Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

Interleukin-1β induces the synthesis and activity of cytosolic phospholipase A2 and the release of prostaglandin E2 in human amnion-derived WISH cells

The objective of this study was to examine the expression and activity of cytosolic phospholipase A₂ (cPLA₂) in relation to prostaglandin E₂ (PGE₂) synthesis in human amnion-derived WISH cells in response to stimulation by interleukin-1β (IL-1β). cPLA₂ activity was characterized by sensitivity to heat and acid treatment, stability to dithiothreitol, and inhibition by the specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃). Treatment of WISH cells with IL-1β (0.01–1 ng/mL) for up to 24 h resulted in a significant increase in PGE₂ release in a concentration- and time-dependent manner accompanied by increases both in total cellular cPLA₂ activity and in cPLA₂ protein levels detected by Western blot analysis. The parallel increase in total cellular cPLA₂ activity and cPLA₂ protein level indicates that IL-1β may induce the synthesis of CPLA₂. Incubation of the cells with 10 μM AACOCF₃ for 24 h significantly inhibited IL-1β-induced PGE₂ production strongly suggesting that cPLA₂ mediates IL-1β-induced PGE₂ formation. In unstimulated cells, there is appreciable total cellular cPLA₂ activity and protein, but these cells produce low amounts of PGE₂ until stimulated by IL-1β, suggesting that cPLA₂ translocation from cytosol to the membrane is necessary for its bioactivity. In contrast to IL-1β, treatment with phorbol ester (12-O-tetradecanoyl phorbol-13-acetate, TPA, 10⁻¹⁰−10⁻⁶ M) for 24 h significantly inhibited total cellular cPLA₂ activity in a concentration-dependent manner. The amount of total cellular cPLA₂ protein seen on Western blot remained unchanged following TPA treatment. These data suggest that in WISH cells, IL-1β induces both translocation to the membrane and de novo synthesis of cPLA₂ protein to sustain prostaglandin (PG) synthesis. In contrast, TPA may only cause cPLA₂ translocation but no increase in cPLA₂ protein synthesis, resulting in limited PFG synthesis. Our results provide a mechanism for the effect of IL-1β on prostaglandin synthesis in human amnion cells and provide support for a role of cPLA₂ in the mechanism initiating human parturition.     

Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria.

Murine epidermal growth factor (EGF) stimulated the production of prostaglandin E₂ (PGE₂) and bone resorption in neonatal mouse calvaria in organ culture. The effect of EGF on bone resorption occurred at low concentrations of the polypeptide (half-max stimulation = 0.4 ng/ml, 6.6 × 10^?11 M). All concentrations of EGF which stimulated resorption also stimulated the production of PGE₂ by bone; concentrations of EGF which did not stimulate resorption did not enhance PGE₂ production. EGF-induced formation of PGE₂ and bone resorption were inhibited completely by indomethacin (200 ng/ml) and hydrocortisone (3 × 10^?6 M). Indomethacin did not inhibit the bone resorption-stimulating activity of exogenous PGE₂. The time courses of action of EGF, parathyroid hormone and exogenous PGE₂ on bone resorption were similar. Brief exposure (15 or 60 min) to EGF (10 ng/ml) did not cause bone resorption or an increase in PGE₂ accumulation in a subsequent 48-h incubation in the absence of EGF. High concentrations (30 to 100 ng/ml) of bovine fibroblast growth factor (FGF) also stimulated the production of PGE₂ and bone resorption. We conclude that concentrations of EGF equal to or less than those present in mouse plasma stimulate the resorption of mouse bone in organ culture by a mechanism that involves the enhanced local production of PGE₂.     

