Prostaglandin E2 production during hepatic regeneration downregulates Kupffer cell IL-6 production.

The liver possesses the remarkable ability to regenerate to its original size after a 70% partial hepatectomy. There has been little effort to characterize the Kupffer cells' role in this unique mammalian reparative physiologic phenomenon. The capacity of rat Kupffer cells (KC) isolated at specific intervals after partial hepatectomy to produce interleukin-6 (IL-6) and prostaglandin E2 (PGE2) in response to endotoxin was evaluated in standard RPMI-1640 (1200 microM L-arginine) and arginine-depleted RPMI-1640 (10 microM L-arginine) media. Regenerating liver KC 48 to 120 hours after partial hepatectomy responded to endotoxin stimulation with a significantly greater (p less than 0.05) production of IL-6 in standard RPMI-1640. Because Kupffer cells function in an environment where high arginase activity results in negligible L-arginine levels, the 10 microM L-arginine RPMI-1640 was used to simulate the true hepatic microenvironment. Production of IL-6 by regenerating liver KC was further increased (p less than 0.05) by placing these same KC in 10 microM L-arginine RPMI-1640 tissue culture media. During the same period, regenerating liver KC produced significantly (p less than 0.01) more PGE2 than sham-operated KC in both standard and low-arginine media. When the cyclo-oxygenase inhibitor indomethacin (1 x 10(-5) M) was added to cultures, the PGE2 production was inhibited, and IL-6 production was upregulated (p less than 0.05) in arginine-depleted cultures. The authors conclude that during hepatic regeneration KC IL-6 production is elevated but controlled in an autoregulatory fashion by KC PGE2 production.     

Protection of sinusoidal endothelial cells against storage/reperfusion injury by prostaglandin E2 derived from Kupffer cells.

BACKGROUND: In clinical liver transplants, grafts are frequently exposed to endotoxin (lipopolysaccharide, LPS) before harvest and may be predisposed to dysfunction. Because graft failure is linked to sinusoidal endothelial cell injury after storage/reperfusion, we investigated the effect of donor exposure to LPS on graft survival in relation to sinusoidal endothelial cell injury after storage/reperfusion in rats. METHODS: Rats were injected with 0.5 mg/kg LPS. In some rats, 20 mg/kg GdCl3 or 5 mg/kg indomethacin was injected before LPS to ablate Kupffer cells and inhibit prostaglandin (PG) synthesis, respectively. Other rats were injected with 100 microg/kg dimethyl PGE2, a stable PGE2 analog. Rat livers were harvested, stored in cold UW solution and transplanted to non-treated rats for determination of survival and liver injury in recipients. Otherwise, after cold storage, the livers were reperfused briefly with physiological buffer containing trypan blue for determination of sinusoidal endothelial cell injury by counting trypan blue-positive nuclei in histological sections. RESULTS: Donor treatment with LPS increased hepatic PGE2 production before storage and decreased recipient survival, but paradoxically decreased killing of sinusoidal endothelial cells after storage and reperfusion. Pretreatment of donors with GdCl3 or indomethacin prevented the protective preconditioning of sinusoidal endothelial cells by LPS, whereas pretreatment with dimethyl PGE2 protected sinusoidal endothelial cells to the same extent as LPS. Unlike LPS, however, PGE2 attenuated graft injury after liver transplants. CONCLUSION: PGE2 derived from LPS-stimulated Kupffer cells protects sinusoidal endothelial cells against storage/reperfusion injury. Unlike LPS, PGE2 improves graft function after liver transplants. Thus, donor preconditioning with PGE2 may be beneficial in liver transplants.     

