Involvement of a pertussis toxin-sensitive G-protein-coupled phospholipase A2 in lipopolysaccharide-stimulated prostaglandin E2 synthesis in cultured rat mesangial cells

E. coli lipopolysaccharide (LPS) stimulated a dose- and time-dependent release of prostaglandin E2 (PGE2) in cultured rat glomerular mesangial cells. Pertussis toxin, an inhibitor of several GTP-binding proteins (G proteins), blocked nearly 80% of the LPS-stimulated PGE2 formation, while having virtually no effect on calcium ionophore-stimulated PGE2 production. We tested the possibility that a G protein-coupled activation of phospholipase A2 mediated the LPS-stimulated PGE2 production. Evidence for LPS activation of phospholipase A2 included a time-dependent LPS-induced generation of [32P]lysophosphatidylcholine and the inhibitory effects of a phospholipase A2 inhibitor, mepacrine, on LPS-induced PGE2 formation. Possible roles for phospholipase C-dependent activation of PGE2 synthesis by LPS seemed unlikely, as LPS did not elevate the cytosolic free calcium concentration or augment the appearance of water-soluble inositol phosphates. We conclude that LPS-induced PGE2 synthesis in rat glomerular mesangial cells is mediated through a G-protein-coupled phospholipase A2 activation. The activation of phospholipase A2 releases arachidonic acid and stimulates PGE2 synthesis preferentially, thereby improving glomerular hemodynamic events in endotoxemia.     

Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and L-N-monomethylarginine (L-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, L-NMMA at low concentrations (< or = 100 microM) inhibited the spontaneous release of No in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, L-NMMA (500 microM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.     

Identification of two neutrophil chemotactic peptides produced by porcine alveolar macrophages.

We have purified to homogeneity two distinct 10-kDa proteins with potent chemotactic activity for neutrophils from porcine alveolar macrophages incubated for 24 h with Escherichia coli endotoxin (lipopolysaccharide (LPS), 10 micrograms/ml). Neutrophil chemotactic activity in alveolar macrophage supernatants was concentrated by adsorption to SP-Sephadex, and purified by cation exchange and reversed phase high performance liquid chromatography. The first peptide, alveolar macrophage chemotactic factor (AMCF)-I, had chemotactic activity for both porcine and human neutrophils. The chemotactic activity for porcine neutrophils was detectable at 3 x 10(-10) M, peaked at 3 x 10(-8) M, and was comparable to that of zymosan-activated porcine serum. Segmental instillation of AMCF-I into porcine lungs caused marked neutrophil accumulation at 4 h in both bronchoalveolar lavage fluid and in lung tissue. The second peptide, AMCF-II, was active at 1.4 x 10(-9) M for porcine neutrophils, but it was less active for human polymorphonuclear neutrophils than was AMCF-I. Oligonucleotide probes to regions of the N-terminal sequences of AMCF-I and AMCF-II hybridized to mRNA recovered from LPS-stimulated alveolar macrophages. The N-terminal sequences and amino acid compositions indicate that AMCF-I and AMCF-II are distinct proteins, but that both have homologies with a family of peptide chemoattractants produced by human blood monocytes and platelets. Thus, alveolar macrophages stimulated with LPS produce two distinct 10-kDa cytokines with potent chemotactic activity for neutrophils. This indicates that there are two different peptide pathways by which alveolar macrophages can recruit neutrophils into the lung.     

Lipopolysaccharide and silica-stimulated mononuclear cell prostaglandin production in ulcerative colitis

Basal, lipopolysaccharide (LPS) and silica-stimulated prostaglandin (PG) production were compared between peripheral blood mononuclear cells (PBMNC) from UC patients and healthy subjects (HS). Basal and LPS-stimulated PBMNC PGI2, but not PGE2, production was greater in UC. LPS stimulated both PGE2 and PGI2 by PBMNC from HS and UC patients. Silica stimulated production of both PGs by cells from HS but only PGE2 by cells from UC patients. The differences in responses to silica and LPS may result from differences in activation of NFkappaB or, alternatively, prior sensitisation to one of these agents. That PBMNC PGE2 production is not increased in UC, as it is in Crohn's disease, suggests that there are differences in PBMNC behaviour between these two diseases.     

Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by /=85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10-15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.     