Dose-related action of estradiol on placental prostanoid prediction

Prostanoids play an important role throughout all of pregnancy and during the initiation and progress of labor. The human placenta at term produces large quantities of prostanoids, yet little is known of the factors that regulate their biosynthesis. Herein, we report the effect of estradiol or estradiol and progesterone on the basal release of placental prostanoids from fresh human term placental explants using a perifusion system.The basal release of prostaglandin E₂ (PGE₂, prostaglandin F_2α (PGF_2α), thromboxane (TxB₂) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) increased about 50% from the fifth to the ninth hour in culture, while the release of 13, 14-dihydro-15-keto-PGF (PGFM) remained constant and hCG release decreased. The dose-related effect of estradiol (20–2,000 ng/ml) in the perifusing medium starting at the fifth hour of perifusiOn (i.e., the zero treatment time) effected no change in the release of TxB₂, PGF_2α, PGFM or hCG. A biphasic action on the release of 6-keto-PGF,. was observed, i.e. it was significantly decreased when incubated with 20 ng/ml of estradiol, but effected an increase after exposure to 200 ng/ml. The concomitant addition of progesterone (2,000 ng/ml) with estradiol (200 ng/ml) significantly inhibited the stimulatory action of estradiol at this dose. The release of PGE₂ was inhibited in a dose-related fashion with increasing dose of estradiol. The addition of progesterone with estradiol (2,000 and 200 ng/ml, respectively) reversed the inhibition of PGE₂ by estradiol alone.These data demonstrate that physiologic levels of estradiol affect 6-keto-PGF_α and PGE₂ release from the human term placenta, but do not significantly alter production of TxB₂, PGFM or hCG under these conditions.     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

The in vivo cytokine release profile following implantation

There has been no comprehensive study that maps the production of the range of inflammatory cytokines following implantation of a material. There is an urgent requirement for specific data on the real time production of biological markers in order to study their effects in vitro and more accurately predict the in vivo response. This study determined the production of IL-1β, IL-2, IL-4, IL-6, IL-10, IFNγ and TNF-α in response to a synthetic material implanted in a rat soft tissue model for up to 90 days. IL-1 β and TNF-α were elevated over the total experimental time course with values in the order of 500 pg/ml for IL-1β and 40 pg/ml for TNF-α. The cytokines IL-2, IL-4, IL-6 and IL-10 were also detected and their production reduced with increasing time.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Decidual adenylate cyclase and prostaglandin production in vitro

Human decidua contains an active adenylate cyclase, and a number of studies indicate that adenylate cyclase is functionally linked to increased in vitro prostaglandin synthesis. Increased decidual prostaglandin synthesis is associated with parturition, and therefore activation of adenylate cyclase may be involved in the control of human parturition. In this study, third trimester human decidual cells were preincubated for no more than 24 h prior to stimulation with a number of reagents which increase cellular cyclic AMP levels. Forskolin rapidly increased intracellular and extracellular cyclic AMP levels, but there was no increase in prostaglandin E₂ biosynthesis during incubations ranging from 5 min up to 24 h. Dibutyryl cyclic AMP or 8-bromo-cyclic AMP were also without effect on PGE₂ production, which suggests that the adenylate cyclase was not linked to the mechanisms regulating prostaglandin production. Cholera toxin increased basal cyclic AMP and PGE₂ synthesis, and was without effect on IL-1β-stimulated PGE₂ levels. PGE₂ synthesis was increased by 24 h culture with IL-1β in all the cell preparations, indicating that the cells were biologically active, and that the lack of effect of changes in cyclic AMP synthesis on PGE₂ levels could not be attributed to a defect in the prostaglandin synthetic pathway. Our findings did not agree with earlier work which showed that changes in cyclic AMP were correlated with changes in PGE₂ production by human decidual cells. It is clear that in the previous studies the decidual cells were preincubated for 4–7 days prior to stimulation, in contrast with 24 h in our investigation. We suggest that the functional link between cyclic AMP and PGE₂ synthesis reported previously may develop during culture, and not be a part of normal decidual cell function, but further studies are needed to test this hypothesis.     