Prostanoid production by lipopolysaccharide-stimulated Kupffer cells

Although some data suggest that macrophages in the reticuloendothelial system (RES) are important sources of thromboxane A2 (TxA2) and prostacyclin (PGI2) during endotoxic shock, we are unaware of data documenting the ability of hepatic macrophages (Kupffer cells) to release either TxA2 or PGI2 when exposed to lipopolysaccharide (endotoxin, LPS). In this study, Kupffer cells were examined for their ability to release prostaglandin E2 (PGE2), TxA2, and PGI2 following stimulation with 0, 1.0, 50.0, and 100.0 micrograms/ml of Escherichia coli LPS. Kupffer cells were obtained from rat livers by enzymatic digestion with 0.05% collagenase followed by enrichment of the macrophage population on the basis of differences in density and adherence among the various cell populations isolated. Based on several criteria (phagocytosis of opsonized sheep erythrocytes, positive staining for esterase and peroxidase, failure to replicate), 95% of adherent cells were Kupffer cells. After 4 days of incubation, cells were stimulated with various doses of LPS for 4 and 8 hr. Prostanoid concentrations in culture supernatants were determined by radioimmunoassay. Increasing doses of LPS significantly (P less than 0.001) increased the concentration of immunoreactive PGE2 (iPGE2) and iTxB2 (the stable metabolite of TxA2). The concentration of i6-keto-PFG1 alpha (stable metabolite of PGI2) increased following stimulation with 1.0 microgram/ml of LPS, but declined as the dose of LPS was increased. The results provide evidence that endotoxin-activated Kupffer cells, like other macrophage populations, release several metabolites of arachidonic acid. Kupffer cell-derived prostanoids, particularly TxA2, may be important mediators of some of the pathophysiologic manifestations of acute endotoxemia.     

Modulation of interleukin-6 by beta2-adrenoceptor in endotoxin-stimulated renal macrophage cells.

BACKGROUND: Activation of the cAMP signaling pathway by means of beta2-adrenoceptor agonists has been shown to up-regulate interleukin-6 (IL-6) gene expression and to stimulate IL-6 production in macrophage cells. However, whether beta2-adrenoceptor activation can also modify the rate of IL-6 production in macrophage cells activated by the bacterial endotoxins has not yet been determined. Using renal resident macrophage cells treated with endotoxin, lipopolysaccharide (LPS), and beta2-adrenoceptor agonist, terbutaline, we investigated the role of cAMP pathway, tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinase (MAPK) pathway (p42/p44) in regulating IL-6 production. METHODS: IL-6 protein, mRNA, and promoter activity were measured in these cells exposed to LPS (1 microg/ml) and/or terbutaline (10(-9) to 10(-6) M). Furthermore, the time course effects of terbutaline on cAMP, MAPK (p42/p44), and TNF-alpha release were evaluated in the cells. RESULTS: Terbutaline at high concentrations (10(-6) M) significantly up-regulated IL-6 by approximately 25% (P<0.05), whereas at a lower concentration (10(-8) M), it down-regulated IL-6 production by 42% (P<0.05). Terbutaline (10(-8) and 10(-6) M) caused a concentration- and time-dependent stimulation of cAMP (P<0.05) and TNF production (P<0.05) and a time-dependent decrease in MAPK activity (P<0.05). Following the addition of a cAMP inhibitor, IL-6 promoter activity was correlated with TNF-alpha levels and MAPK activity. CONCLUSIONS: A biphasic effect of beta2-adrenoceptor agonist on IL-6 production in renal resident macrophage cells became apparent when LPS was exposed to the cells. The terbutaline-induced down-regulation of IL-6 gene production was mediated by an inhibitory effect of terbutaline on TNF-alpha, which was exerted through the MAPK and cAMP pathways, whereas the up-regulation appeared to be due to a direct action of intracellular cAMP.     

Stimulation of cAMP production and cyclooxygenase-2 by prostaglandin E(2) and selective prostaglandin receptor agonists in murine osteoblastic cells