Cytokine-mediated inflammatory hyperalgesia limited by interleukin-13

The effect of interleukin-13 (IL-13) on hyperalgesic responses to intraplantar (i.pl.) injection of carrageenin, E. coli endotoxin (LPS), bradykinin, tumour necrosis factor a (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) was investigated in a model of mechanical hyperalgesia in rats. Also, the cellular source of the IL-13 was investigated. IL-13, administered 30 min before the stimulus, inhibited responses to carrageenin, LPS, bradykinin, and TNF-alpha, but not responses to IL-1 beta, IL-8 and PGE2. IL-13, administered 2 hours before the injection of IL-1b, did not affect the response to IL-1b, whereas IL-13, administered 12 hours or 12 + 2 hours before the IL-1 beta, inhibited the hyperalgesia (- 35%, - 77%, respectively). In murine peritoneal macrophages, IL-13 administered 2 hours before stimulation with LPS, inhibited the production of IL-1 beta (- 67%) and PGE(2) (- 56%). IL-13 administered 12 hours before stimulation with LPS inhibited LPS-stimulated PGE(2) but not IL-1 beta. An anti-IL-13 serum potentiated responses to carrageenin, LPS, bradykinin and TNF-alpha (but not IL-1 beta and IL-8), as well as responses to bradykinin in rats depleted of mast cells with compound 40/80, but not in athymic rats. These data suggest that IL-13, released by lymphocytes, limits inflammatory hyperalgesia by the inhibition of the production TNF-alpha, IL-1 beta, IL-8 and PGs.     

Regulation of leptin release and lipolysis by PGE2 in rat adipose tissue

The role of eicosanoids formed by adipose tissue from rats was examined in the presence of the specific cyclooxygenase-2 inhibitor NS-398. This agent totally blocked the release of prostaglandin E2 (PGE2) by rat adipose tissue over a 24-h incubation in primary culture. The final concentration of PGE2 after 24 h was 12 nM, and half-maximal inhibition of PGE2 formation required 35 nM NS-398. While inhibition of PGE2 formation by NS-398 had no effect on basal leptin release or lipolysis, it enhanced the lipolytic action of 10 nM isoproterenol by 36%. The in vivo administration of PGE2 doubled serum leptin. PGE2 also directly stimulated leptin release by rat adipose tissue incubated in the presence of 25 nM dexamethasone, which inhibited endogenous PGE2 formation by 94%. The inhibition of lipolysis as well as the stimulation of leptin release by PGE2 were mimicked by N6-cyclopentyladenosine (CPA). These data indicate that exogenous PGE2 can stimulate leptin release by adipose tissue when the basal formation of PGE2 is blocked by dexamethasone. However, while the endogenous formation of PGE2 does not appear to regulate basal lipolysis or leptin release, it may play a role in the activation of lipolysis by catecholamines.     

Inhibition by 17 beta-estradiol of PTH stimulated resorption and prostaglandin production in cultured neonatal mouse calvariae

Previous attempts to show a direct effect of physiological concentrations of 17 beta-estradiol (beta E2) on bone in vitro have been unsuccessful. We describe a culture system using neonatal mouse calvariae in which beta E2 in the range 1 pM to 1 nM inhibited parathyroid hormone (PTH) stimulated prostaglandin E2 (PGE2) release by 50 to 70% in the presence and absence of cortisol. In addition, beta E2 reduced medium calcium concentration and release of previously incorporated 45Ca by 10 and 20%, respectively, in PTH stimulated cultures. Indomethacin did not block beta E2 effects on resorption. 17 alpha-Estradiol (alpha E2) reduced PTH stimulated 45Ca release but not PGE2 release. Thus, beta E2 has direct effects on bone consistent with its known effects to decrease bone resorption in vivo.     

