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Placental ATP-binding cassette (ABC) transporters limit fetal exposure to xenobiotics by regulating transplacental passage into the fetal circulation; their expression and function in fetal membranes, however, has not been studied. In the present study the expression, localisation and function of ABC transporters in human amnion was examined to explore their potential role in modulating amniotic fluid drug disposition in pregnancy. Single-assay oligo-microarrays were used to profile amnion gene expression, and drug transporters expressed at significant levels were identified and selected for further studies. The expression of ABCG2/breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRP) 1 (ABCC1), 2 (ABCC2) and 5 (ABCC5) was detected on the arrays, and verified by RT-PCR and immunoblotting. On confocal microscopy of fetal membrane cryosections, MRP1 and MRP5 were immunolocalised to both apical and basolateral surfaces of the amniotic epithelium, while MRP2 was expressed at low levels only in the apical membrane. BCRP in contrast showed cytoplasmic staining throughout the amniotic epithelium. In addition to the amnion, MRP1 and BCRP immunostaining was observed in the chorion and the decidua. Cell accumulation studies using selective MRP and BCRP inhibitors showed the transporters to be functionally active in amnion epithelial monolayer cultures. In contrast, transwell transport studies using intact amnion membranes did not show significant vectorial transport. These findings identify the amnion as a novel site of ABC drug transporter expression. Functional studies indicate that they may act primarily to prevent cellular xenobiotic accumulation, rather than to confer fetal protection through reduced accumulation in amniotic fluid.

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.

Problem: To determine whether leptin exhibits cytokine-like properties in gestational tissues in light of its homologies with the class I family of cytokines., Method of Study: WISH and JEG3 cells, and amnion and choriodecidua explants, were treated inflammatory modulators (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha] and bacterial lipopolysaccharide [LPS]) and leptin production was measured by immunoassay. Other agents known to regulate adipocyte leptin production were also tested for comparative purposes. In addition, WISH cells, JAR cells and placental explants were treated with leptin to assess its effects on production of IL-8, IL-6 and prostaglandin E2 (PGE2)., Results: Leptin production by all cells and tissues studied was unaffected by treatment with IL-1beta (2.5 ng/mL), TNF-alpha (25 ng/mL) and LPS (2.5 microg/mL). Dexamethasone stimulated leptin production over two-fold by WISH and JEG3 cells, whereas insulin also stimulated a two-fold increase in leptin production in JEG3 cells. IL-6 production by JAR cells and placental explants was stimulated (two- to three-fold) by leptin (300 ng/mL). PGE2 production was unaffected., Conclusions: Leptin derived from gestational tissues is unlikely to play a role in inflammatory reactions within the placenta, but may regulate placental cytokine production. The physiological significance of amnion-derived leptin remains to be established.

To evaluate the association between intercellular adhesion molecule-1 (ICAM-1) in the amnion and preterm labour and delivery, we have assessed ICAM-1 mRNA abundance by Northern analysis and protein levels by enzyme-linked immunosorbent assay (ELISA), in samples of this tissue after term and preterm delivery. The median ICAM-1 mRNA expression following preterm delivery (PTD, n=30) was 24 times greater (P< 0.05) than following elective caesarean section prior to labour at term (CST, n=14). ICAM-1 expression following vaginal delivery after spontaneous labour at term (SLT, n=11) was seven times greater than in the CST group (P< 0.05). The concentration of ICAM-1 protein in the PTD samples (n=31) was four-fold greater than (P< 0.05) in CST (n=14). It was also three-fold greater than in the SLT (n=15) samples (P< 0.05). The results were substantially the same when a preterm spontaneous labour group (PTL) (n=26), exclusive of deliveries complicated by pre-eclampsia (n=1) or intrauterine growth restriction (n=3), was compared to the CST and SLT groups. The ICAM-1 mRNA expression did not differ significantly (P=0.93) between PTL with (n=12) or without (n=14) indicators of intrauterine infection. The results were similar when ICAM-1 protein concentrations were compared (P=0.43) between these two groups. These findings indicate that ICAM-1 is expressed by the human amnion and that this expression is elevated with preterm labour and delivery., (Copyright 2000 Harcourt Publishers Ltd.)