Prostaglandins (PGs), particularly PGE(2), can stimulate bone resorption and formation and auto-amplify their effects by inducing cyclooxygenase (COX)-2. We examined the role of different PG receptors in stimulating cAMP production and COX-2 expression in murine calvarial osteoblasts. Cells were obtained from PGE(2) receptor (EP2R and EP4R) wild-type and knockout (KO) mice and from mice transgenic for the COX-2 promoter fused to a luciferase reporter. We analyzed effects of selective agonists, EP2A and EP4A, for EP2R and EP4R, which mediate the increase in cAMP in response to PGE(2). We also tested agonists for other PGE(2) receptors (EP1A and EP3A) and for prostacyclin (IPA), prostaglandin D(2) (DPA), thromboxane (TPA), and prostaglandin F(2alpha) (FPA) receptors. PGE(2) and EP2A were the most effective stimulators of cAMP production. EP4A, IPA, and DPA produced smaller responses, and EP1A, EP3A, FPA, and TPA were ineffective. In EP2R KO cells, cAMP responses to PGE(2) were reduced by 80%, and responses to EP2A were abrogated. In EP4R KO cells, cAMP responses to PGE(2) and EP2A showed a small reduction, while the response to EP4A was abrogated. Pretreatment with PGE(2), EP2A, or EP4A down-regulated the subsequent response to the respective ligands. COX-2 induction was measured by increased luciferase activity and mRNA expression. PGE(2) was the most effective agonist; EP2A and another selective EP2R agonist, butaprost, showed similar efficacy, and EP4A was less effective. EP2A and EP4A effects on luciferase activity were additive, and effects of the combination were similar to PGE(2) itself. IPA, TPA, and DPA produced 2- to 6-fold increases in COX-2 expression. FPA was a weak agonist, while EP1A and EP3A were inactive. Treatment with specific inhibitors indicated that PGE(2), EP2A, and EP4A induced COX-2 expression largely through protein kinase A (PKA). We conclude that the PG induction of COX-2 in this system generally paralleled effects on cAMP production and was mediated predominantly via the PKA pathway.     

Recombinant interleukin-1 stimulates prostaglandin E2 production by osteoblastic cells: Synergy with parathyroid hormone

Summary Recombinant mouse IL-1 (Interleukin-1) has been shown to be capable of stimulating prostaglandin E₂ (PGE₂) production by isolated rat osteoblastic cells in a dose-dependent manner. The rapidity of the effect (1 hour) and the potency of IL-1 (5×10⁻¹² M) in producing this effect suggest that IL-1 may exert some of its effects on bone via PGE₂. Parathyroid hormone (PTH) appears to have a strong synergistic effect with IL-1. These data further substantiate the role of IL-1 in bone physiology.     

Induction of macrophagic prostaglandin E2 synthesis by glioma cells

OBJECT: It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity, and that cyclooxygenase (COX) inhibitors impede tumor growth and infiltration. Macrophages in tumor-bearing hosts are activated to produce PGE2, which induces an immunosuppressive state. Note, however, that the precise mechanism by which PGE2 induces an immunosuppressive state is still unclear. In this study, the authors investigated the mechanism of PGE2 production in glioma-bearing hosts. METHODS: The human and murine glioma cells that were studied did not produce a significant amount of PGE2. However, the coculture of human peripheral blood mononuclear cells or murine peritoneal macrophages with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. In contrast, production of tumor necrosis factor and interleukin (IL)-12p70 by macrophages and cytotoxic T lymphocyte induction were suppressed by culturing with conditioned glioma medium; this suppression was abrogated by the addition of the COX inhibitor indomethacin. The macrophagic expression of COX-2, and particularly the expression of microsomal PGE synthase (mPGES)-1, a terminal enzyme of the arachidonate cascade, was enhanced by the glioma-derived soluble factors. Furthermore, IL-12p70 production was not clearly suppressed in macrophages from mPGES-1-deficient mice. The glioma-derived soluble factors were sensitive to treatment with heat and papain. CONCLUSIONS: These results indicated that PGE2 production by macrophages is enhanced by glioma-derived soluble factors, which induce an immunosuppressive state in glioma-bearing hosts. Therefore, the inhibition of PGE2 synthesis, targeting COX-2 and mPGES-1, is an effective treatment for the induction of antiglioma immune responses.     