Regulation of interleukin 1 production by mouse peritoneal macrophages. Effects of arachidonic acid metabolites, cyclic nucleotides, and interferons

The effects of prostaglandin E2 (PGE2), cyclic nucleotides, leukotriene B4 (LTB4), and interferons on interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated C3H/HeNCrl mouse peritoneal macrophages were studied. IL 1 production was inhibited by PGE2, the adenosine 3':5'-monophosphate analog dibutyryl cAMP, the cAMP agonist isoproterenol, and the phosphodiesterase inhibitor isobutylmethylxanthine. These agents were more inhibitory when added early in the latent phase of IL 1 synthesis following stimulation with LPS rather than just prior to release of IL 1 into the medium. Production of both the intracellular and extracellular forms of IL 1 was blocked by PGE2 and cAMP. Suppression of LPS-induced IL 1 production by PGE2 was prevented by leukocyte alpha-interferon. Moreover, alpha-interferon augmented LPS-induced IL 1 production but did not stimulate IL 1 production in the absence of LPS. Immune gamma-interferon markedly inhibited LPS-stimulated IL 1 production. The lipoxygenase inhibitor eicosa-5,8,11,14-tetraynoic acid suppressed, whereas 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline augmented, LPS-induced IL 1 production. The opposing effects of these agents suggested that lipoxygenase metabolites do not act as inducers of IL 1 production. Purified LTB4 did not stimulate base-line or augment LPS-induced IL 1 production (both intracellular and extracellular forms). Moreover, calcium ionophore A23187 (a lipoxygenase activator) did not stimulate IL 1 production, alone or in combination with LTB4. Thus, net IL 1 production by macrophages may be regulated by a balance between the effects of PGE2, cAMP, alpha-interferon, and gamma-interferon, but not LTB4.     

Prostanoids stimulate K secretion and Cl secretion in guinea pig distal colon via distinct pathways

Short-circuit current (I(sc)) and transepithelial conductance (Gt) were measured in guinea pig distal colonic mucosa isolated from submucosa and underlying muscle layers. Indomethacin (2 microM) and NS-398 (2 microM) were added to suppress endogenous production of prostanoids. Serosal addition of PGE2 (10 nM) stimulated negative I(sc) consistent with K secretion, and concentrations >30 nM stimulated positive I(sc) consistent with Cl secretion. PGE2 also stimulated Gt at low and high concentrations. Dose responses to prostanoids specific for EP prostanoid receptors were consistent with stimulating K secretion through EP2 receptors, based on a rank order potency (from EC50 values) of PGE2 (1.9 nM) > 11-deoxy-PGE1 (8.3 nM) > 19(R)-hydroxy-PGE2 (13.9 nM) > butaprost (67 nM) > 17-phenyl-trinor-PGE2 (307 nM) > sulprostone (>10 microM). An isoprostane, 8-iso-PGE2, stimulated K secretion with an EC50 of 33 nM. Cl secretory response was stimulated by PGD2 and BW-245C, a DP prostanoid receptor-specific agonist: BW-245C (15 nM) > PGD2 (30 nM) > PGE2 (203 nM). Agonists specific for FP, IP, and TP prostanoid receptors were ineffective in stimulating I(sc) and Gt at concentrations <1 microM. These results indicate that PGE2 stimulated electrogenic K secretion through activation of EP2 receptors and electrogenic KCl secretion through activation of DP receptors. Thus stimulation of Cl secretion in vivo would occur either via physiological concentrations of PGD2 (<100 nM) or pathophysiological concentrations of PGE2 (>100 nM) that could occur during inflammatory conditions.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are known to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O-2) production as well as the generation of PGE2, PGF2 alpha, and TXB2 from resident, oil-elicited and thioglycollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O-2, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O-2, these cells did secrete significant levels of PGE2, PGF2 alpha, and TXB2 in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE2 and PGF2 alpha when incubated with C5a. However, production of TXB2 was not stimulated. FMLP was inactive in stimulating PGE2, PGF2 alpha, and TXB2 in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

Distinct protein kinase A anchoring proteins direct prostaglandin E2 modulation of Toll-like receptor signaling in alveolar macrophages

Toll-like receptors (TLRs) direct a proinflammatory program in macrophages. One mediator whose generation is induced by TLR ligation is prostaglandin E(2) (PGE(2)), which is well known to increase intracellular cAMP upon G protein-coupled receptor ligation. How PGE(2)/cAMP shapes the nascent TLR response and the mechanisms by which it acts remain poorly understood. Here we explored PGE(2)/cAMP regulation of NO production in primary rat alveolar macrophages stimulated with the TLR4 ligand LPS. Endogenous PGE(2) synthesis accounted for nearly half of the increment in NO production in response to LPS. The enhancing effect of PGE(2) on LPS-stimulated NO was mediated via cAMP, generated mainly upon ligation of the E prostanoid 2 receptor and acting via protein kinase A (PKA) rather than via the exchange protein activated by cAMP. Isoenzyme-selective cAMP agonists and peptide disruptors of protein kinase A anchoring proteins (AKAPs) implicated PKA regulatory subunit type I (RI) interacting with an AKAP in this process. Gene knockdown of potential RI-interacting AKAPs expressed in alveolar macrophages revealed that AKAP10 was required for PGE(2) potentiation of LPS-induced NO synthesis. AKAP10 also mediated PGE(2) potentiation of the expression of cytokines IL-10 and IL-6, whereas PGE(2) suppression of TNF-α was mediated by AKAP8-anchored PKA-RII. Our data illustrate the pleiotropic manner in which G protein-coupled receptor-derived cAMP signaling can influence TLR responses in primary macrophages and suggest that AKAP10 may coordinate increases in gene expression.     

