Does formyl-methionyl-leucyl-phenylalanine exert a physiological role in labor in women?

The classical chemotactic receptor for N-formyl peptides has traditionally been associated with polymorphonuclear and mononuclear phagocytes; however, several recent reports indicate that this receptor is also expressed in non-myeloid cells. In this study we have investigated the presence of binding sites for formyl-methionyl-leucyl-phenylalanine (fMLP) in human amniotic membranes of laboring and nonlaboring women; we have also evaluated the effect of the peptide on prostaglandin E (PGE) release from the same tissue. Our results demonstrate the presence of specific, saturable binding sites for 3H-fMLP; Scatchard plot analysis suggests the presence of both high- and low-affinity binding sites in laboring amnion, while only the low-affinity receptors were evident in nonlaboring tissue. N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine (Boc-MLP), a formyl peptide receptor antagonist, inhibited 3H-fMLP binding in both preparations. In addition, fMLP was able to significantly increase PGE synthesis in perifused amnion fragments from laboring and nonlaboring women. This effect was counteracted by Boc-MLP treatment. The presence of specific binding sites for fMLP in amniotic tissue and their differing expression in laboring versus nonlaboring membranes, together with the action of the peptide on PGE synthesis, all suggest a physiological role for fMLP in labor.     

A product of activated human granulocytes stimulates prostaglandin E2 synthesis in human amnion cells

Cell-free supernatant from formylmethionyl-leucyl-phenylalanine (fMLP)-activated granulocytes causes a time- and concentration-dependent stimulation of prostaglandin E2 (PGE2) production in amnion cells. PGE2 concentration in the culture medium after 36 h treatment with granulocyte supernatant (from 40 x 10(6) granulocytes/ml of amnion cell medium), 1.49 +/- 0.71 pg/ng DNA (n = 13), was significantly higher (p = 0.0015) than in control cells (0.33 +/- 0.23 pg/ng DNA, n = 13). Indomethacin abolished this stimulation. Granulocyte supernatant and human epidermal growth factor (hEGF) had an additive effect on amnion cell PGE2 production. Catalase, superoxide dismutase (SOD), protease inhibitors or the platelet-activating factor (PAF) antagonist L-659,989 had no effect. Actinomycin D, cycloheximide and mepacrine reduced the PGE2 production. The phospholipase A2 activity present in granulocyte supernatants was resistant to heating, whereas heating decreased their PGE2-stimulating activity by 92%. Exogenous phospholipase A2 had no effect on PGE2 synthesis. The granulocyte product could be precipitated with ammonium sulphate. On gel filtration of supernatant, two peaks of PGE2-synthesis stimulating activity were obtained (molecular weights 12,000 and 60,000). This data serve to explain the association of chorioamnionitis with preterm labor: activated granulocytes release a protein(s) that induces prostaglandin production in amnion cells, and thus promote labor.     

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)     

17beta-estradiol modulates prostaglandin E2 release from human amnion-derived wish cells

In human amnion-derived WISH cells [(3)H]estradiol-17beta binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17beta-estradiol stimulates prostaglandin (PG)E(2) release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17beta-estradiol modulates PGE(2) release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17beta-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE(2) release observed in the presence of Ro 20-1724.     

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.     

Annexin I and dexamethasone effects on phospholipase and cyclooxygenase activity in human synoviocytes

Annexin I is a glucocorticoid-induced mediator with anti-inflammatory activity in animal models of arthritis. We studied the effects of a bioactive annexin I peptide, ac 2-26, dexamethasone (DEX), and interleukin-1beta (IL-1beta) on phospholipase A2 (PLA2) and cyclooxygenase (COX) activities and prostaglandin E2 (PGE2) release in cultured human fibroblast-like synoviocytes (FLS). Annexin I binding sites on human osteoarthritic (OA) FLS were detected by ligand binding flow cytometry. PLA2 activity was measured using 3H-arachidonic acid release, PGE2 release and COX activity by ELISA, and COX2 content by flow cytometry. Annexin I binding sites were present on human OA FLS. Annexin I peptide ac 2-26 exerted a significant concentration-dependent inhibition of FLS constitutive PLA2 activity, which was reversed by IL-1beta. In contrast, DEX inhibited IL-1beta-induced PLA2 activity but not constitutive activity. DEX but not annexin I peptide inhibited IL-1beta-induced PGE2 release. COX activity and COX2 expression were significantly increased by IL-1beta. Annexin I peptide demonstrated no inhibition of constitutive or IL-1beta-induced COX activity. DEX exerted a concentration-dependent inhibition of IL-1beta-induced but not constitutive COX activity. Uncoupling of inhibition of PLA2 and COX by annexin I and DEX support the hypothesis that COX is rate-limiting for PGE2 synthesis in FLS. The effect of annexin I but not DEX on constitutive PLA2 activity suggests a glucocorticoid-independent role for annexin I in autoregulation of arachidonic acid production. The lack of effect of annexin I on cytokine-induced PGE2 production suggests PGE2-independent mechanisms for the anti-inflammatory effects of annexin I in vivo.     