The effect of adrenergic stimulation on urinary prostaglandin E2 and 6 keto PGF1α in man

To evaluate the details of the adrenergic stimulation of urinary prostaglandins in man, ten normal volunteers were given various agonists and antagonists. The effect of 4 hour IV infusions of norepinephrine (NE), NE + phentolamine (PHT), NE + phenoxybenzamine (PHB), NE + prazosin (PZ), isoproterenol (ISO), and PHT alone on urinary PGE₂ and PGI₂ (6 keto PGF_1α) were determined. PGE₂ and 6 keto PGF_1α were measured by radioimmunoassay from 4 hour urine samples. NE stimulated both PGE₂ (196±40 to 370±84 ng/4 hrs/g creatinine and 6 keto PGF_1α(184±30 to 326±36), both p<0.01. In contrast, ISO had no effect on either PGE₂ or 6 keto PGF_1α excretion. Alpha blockade with PHT. PHB, or PZ inhibited the NE induced systemic pressor effect. However, the effect of the alpha blockers on the NE induced stimulation of PGE₂ and 6 keto PGF_1α varied. PHT did not alter the NE stimulated PGE₂ or 6 keto PGF_1α release (370±84 vs. 381±80) PGE₂ and (326±50 vs. 315±40) 6 keto PGF_1α, both p>0.2). PHT alone stimulated only 6 keto PGF_1α. PHB and the specific α₁ antagonist PZ similarly eliminated the NE induced prostaglandin release. These results suggest that adrenergically mediated urinary prostaglandin release in man is via an alpha receptor with α₁ characteristics.     

Actions of interleukin-2 on amnion prostaglandin biosynthesis

Preterm labor is associated with increased intrauterine prostaglandin (PG) production. Intrauterine infections are frequently associated with preterm labor and increased cytokine production. The cytokine interleukin-2 (IL-2) is a potent T-cell growth factor necessary for effective cell-mediated immunity. In this study we evaluated the effects of IL-2 on PGE₂ biosynthesis by human amnion cells. IL-2 alone modestly but significantly inhibited amnion PGE₂ production. Moreover, IL-2 also attenuated, in a concentration-related fashion, the stimulatory actions of IL-1β on PGE₂ production by amnion cells. These data suggest that IL-2 could potentially represent a negative regulatory element in the mechanisms of preterm labor in association with intrauterine infection.     

Effects of hydrocortisone, oestradiol and progesterone on A23187-stimulated prostaglandin output from the guinea-pig uterus superfused in vitro

Hydrocortisone (10 μg/ml) had no effect on the basal outputs and A23187-stimulated outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α from the Day 15 guinea-pig uterus superfused . These findings indicate that the high output of PGF_2α from the guinea-pig uterus during the last one-third of the oestrous cycle is not modulated by the adrenal glucocorticoid hormones. Progesterone (10 gmg/ml) had no effect on the A23187-induced increases in PG output from the Day 15 guinea-pig uterus. However, oestradiol (10 gmg/ml but not 1 μg/ml) significantly reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by A23187 from the Day 15 guinea-pig uterus, without affecting basal PG outputs. The increase in uterine tone induced by A23187 in the Day 15 guinea-pig uterus was reduced by 20–50% by oestradiol (10 μg/ml). The addition of oestradiol (10 μg/ml) and progesterone together (10 gmg/ml) produced the same effects on the Day 15 guinea-pig uterus as oestradiol alone. Oestradiol (10 μg/ml) also reduced the A23187-induced increases in PG output from the Day 7 guinea-pig uterus, but did not reduce the increase in uterine tone. Oestradiol (10 gmg/ml) reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by exogenous arachidonic acid from the Day 7 and Day 15 guinea-pig uterus. Previous studies have shown that oestradiol is not a cyclo-oxygenase inhibitor. The present findings suggest that oestradiol, at a relatively high concentration, may interfere with the access of arachidonic acid to the cyclo-oxygenase enzyme. This action of oestradiol may explain its anti-luteolytic action when administered to guinea-pigs in large doses after Day 9 of the cycle.     