MAPK regulation of prostaglandin E2 production by lipopolysaccharide-stimulated macrophages is not dependent on nuclear factor kappaB

BACKGROUND: Prostaglandin E2 (PGE2) is a major contributor to the production and maintenance of immunosuppression in injured and septic patients. Although the synthesis of PGE2 by various enzymes has been elucidated, the regulatory mechanism of these enzymes is not clear. The purpose of this study was to determine the role of MAPK cascades in COX-2 gene activation by lipopolysaccharide (LPS)-stimulated macrophages (Mphi). MATERIALS AND METHODS: RAW 264.7 cells, a mouse Mphi cell line, were exposed to Escherichia coli LPS (10 microg/ml) in the presence of PD98059, a selective inhibitor of MAPKP44/P42, and SB202190, a selective inhibitor of MAPKP38. COX-2 mRNA expression and PGE2 production were measured by Northern Blot assay and ELISA, respectively. Mphi nuclear factor (NF)kappaB and cAMP-response element (CRE) activities were determined by electrophoretic mobility shift assays. RESULTS: LPS stimulation increased Mphi COX-2 mRNA expression and PGE2 production. PD98059 or SB202190 attenuated LPS-induced COX-2 mRNA as well as PGE2 production in a dose-dependent fashion. Inhibition of MAPKP44/P42 or MAPKP38 had no effect on NFkappaB activation but reduced CRE activity induced by LPS stimulation. CONCLUSION: Our data show that MAPK cascades regulate COX-2 gene expression and PGE2 production in LPS-stimulated Mphi through NFkappaB independent pathways. The regulatory mechanism is likely to be mediated through CRE.     

Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts

We have previously shown that the therapeutic range of ultrasound heals osteoradionecrotic bone and induces bone formation in vitro. It is well established that nitric oxide (NO) and prostaglandins are crucial early mediators in mechanically induced bone formation. The therapeutic range of ultrasound may act in the same way; therefore, we have investigated the effect of the therapeutic range of ultrasound on NO induction and prostaglandin E(2) (PGE(2)) production in vitro. Two ultrasound machines were evaluated, "traditional" (1 MHz, pulsed 1:4, tested at four intensities) and a "long-wave" (45 kHz, continuous, also tested at four intensities) devices. Ultrasound was applied to human mandibular osteoblasts for 5 min, and incubated at 37 degrees C for up to 24 h. The control group (sham insonated) was treated in the same way. NO was determined by measuring the nitrite concentration in the culture media colorimetrically, and PGE(2) was assayed by radioimmunoassay. Ultrasound produced a significant increase in both induced nitrite and PGE(2) production. The NO synthesis appeared to be via inducible NO synthase (iNOS) on the basis of the time course and levels of nitrite obtained, although the inhibition of other NOS isoforms by aminoguanidine cannot be excluded. PGE(2) synthesis appeared to be via COX-2. With the 45 kHz machine, a significant increase in NO was achieved at three intensities, 5, 30, and 50 mW/cm(2). The 1 MHz machine stimulated the synthesis of both NO and PGE(2), but was significant at only one dose (0.1 W/cm(2(SAPA))). There was no difference between the two machines with regard to PGE(2) synthesis. The time-course experiment revealed peak production to be 12-18 h for both NO and PGE(2). The therapeutic range of ultrasound stimulates both NO and PGE(2) synthesis by human osteoblasts, and the 45 kHz machine appeared to be more effective than the traditional short-wave length. These results may reflect the healing effect of ultrasound on fractures and osteoradionecrosis.     

Dietary omega-3 fatty acids decrease mortality and Kupffer cell prostaglandin E2 production in a rat model of chronic sepsis

We tested the hypothesis that substitution of omega-3 fat for dietary omega-6 fat would reduce mortality and decrease Kupffer cell prostaglandin E2 (PGE2) production in a rat model of chronic sepsis. Rats were fed via gastrostomy for 12 days with isonitrogenous, isocaloric diets containing 15% of calories as either safflower oil (omega-6) or a 10:1 mixture of menhaden oil (omega-3) and safflower oil. After five days of feeding, animals received an intra-abdominal abscess of defined bacterial content. Survivors were killed on post-laparotomy day 6 in conjunction with liver perfusion and protease liver digestion for Kupffer cell isolation. Kupffer cell PGE2 production was measured by radioimmunoassay after 18 hours of cell culture and again after stimulation with 0 LPS, 10 ng/ml LPS, and 10 micrograms/LPS. Mortality was decreased in menhaden oil-fed animals compared with safflower oil-fed animals (16% vs. 35%). Kupffer cell PGE2 production was decreased in menhaden oil-fed animals at 18 hours (354 +/- 54 vs. 570 +/- 95 pg/0.1 ml; p = 0.09) and after stimulation with 10 micrograms/ml LPS (140 +/- 41 vs. 288 +/- 45 pg/0.1 ml; p = 0.03) compared with safflower oil-fed animals.     