Objective: To test the hypothesis that amnion cytokine production might be regulated by prostanoids., Methods: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effects of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined., Results: Prostaglandin (PG)A2, PGD2, PGF2 alpha, PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonist U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations > or = 8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1 beta-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effects of IL-1 beta on cytokine production., Conclusion: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.

Increased production of prostaglandins and cytokines by amnion, particularly prostaglandin (PG) E2, interleukin (IL)-6 and IL-8, is thought to be an important event in infection-associated preterm labour. We characterized the amnion-derived AV3 cell line to determine its appropriateness as a model for investigation of the regulation of amnion cytokine and PG production. Amnion-derived AV3 cells were treated with tumour necrosis factor-alpha (TNF-alpha, interleukin-1beta (IL-1beta), epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) and IL-6, IL-8 and prostaglandin production was determined by immunoassay. Production of IL-6 and IL-8 rose dramatically with all treatments. PGE2, but not PGF2alpha or 6-keto-PGF1alpha, biosynthesis was also increased in a concentration-dependent manner with all treatments. A rapid increase in PGHS-2 (but not PGHS-1) mRNA expression was observed in response to TNF-alpha and IL-1beta. We conclude that the AV3 cell line inflammatory response profile is similar to those observed in primary amnion and other amnion-derived cell lines, and is an appropriate model for human amnion.

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.

The metabolism of arachidonic acid results in the production of prostaglandins (PGs), which are involved in the initiation of labour at term and preterm. The fetal membranes are a source of pro-inflammatory cytokines which promote increased PG biosynthesis via increased release of arachidonic acid and its conversion to biologically active metabolites such as PGE2 and PGF2alpha. In the amnion, the liberation of arachidonic acid from membrane glycerophospholipid stores can be catalysed by cytosolic phospholipase A2 (cPLA2). In amnion-derived WISH cells, the addition of tumour-necrosis factor alpha (TNF-alpha) (50 ng/ml) provoked a time-dependent increase in the expression of the cPLA2 mRNA which was greatest at 8 and 16 h post-treatment (3.62+/-0.52 and 3.15+/-0.45-fold of control, n=3). The increase in cPLA2 mRNA expression by TNF-alpha was unaffected by the prior addition of interleukin-4 (IL-4) (10 ng/ml), a known inhibitor of prostaglandin endoperoxide H synthase (PGHS)-2 mRNA and protein expression in WISH cells. TNF-alpha also increased the level of immunoreactive cPLA2 protein in a time-dependent manner with the highest levels evident after 8 and 16 h. As with the mRNA, cPLA2 protein levels were unaffected by pre-incubation with IL-4. The inclusion of the cPLA2-specific inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) resulted in a concentration-dependent inhibition of PGE2 biosynthesis in WISH cells treated with TNF-alpha (>95 per cent at 2 microM). We conclude that TNF-alpha increases the abundance of the cPLA2 mRNA and protein in amnion epithelial cells, an effect which plays an important role in amnion PG biosynthesis in the presence of intrauterine infection.

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.

Problem: In hematopoietic cells, interleukin (IL)-13 shares many actions with IL-4. The effects of IL-13 in gestational tissues have yet to be reported, however. We compared the effects of IL-4 and IL-13 on the production of cytokines and prostaglandin E2 (PGE2) in epithelial amnion-derived WISH cells., Method of Study: WISH cells were treated with IL-4 or IL-13 (0.08-10 ng/ml) with/without cotreatment with IL-1 beta (0.2 ng/ml), tumor necrosis factor-alpha (10 ng/ml) or epidermal growth factor (5 ng/ml). The production of IL-6, IL-8, and PGE2 was measured by immunoassay after 16 hr., Results: Both IL-4 and IL-13 inhibited PGE2 production with indistinguishable concentration-response curves, under basal or stimulated conditions. The maximal inhibition of IL-1 beta-stimulated PGE2 production (to 28% +/- 10% of control) was seen at 10 ng/ml of IL-4 or IL-13. Basal IL-6 production was stimulated approximately twofold by IL-4 and IL-13, whereas IL-4 and IL-13 both inhibited cytokine-stimulated (but not basal) IL-8 production by approximately 50%. In the presence of 1 ng/ml of IL-4, IL-13 was unable to further inhibit PGE2 production., Conclusions: The inhibition of PGE2 and IL-8 production by IL-4 in WISH cells is mimicked by IL-13. Both cytokines, probably through binding to a common receptor complex, may share a role in suppressing inflammatory reactions within intrauterine tissues.