Cyclo-oxygenase products in carrageenin-induced inflammation

Prostaglandin (PG) E₂, thromboxane (TX) B₂ and the stable breakdown product of prostacyclin, 6-oxo-PGF_1α are present in carrageenin-induced inflammatory exudates. Carrageenin-impregnated polyester sponges were implanted subcutaneously in rats and inflammatory exudates were collected 4–192 h after implantation. The concentrations of cyclo-oxygenase products in sponge fluids was measured by radioimmunoassay after extraction and purification. All three products were detectable after 4 h and reached a peak at 12–24 h. Mean TXB₂ concentrations reached 74 ng/ml at 12 h but decreased to less than 10 ng/ml after 24 h. PGE₂ concentrations were 65 ng/ml at 24 h, after which there was no significant increase and then dropped to about 20 ng/ml between 96 and 192 h. 6-oxo-PGF_1α reached a concentration of 33 ng/ml at 24 h which did not change significantly until levels fell to less than 10 ng/ml between 96 and 192 h. The presence of PGE₂, TXB₂ and 6-oxo-PGF_1α was confirmed by gas-liquid chromatography and mass spectrometry. Total leukocyte numbers increased steadily and were at their highest (116.0 × 10⁶ cells/ml) at 192 h. These results suggest that thromboxanes and prostacyclin, as well as PGE₂, contribute to the acute inflammatory response.     

Stimulation of prostaglandin production in bone by phorbol diesters and melittin

The production of prostaglandin E₂ (PGE₂) and bone resorption were studied in neonatal mouse calvaria in organ culture. Two tumor promoters 12- -tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12, 13-di-decanoate, but not the non-tumor promoters 4α-phorbol-12,13-didecanoate and phorbol, stimulated both PGE₂ synthesis in bone and bone resorption. The effect of TPA was maximum at about 25 ng/ml, and half-maximum stimulation occurred at about 8 ng/ml TPA. The effects of TPA on the production of PGE₂ and bone resorption were inhibited completely by indomethacin (5.6 × 10⁻⁸ to 5.6 × 10⁻⁷ M). The bee venom toxin, melittin, was also a potent stimulator of prostaglandin synthesis in bone and bone resorption. The effect of melittin was maximum at about 25 ng/ml, and the dose-response curve was biphasic. The effects of melittin on the production of PGE₂ and bone resorption were also inhibited by indomethacin. Indomethacin did not inhibit the bone resorption-stimulating activity of exogenously added PGE₂. We conclude that phorbol diesters, which have irritant and tumor-promoting activity in mouse skin, and the polypeptide melittin can act directly on bone to stimulate resorption by a mechanism involving the local production of PGE₂ or possibly other indomethacin-inhibited metabolites of arachidonic acid.     

7beta-hydroxy-epiandrosterone modulation of 15-deoxy-delta12,14-prostaglandin J2, prostaglandin D2 and prostaglandin E2 production from human mononuclear cells

7β-hydroxy-epiandrosterone (7β-OH-EPIA) has been shown to be cytoprotective in various organs including the brain. It has also been shown that prostaglandin D₂ (PGD₂) and its spontaneous metabolite 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) are also cytoprotective. It is possible that these prostaglandins derived from circulating mononuclear cells may mediate the actions of 7β-OH-EPIA. The aim of this study, therefore, was to ascertain the effect of 7β-OH-EPIA (in the absence or presence of tumour necrosis factor-α (TNF-α)), a pro-inflammatory stimulus, on the biosynthesis of PGD₂, PGE₂ and 15d-PGJ₂ from human mononuclear cells. Prostaglandins were measured by enzyme immunoassay (EIA). 7β-OH-EPIA alone induced a concentration-dependant increase in the production of PGD₂. TNF-α increased PGD₂ levels which were enhanced by 7β-OH-EPIA. 7β-OH-EPIA increased 15d-PGJ₂ levels both in the absence and presence of TNF-α. 7β-OH-EPIA alone had no effect on PGE₂ biosynthesis but suppressed TNF-α-induced PGE₂ circa 50%. 7β-OH-EPIA also increased the level of free arachidonic acid and radiolabelled prostaglandins in cells pre-incubated with radiolabelled arachidonic acid, indicating that the increase may occur via the enhanced release of substrate arachidonic acid. 7β-OH-EPIA did not affect levels of the anti-inflammatory cytokine IL-10 indicating that this is an unlikely mechanism by which 7β-OH-EPIA induces its actions but more likely exerts its effects via the production of cytoprotective prostaglandins.