Cyclosporine decreases prostaglandin E2 production in mouse medullary thick ascending limb cultured cells

Intrarenal vasoconstriction is thought to be the major pathogenesis of cyclosporine (CsA) nephrotoxicity. Nitric oxide (NO) and prostaglandin E2 (PGE2) are two of the major intrarenal vasodilators, which protect kidney from ischemia. CsA inhibited NO production in renal epithelial cells. The interaction between CsA and intrarenal PGE2 and NO production is still unclear. The aim of the study is to evaluate the interaction of CsA with intrarenal PGE2 and NO production in renal epithelial cells. Models of cultured mouse thick ascending limb (TAL) cells are chosen to perform the experiments, as TAL cells are the major site of intrarenal PGE2 production and target of CsA nephrotoxicity. We investigated the PGE2 production by enzyme-linked immunosorbent assay, and cyclooxygenase (COX-1 and COX-2) mRNA expression by RT-PCR in cultured cells treated with or without CsA. TAL cells maintained the main characteristics of their parental cells. TAL cells produce PGE2 mainly by COX-1 in steady state and by COX-2 in stimulated state by lipopolysaccharide (LPS). CsA (100 ng/ml) significantly reduced the PGE2 production up to 43% in TAL cells in LPS stimulated status (control versus CsA: 375.1 +/- 15.5 vs. 187.2 +/- 12.2 nm/mg protein, n = 7, P < 0.001). The effects were dose-dependent. The mRNA expression of COX1 is not affected and COX-2 is decreased in CsA-treated TAL cells. NO donor could prevent the inhibitory effects of CsA. We concluded that CsA decreased intrarenal PGE2 production in stimulated status mainly by decreasing COX-2 expression. NO might play a role in the CsA effect. The results suggested the role possible of PGE2 in CsA nephrotoxicity.     

Early interleukin 6 production by leukocytes during ischemic acute kidney injury is regulated by TLR4

Although leukocytes infiltrate the kidney during ischemic acute kidney injury (AKI) and release interleukin 6 (IL6), their mechanism of activation is unknown. Here, we tested whether Toll-like receptor 4 (TLR4) on leukocytes mediated this activation by interacting with high-mobility group protein B1 (HMGB1) released by renal cells as a consequence of ischemic kidney injury. We constructed radiation-induced bone marrow chimeras using C3H/HeJ and C57BL/10ScNJ strains of TLR4 (-/-) mice and their respective TLR4 (+/+) wild-type counterparts and studied them at 4?h after an ischemic insult. Leukocytes adopted from TLR4 (+/+) mice infiltrated the kidneys of TLR4 (-/-) mice, and TLR4 (-/-) leukocytes infiltrated the kidneys of TLR4 (+/+) mice but caused little functional renal impairment in each case. Maximal ischemic AKI required both radiosensitive leukocytes and radioresistant renal parenchymal and endothelial cells from TLR4 (+/+) mice. Only TLR4 (+/+) leukocytes produced IL6 in vivo and in response to HMGB1 in vitro. Thus, following infiltration of the injured kidney, leukocytes produce IL6 when their TLR4 receptors interact with HMGB1 released by injured renal cells. This underscores the importance of TLR4 in the pathogenesis of ischemic AKI.     