Activin-betaA subunits are expressed by the human placenta and extraplacental membranes at term and preterm. The regulation of activin-A production by these tissues has not been characterized to date, however. To determine the effects on activin-A production of pro-inflammatory cytokines, amnion, decidual and placental cells were isolated by enzyme dispersion and treated in primary culture with interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). Activin-A production (determined by ELISA) by amnion, decidual and placental cultures was 1.2 +/-0.27, 31.1+/-9.9, and 50.7+/-28.5 pg/microg protein/16 h, respectively (mean+/-SEM; n=5-7 experiments). Both IL-1beta and TNF-alpha stimulated activin-A production in a concentration-dependent fashion in all cultures; maximal stimulation was achieved at 0.25-1.0 ng/ml IL-1beta and 25-50 ng/ml TNF-alpha, respectively. In amnion, decidual and placental cultures IL-1beta stimulated activin-A production to 747+/-274, 190+/-11, and 254+/-60.2 per cent of controls, while TNF-alpha stimulated production to 312+/-81.5, 194+/-22.5, and 193+/-12.5 per cent, respectively (mean+/-SEM; n=5; P<0.05 by ANOVA). These studies show for the first time that pro-inflammatory cytokines are potent stimulators of activin-A production by intrauterine tissues. This may provide an explanation for the elevated concentrations of activin-A measured in the sera of some women in preterm labour.

Amniotic fluid at term contains high concentrations of interleukin (IL)-6 and IL-8. The source of these cytokines has not been identified, although the fetal membranes (amnion and chorion) are likely contributors. Amnion cytokine production was investigated by using amnion cells isolated by enzymatic digestion (from placentas delivered at term before labor) and cultured in vitro. IL-6 and IL-8 were measured in conditioned media by ELISA. Amnion cells produced detectable amounts of both IL-6 and IL-8 throughout the 7-day culture period. The ratio of IL-8 to IL-6 was approximately 5:1, similar to the ratio found in amniotic fluid. Production of both IL-6 and IL-8 was stimulated in a concentration-dependent fashion by interleukin-1beta (0.1-10 ng/ml), tumor necrosis factor-alpha (1-100 ng/ml), and bacterial lipopolysaccharide (0.1-10 microg/ml), and also by 100 nM phorbol 12-myristate 13-acetate. Epidermal growth factor (1-25 ng/ml) had only minimal effects on amnion cytokine production. Dexamethasone (10 nM) inhibited IL-6/-8 production by approximately 50% throughout the culture period. Production of IL-6/-8 by cultured amniotic fibroblasts, which under basal conditions was much lower than that by epithelial cells, was regulated by all the agents tested in a fashion similar to that of the epithelial cells. These findings suggest that the amnion contributes to the pool of IL-6 and IL-8 in amniotic fluid. We speculate that amnion-derived cytokines might have functions during normal human parturition that are distinct from their conventional roles as inflammatory mediators.

Problem: To determine whether amnion cells produce interleukin (IL)-6 and -8 and thus may contribute to the high concentrations of these cytokines in amniotic fluid at term., Method of Study: Amnion-derived WISH cells were treated in culture with stimuli over 16 hr, and IL-6 and IL-8 concentrations in the conditioned media were measured by enzyme-linked immunosorbent assay or bioassay (IL-6 only)., Results: IL-8 production was approximately 5-fold higher than that of IL-6 under basal and stimulated conditions. Significant (by Dunnett's test after analysis of variance) stimulation of production of both cytokines was achieved by IL-1 beta (> 0.2 ng/ml), TNF alpha (> 10 ng/ml), and the phorbol ester, phorbol 12-myristate 13-acetate (> 2 nM), over a 16-hr culture period. Epidermal growth factor at 10 ng/ml induced a small increase in production of IL-8, but not of IL-6, whereas bacterial lipopolysaccharide had minimal effects on production of either cytokine. Basal and cytokine-stimulated IL-6 and IL-8 production was inhibited by dexamethasone at concentrations equal to or greater than 1 nM., Conclusion: These findings suggest that amnion may be a significant contributor to the IL-6 and IL-8 content of amniotic fluid, and that WISH cells may be a suitable model for the study of cytokine production by amnion epithelial cells.