated prostaglandin production from human decidual cells. Very high levels of interleukin-1 receptor antagonist (>1000 pg/ml) had limited inhibitory effects on IL-1β-stimulated PGE2 synthesis, and lower levels of antagonist (<1000 pg/ml) increased the effects of IL-1β. Low concentrations of the antagonist alone (1–100 pg/ml) increased basal PGE2 production, whereas higher levels (10–100 ng/ml) had less effect. It seems, therefore, that in human decidua the “antagonist” is more accurately described as a partial agonist. It has been suggested that the IL-1 receptor antagonist could be used to inhibit decidual prostaglandin synthesis and thereby prevent preterm labor, but this report shows that caution should be exercised before using the receptor antagonist.     

The effects of interleukins 1α and 1β on prostaglandin production by cultured human fetal membranes

The effects of IL-1α and IL-1β on cultured human fetal membranes were studied. These cytokines are known to regulate prostaglandin synthesis by the separated components of the fetal membranes (amnion, chorion and decidua), but their effects on intact tissue are unknown. IL-1α increased PGE2 levels on the fetal side of the membrane, indicating increased production of prostaglandin from the amnion, but had little effect on levels of PGE2 on the maternal side of the membrane. Low levels of IL-1β (0.1 - 1.0 ng/ml) increased PGE2 levels on the fetal side of the meembrane, and also increased the production of PGE2 metabolites and PGF2α, suggesting that this cytokine stimulated the decidua as well as the amnion. High concentrations of both cytokines appeared able to stimulate prostaglandin production by the side of the membrane opposing that to which they were added, but it is not clear whether this was mediated by factors released by the stimulated membrane, or by direct transfer of small quantities of cytokines through the membrane. Taken together, these results indicate that IL-1β was a potent stimulator of the synthesis of prostaglandins by decidua and by amnion, whereas IL-1α only stimulated the amnion.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Cannabinoids stimulate prostaglandin production by human gestational tissues through a tissue- and CB1-receptor-specific mechanism

Endocannabinoids have been implicated in the mechanisms of implantation, maintenance of pregnancy, and parturition in women. Intrauterine prostaglandin production and actions are also critical in each of these mechanisms. Hence, we have evaluated the effects of cannabinoids on prostaglandin biosynthesis by human gestational membranes. Explants of term amnion and choriodecidua were established and treated with the endogenous endocannabinoids 2-arachidonoyl glycerol and anandamide, as well as the synthetic cannabinoid CP55,940, to determine their ability to modulate PGE(2) production. The explants were also treated with CP55,940 in the presence of either SR141716A (a potent and selective antagonist of the cannabinoid receptor CB1) or NS398 [a cyclooxygenase (COX)-2 inhibitor] to determine whether any observed stimulation of PGE(2) production was mediated through the CB1-receptor and/or COX-2 activity. All three cannabinoids caused a significant increase in PGE(2) production in the amnion but not in the choriodecidua. However, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. The enhanced PGE(2) production caused by CP55,940 was abrogated by cotreatment with either SR141716A or NS398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by CB1 agonism and COX-2. Data from Western blotting show that cannabinoid treatment results in the upregulation of COX-2 expression. This study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labor and membrane rupture.     

Labour-associated changes in the regulation of production of immunomodulators in human amnion by glucocorticoids, bacterial lipopolysaccharide and pro-inflammatory cytokines

Parturition is associated with changes in the production of inflammatory mediators by gestational tissues. An explant system was established to study the change in response of human amnion to various regulating factors during labour. Disks of tissue (6 mm) were excised from amnion membranes obtained either at term by Caesarian section before labour (n = 5-6) or after spontaneous vaginal delivery (n = 3-7). After 24 h equilibration in media, the tissues were treated with interleukin 1 beta (10 ng ml-1), tumour necrosis factor alpha (100 ng ml-1), lipopolysaccharide (5 micrograms ml-1) and dexamethasone (1 mumol l-1) or an appropriate vehicle control for 24 h (n = 3 wells per treatment). Media were harvested and interleukin 10, interleukin 6 and prostaglandin E2 concentrations were determined by immunoassay. In tissues taken both before and after the onset of labour, basal interleukin 10 production by amnion explants was near to the limit of detection. Basal production rates of PGE2 by amnion explants were significantly higher (P < 0.0012; Mann-Whitney U test) in tissues taken during labour than in tissues taken before the onset of labour, while interleukin 6 production was not significantly altered by labour. Production rates of interleukin 6 and prostaglandin E2 were significantly increased by interleukin 1 beta, tumour necrosis factor alpha and lipopolysaccharide in explants from tissues taken during and before labour, while the responsiveness of interleukin 10 production to these treatments was inconsistent. Dexamethasone had no effect on interleukin 6 production by amnion explants, but significantly inhibited prostaglandin E2 production, although this inhibition was approximately 30% lower in tissues obtained after the onset of labour. These results support the presence of inflammatory positive feedback cycles, coincident with a deficiency of an anti-inflammatory factor within gestational tissue, which may be involved in the progression or maintenance of labour.