Elevated tumor necrosis factor alpha production concomitant to elevated prostaglandin E2 production by trauma patients' monocytes

The level of tumor necrosis factor alpha (TNF alpha), a monokine implicated in mediating septic shock, is elevated in the blood of some patients with sepsis. Monocytes from 11 trauma patients and 11 burn patients were suboptimally stimulated with interferon gamma and muramyl dipeptide, an analogue of bacterial wall products. The patients with sepsis showed significantly greater total TNF alpha levels (secreted in combination with cell-associated) 3 days before septic episodes, as compared with normal controls (32.38 to 2231.76 ng/10(6) monocytes per milliliter, median = 121.03 ng/10(6) monocytes per milliliter; normal control: 0.00 to 18.20 ng/10(6) monocytes per milliliter, median = 5.93 ng/10(6) monocytes per milliliter). Increases in patients' total monocyte TNF alpha levels greater than 30 ng/10(6) monocytes per milliliter correlated with septic episodes. In patients with sepsis, the total monocyte TNF alpha levels were increased despite a concomitant increase in their prostaglandin E2 levels in both stimulated (interferon gamma plus muramyl dipeptide) and unstimulated in vitro assays (9 patients: stimulated prostaglandin E2 range, 30.1 to 123.6 ng/10(6) monocytes per milliliter). Massively elevated monocyte TNF alpha and prostaglandin E2 production occurred simultaneously in patients with sepsis.     

前列腺素E2受体在前列腺素E2诱导H9c2心肌细胞肥大中的作用

目的 评价前列腺素E2受体(EP受体)在前列腺素E2(PCE2)诱导H9c2心肌细胞肥大中的作用.方法 培养H9c2心肌细胞,以4×104个/ml的密度接种于培养瓶(每瓶3ml)、24孔(每孔1 ml)或6孔(每孔2 ml)培养板.采用随机数字表法,将细胞随机分为4组(n=24):空白对照组(C组)不予任何处理,继续培养48 h;PGE2组在细胞培养液中加入PGE2(终浓度1μmol/L);AH6809组(A组)在细胞培养液中加入PGE2(终浓度1μmol/L)和AH6809(EP1及EP2受体拮抗剂,终浓度10μmol/L);GW627368X组(G组)在细胞培养液中加入PGE2(终浓度1μmol/L)和GW627368X(EP4受体拮抗剂,终浓度10 μmol/L).孵育48 h后采用免疫荧光观察心肌细胞形态,Image J医学图像分析系统测量心肌细胞直径,BCA法检测心肌细胞总蛋白含量,RT-PCR法测定胞浆ANP mRNA及BNP mRNA的表达水平.结果 与C组比较,PGE2组、A组和G组心肌细胞总蛋白含量和心肌细胞直径增加,胞浆ANPmRNA及BNPmRNA表达上调(P＜0.05).与PGE2组比较,G组心肌细胞总蛋白含量和心肌细胞直径降低,胞浆ANP mRNA及BNP mRNA表达下调(P＜0.05),A组上述各指标差异无统计学意义(P＞0.05).结论 EP4受体介导了PGE2诱导的心肌细胞肥大效应,而该效应与EP1和EP2受体无关.     

Cyclosporin A inhibits prostaglandin E2 formation by rat mesangial cells in culture

A reversible reduction in glomerular filtration rate (GFR) is a frequent side effect in patients treated with the immunosuppressant cyclosporin A (CsA). The pathophysiology of acute CsA nephrotoxicity, however, is unclear. Since eicosanoids are local mediators of glomerular hemodynamics, they might be involved in CsA induced changes in GFR. We therefore studied the effect of CsA on prostaglandin E2 (PGE2) production by rat mesangial cells in culture. PGE2 production by mesangial cells following stimulation with angiotensin II (AII) (10(-6) M) or the Ca2+-ionophore A23187 (1 microgram/ml) was significantly inhibited when cells were grown for 24 hours in media which contained CsA (800 to 3200 ng/ml). CsA did not affect viability of mesangial cells as determined by 51Cr release or by cell proliferation measured by 3H-thymidine incorporation. CsA (3200 ng/ml) did not inhibit PGE2 formation by rat MC microsomes incubated with arachidonic acid. However, CsA, in a dose dependent manner, inhibited A23187 and angiotensin II induced release of 3H-labelled arachidonic acid from rat mesangial cells. These data demonstrate that CsA reduces PGE2 formation by rat mesangial cells in culture, probably by inhibiting the release of substrate arachidonic acid from cell membranes rather than by inhibition of cyclooxygenase. This effect may contribute to the reduction in GFR which accompanies CsA therapy.