Substance P acts directly upon cloned B lymphoma cells to enhance IgA and IgM production.

The IgA producing murine B lymphoma, CH12.LX.C4.4F10 (4F10) and the IgM producing murine lymphoma, CH12.LX.C4.5F5 (5F5) were found to express substantial numbers of substance P (SP) receptors having dissociation constants equal to 0.69 nM. Binding of SP by these B lymphoma cells was via the tachykinin-specific C-terminus sequence, Phe-X-Gly-Leu-Met-NH2, because SP, SP antagonist (D-Pro2-D-Phe7-D-Trp9-SP), eledoisin, and substance K could effectively inhibit radiolabeled SP binding, whereas the SP N-terminus fragment, SP (1-4), could not. The functionality of these receptors could be demonstrated by the ability of subnanomolar concentrations of SP to induce Ig secretion in a dose-dependent fashion. However, the presence of a second stimulus in these cultures was required to obtain maximal increases. IgA secretion by 4F10 cells was elevated only 25 to 37%, and IgM secretion by 5F5 cells was not significantly increased in cultures in which nanomolar concentrations of SP were present. Conversely, coculturing 5F5 cells with a suboptimal concentration of LPS (50 ng/ml) and 10(-10)M SP resulted in an approximate threefold increase in supernatant IgM when compared to control cultures stimulated with LPS alone. While not as dramatic, 10(-10) M SP also enhanced IgA secretion of LPS-stimulated 4F10 cells by approximately 45%. This enhancement of Ig secretion was SP-specific, as evidenced by the ability of 1000-fold excess of SP antagonist to block SP-induced, but not LPS-induced, Ig production. Clearly, SP could act synergistically with LPS to enhance Ig secretion; therefore, we questioned whether this augmentation was also reflected at the level of H chain mRNA expression. 10(-9)M SP induced modest increases (50 to 60%) in mu-chain mRNA expression by LPS-stimulated 5F5 cells when compared with cells stimulated with LPS alone. The 4F10 cells did not display this magnitude of difference for alpha-chain mRNA expression. Thus, although SP-induced increases of mu-chain mRNA by 5F5 cells may contribute to the increased Ig secretion observed by these LPS-activated lymphocytes, it is unlikely that increased mRNA expression can totally account for the threefold increases in secretion that were observed.     

Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.     

The induction of prostaglandin E synthase and upregulation of cyclooxygenase-2 by 9-cis retinoic acid

9-cis Retinoic acid (9cRA) is a promising lead compound to design the retinoid X receptor (RXR) ligands with the ability to simultaneously activate RXR heterodimers with the selectivity to their nuclear receptor partners. In this study, we investigated the effects of 9cRA on the prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) production. 9cRA increased the PGE2 and TXA2 productions in the presence of lipopolysaccharide (LPS). All-trans retinoic acid, the retinoic acid receptor ligand, also increased their production. We revealed that cyclooxygenase (COX)-2 was clearly induced by 9cRA in the presence of LPS. The induction was not suppressed by indomethacin, which completely inhibited the increase in the LPS-stimulated prostanoid production by 9cRA. The expression levels of the toll-like receptor 4 and CD14, which were components of the LPS receptor complex, were increased by 9cRA in the presence and absence of LPS. PGE synthase was also clearly increased by 9cRA in the presence and absence of LPS. In this study, we noted that 9cRA increased the production of PGE2 and TXA2 by the induction of COX-2 and PGE synthase in the presence of LPS. The induction of the LPS receptor complex by 9cRA is able to upregulate the induction of COX-2 by LPS.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.