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.     

Sphingosine 1-phosphate in amniotic fluid modulates cyclooxygenase-2 expression in human amnion-derived WISH cells

The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several S1P receptors including S1P1, S1P2, and S1P3. When WISH cells were stimulated with S1P, PGE2 synthesis increased in a concentration-dependent manner, showing maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that S1P stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P1 or S1P3 but not of S1P2 antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE2 production in WISH cells, indicating the involvements of S1P1 and S1P3 in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells.     

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.     

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.     

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.     

Corticotropin-releasing hormone increases the expression of the prostaglandin E(2) receptor subtype EP1 in amnion WISH cells

The purpose of this study was to investigate the effect of corticotropin-releasing hormone (CRH) on the expression of the prostaglandin (PG) E(2) EP1 receptor subtype and PGE(2) production in amnion WISH cells (AWC). AWC cultures were incubated with CRH. Culture fluid was collected for PGE(2) measurement, and the cells were collected and analyzed for EP1 protein and mRNA. Immunohistochemical localization of the EP1 receptor was also performed. Incubation of AWC with CRH resulted in a dose-dependent increase (r = 0.97) in the level of EP1 receptor protein (P < 0.001). Coincubation of AWC with CRH and indomethacin resulted in the decreased production of PGE(2) while having no effect on EP1 receptor expression. A significant but not dose-dependent increase in EP1 mRNA expression was also observed (P < 0.01). Immunohistochemical evaluation verified cell membrane localization of the receptor in both stimulated and unstimulated cells and confirmed the increased expression of EP1 receptor in response to CRH. Incubation of AWC with CRH also resulted in increased culture fluid PGE(2) levels (P < 0.01). These results suggest that the role CRH plays in the initiation of labor may also involve the promotion of elevated PGE(2) levels and increased expression of the EP1 receptor in amnion.     

Binding of prostaglandin E2 to cultured bovine adrenal chromaffin cells and its effect on catecholamine secretion

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E1 and E2. We have now investigated the binding of PGE2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE2 with a dissociation constant of 2 nM and a concentration of about 40,000 binding sites per cell. Low concentrations of PGE2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 microM) of PGE2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.     

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.     

Estradiol-17 beta and 2-hydroxyestradiol-17 beta-induced differential production of prostaglandins by cells dispersed from human intrauterine tissues at parturition

Prostaglandin (PGE, 6-keto PGF1 alpha) output by cells dispersed from human amnion and decidua in the presence of increasing levels (0-5000 ng/ml) of estradiol-17 beta (E2) or 2-hydroxyestradiol-17 beta (2-OH E2) was studied in relation to parturition. Tissues were obtained from women at term either before (CS) or after (SL) spontaneous labor and vaginal delivery. In the absence of estrogens, the output of both PGs from amnion increased significantly with labor. No significant increase in decidua PG output occurred with labor. Neither estrogen influenced CS amnion PG output. However, both E2 and 2-OH E2 stimulated SL amnion PGE output (2-OH E2 greater than E2) while having no affect on 6-keto PGF1 alpha output. Only the highest dose of 2-OH E2 stimulated PGE output in CS decidua, but both estrogens significantly inhibited 6-keto PGF1 alpha output in this tissue. In SL decidua only 2-OH E2 significantly stimulated PGE, and neither estrogen affected 6-keto PGF1 alpha output. These results might suggest that estrogens modulate PG biosynthesis at the level of endoperoxide to primary PG conversion.     

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.     

Labor and inflammation increase the expression of oxytocin receptor in human amnion

The oxytocin/oxytocin receptor (OXT/OXTR) system plays an important role in the regulation of parturition. The amnion is a major source of prostaglandins and inflammatory cytokine synthesis, which increase both before and during labor. Amnion is a noncontractile tissue; therefore, the role played by OXT/OXTR in this tissue will be fundamentally different from the role played in myometrial contractions. In the present study, we demonstrate increased OXTR mRNA and protein concentrations in human amnion epithelial cells associated with the onset of labor. We show that incubation of primary human amnion epithelial cells with IL1B results in a rapid, transient up-regulation of OXTR mRNA expression, which peaks in prelabor samples after 6 h. Incubation of prelabor amnion epithelial cells with OXT results in a marked increase of prostaglandin E(2) synthesis, and we demonstrate that OXT activates the extracellular signal-regulated protein kinase signal transduction pathway to stimulate up-regulation of cyclo-oxygenase 2 in human amnion epithelial cells. The increased ability of human amnion to produce prostaglandins in response to OXT treatment suggests a complementary role for the OXT/OXTR system in the activation of human amnion and the onset of labor.     

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.     

The mechanisms of action of interleukin-1 on rabbit intestinal epithelial cells

Interleukin-1 (IL-1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL-1's action, cells were treated with IL-1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2-activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL-1 caused concentration- and time-dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX-2 and PGE2. The IL-induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX-2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine.