Reduced Prostaglandin E2 Release Research Articles

Membrane lipid composition of bronchial epithelial cells influences antiviral responses during rhinovirus infection

ABSTRACT Lipids and their mediators have important regulatory functions in many cellular processes, including the innate antiviral response. The aim of this study was to compare the lipid membrane composition of in vitro differentiated primary bronchial epithelial cells (PBECs) with ex vivo bronchial brushings and to establish whether any changes in the lipid membrane composition affect antiviral defense of cells from donors without and with severe asthma. Using mass spectrometry, we showed that the lipid membrane of in vitro differentiated PBECs was deprived of polyunsaturated fatty acids (PUFAs) compared to ex vivo bronchial brushings. Supplementation of the culture medium with arachidonic acid (AA) increased the PUFA-content to more closely match the ex vivo membrane profile. Rhinovirus (RV16) infection of AA-supplemented cultures from healthy donors resulted in significantly reduced viral replication while release of inflammatory mediators and prostaglandin E2 (PGE2) was significantly increased. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthases, suppressed RV16-induced PGE2 release and significantly reduced CXCL-8/IL-8 release from AA-supplemented cultures indicating a link between PGE2 and CXCL8/IL-8 release. In contrast, in AA-supplemented cultures from severe asthmatic donors, viral replication was enhanced whereas PTGS2 expression and PGE2 release were unchanged and CXCL8/IL-8 was significantly reduced in response to RV16 infection. While the PTGS2/COX-2 pathway is initially pro-inflammatory, its downstream products can promote symptom resolution. Thus, reduced PGE2 release during an RV-induced severe asthma exacerbation may lead to prolonged symptoms and slower recovery. Our data highlight the importance of reflecting the in vivo lipid profile in in vitro cell cultures for mechanistic studies.

BMP4 micro-immunotherapy increases collagen deposition and reduces PGE2 release in human gingival fibroblasts and increases tissue viability of engineered 3D gingiva under inflammatory conditions.

BackgroundWe aimed to evaluate the effect of low doses (LD) bone morphogenetic protein‐2 (BMP2) and BMP4 micro‐immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration.MethodsWe first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1β. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization‐related enzymes (matrix metalloproteinase‐1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long‐term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release.ResultsLD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re‐establish the impaired collagen metabolism induced by IL1β treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days.ConclusionsLD BMP4 MI treatment shows anti‐inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

Abstract 3867: Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer

Abstract Introduction and Objective: Prostaglandin E2 (PGE2) is a lipid-derived signaling agent that promotes tumorigenesis via increasing stemness, proliferation, and angiogenesis. PGE2 levels are elevated in multiple cancers, including bladder (BCa) where they may play a pathological role. PGE2 is generated in part by cyclooxygenase enzymes (COX-1/COX-2). Clinical trials using the COX-2 specific inhibitor celecoxib to prevent progression and recurrence of non-muscle invasive BCa report hazard ratio reductions; however, celecoxib fails to significantly outperform standard of care. These studies are dose-limited by cardiotoxicity present with COX inhibition, but have suggested higher doses of celecoxib may yield significant results. Microsomal PGE2 synthase-1 (mPGES-1) is one of two terminal enzymes (downstream of COX-1/2) associated with PGE2 production. Inhibition of mPGES-1 does not affect synthesis of the prostaglandins responsible for cardiotoxicity but still reduces PGE2 levels. Using a model of carcinogen induced BCa, we have previously established mPGES1-/- have significantly smaller bladder tumors by weight, reduced pathological stage, reduced PGE-2 metabolites, and reductions in percent tumor involvement in the bladder compared to WT mice. The purpose of this study was to further understand mPGES mediated tumorigenesis using in vitro cellular models. We hypothesized pharmacological inhibition with the mPGES-1 specific inhibitor MF63 would reduce PGE2 levels leading to reduced cellular proliferation and reduced PGE2 release in vitro. Methods: mPGES1 protein and RNA levels were measured in benign (UROtsa) or malignant BCa cell lines using western blotting and qPCR respectively. PGE2 levels were assessed by ELISA. Cell proliferation was assessed using the hexosaminidase assay and colony formation assays. Cell death was assessed using lactate dehydrogenase (LDH) release. Results: In vitro, BCa cell lines HT-1376, RT-4 and HTB-9 significantly upregulate mPGES1 as compared to a benign urothelial cell line (UROtsa). To assess the capacity of MF63 to effect PGE2 production, we treated HT-1376 and HTB-9 cells with MF63 at increasing concentrations and assessed PGE2 levels. MF63 significantly reduced PGE2 production after 24h in both cell lines. Furthermore, pharmacological inhibition of mPGES1 with MF63 at concentrations of 10µM and above significantly reduced cellular proliferation after 48 or 72 hours using both colony formation and the hexosaminidase assay to assess proliferation. Finally, concentrations of 50µM MF63 robustly induced cell death as measured by LDH release. Conclusions: These data show that inhibition of mPGES1 in vitro with the inhibitor MF63 results in decreases in PGE2 levels and reduced cellular proliferation, likely due in part to cell death induction. mPGES1 inhibitors are expected to have improved safety profiles and given our results, may have therapeutic potential in BCa. Citation Format: Benjamin Leeland Woolbright, Jordan Barney, Van Schloegel Schloegel, Erika Abbott, John Arthur Taylor. Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3867.

Acetate Reduces PGE2 Release and Modulates Phospholipase and Cyclooxygenase Levels in Neuroglia Stimulated with Lipopolysaccharide

Acetate supplementation attenuates neuroglial activation, increases histone and non-histone protein acetylation, reduces pro-inflammatory cytokine expression, and increases IL-4 transcription in rat models of neuroinflammation and Lyme's neuroborreliosis. Because eicosanoid signaling is involved in neuroinflammation, we measured the effect acetate treatment had on phospholipase, cyclooxygenase, and prostaglandin E2 (PGE2) levels in BV-2 microglia and primary astrocytes stimulated with lipopolysaccharide (LPS). In BV-2 microglia, we found that LPS increased the phosphorylation-state of cytosolic phospholipase A2 (cPLA2), reduced the levels of phospholipase C (PLC) β1, and increased the levels of cyclooxygenase (Cox)-1 and -2. Acetate treatment returned PLCβ1 and Cox-1 levels to normal, attenuated the increase in Cox-2, but had no effect on cPLA2 phosphorylation. In primary astrocytes, LPS increased the phosphorylation of cPLA2 and increased the levels of Cox-1 and Cox-2. Acetate treatment in these cells reduced secretory PLA2 IIA and PLCβ1 levels as compared to LPS-treatment groups, reversed the increase in cPLA2 phosphorylation, and returned Cox-1 levels to normal. Acetate treatment reduced PGE2 release in astrocytes stimulated with LPS to control levels, but did not alter PGE2 levels in BV-2 microglia. The amount of acetylated H3K9 bound to the promoter regions of Cox-1, Cox-2, IL-1β and NF-κB p65 genes, but not IL-4 in were increased in BV-2 microglia treated with acetate. These data suggest that acetate treatment can disrupt eicosanoid signaling in neuroglia that may, in part, be the result of altering gene expression due chromatin remodeling as a result of increasing H3K9 acetylation.

Flow-induced prostaglandin E2 release regulates Na and K transport in the collecting duct

Fluid shear stress (FSS) is a critical regulator of cation transport in the collecting duct (CD). High-dietary sodium (Na) consumption increases urine flow, Na excretion, and prostaglandin E(2) (PGE(2)) excretion. We hypothesize that increases in FSS elicited by increasing tubular flow rate induce the release of PGE(2) from renal epithelial cells into the extracellular compartment and regulate ion transport. Media retrieved from CD cells exposed to physiologic levels of FSS reveal several fold higher concentration of PGE(2) compared with static controls. Treatment of CD cells with either cyclooxygenase-1 (COX-1) or COX-2 inhibitors during exposure to FSS limited the increase in PGE(2) concentration to an equal extent, suggesting COX-1 and COX-2 contribute equally to FSS-induced PGE(2) release. Cytosolic phospholipase A2 (cPLA2), the principal enzyme that generates the COX substrate arachidonic acid, is regulated by mitogen-activated protein-kinase-dependent phosphorylation and intracellular Ca(2+) concentration ([Ca(2+)](i)), both signaling processes, of which, are activated by FSS. Inhibition of the ERK and p38 pathways reduced PGE(2) release by 53.3 ± 8.4 and 32.6 ± 11.3%, respectively, while antagonizing the JNK pathway had no effect. In addition, chelation of [Ca(2+)](i) limited the FSS-mediated increase in PGE(2) concentration by 47.5 ± 7.5% of that observed in untreated sheared cells. Sheared cells expressed greater phospho-cPLA2 protein abundance than static cells; however, COX-2 protein expression was unaffected (P = 0.064) by FSS. In microperfused CDs, COX inhibition enhanced flow-stimulated Na reabsorption and abolished flow-stimulated potassium (K) secretion, but did not affect ion transport at a slow flow rate, implicating that high tubular flow activates autocrine/paracrine PGE(2) release and, in turn, regulates flow-stimulated cation transport. In conclusion, FSS activates cPLA2 to generate PGE(2) that regulates flow-mediated Na and K transport in the native CD. We speculate that dietary sodium intake modulates tubular flow rate to regulate paracrine PGE(2) release and cation transport in the CD.

Evidence for Interleukin‐10‐Mediated Inhibition of Cyclo‐ oxygenase‐2 Expression and Prostaglandin Production in Preterm Human Placenta

Interleukin-10 (IL-10) is thought to be a key cytokine for the maintenance of pregnancy. Here we examined the expression profiles of IL-10 and cyclo-oxygenase-2 (COX-2), and the effect of IL-10 on COX-2 expression and prostaglandin release in the human placenta from preterm labor deliveries associated with chorioamnionitis. Placental tissues from preterm labor and term labor deliveries were processed for ex vivo placental explant culture system. IL-10 expression was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical (IHC) analysis. COX-2 expression was evaluated by IHC, Western blotting and reverse transcriptase-polymerase chain reaction. Prostaglandin E2 (PGE2) release was measured by ELISA. IL-10 was significantly reduced in chorioamnionitis-associated preterm labor as well as in term labor placental tissues compared with second trimester normal pregnancy samples obtained from elective terminations. Similar results were obtained with freshly isolated cytotrophoblasts from these deliveries. As expected, COX-2 mRNA was detected at significant levels in tissues from term and preterm labor deliveries compared with no labor term deliveries. Importantly, IL-10 inhibited COX-2 expression in cultured placental explants from preterm labor deliveries, but not from term labor samples. Inhibition of COX-2 expression coincided with reduced PGE2 release. These results demonstrate the importance of IL-10 in countering inflammation associated with preterm labor, and suggest that term and preterm parturition may, in part, represent different conditions.

Dietary conjugated linoleic acid reduces PGE2 release and interstitial injury in rat polycystic kidney disease

Conjugated linoleic acid (CLA) describes positional isomers of linoleic acid (LA). Experimental health benefits of CLA include amelioration of malignancy and inflammatory disease and reduction of adiposity. The Han:SPRD-cy rat model of polycystic kidney disease (PKD) features prominent renal interstitial inflammation and fibrosis that is amenable to dietary modification. We studied CLA supplementation in the modification of inflammatory outcomes in the Han:SPRD-cy rat. Male offspring of Han:SPRD-cy heterozygotes were fed diets, using corn oil or corn oil with a CLA enriched oil (1% of diet by weight as CLA). After 8 weeks, measurements included renal function and morphometry, ex vivo release of renal prostaglandin E2 (PGE2), and renal and hepatic tissue fatty acid profiles. Urine creatinine was significantly higher in PKD animals fed CLA (P = 0.004), but differences in serum creatinine and creatinine clearance did not quite reach significance in PKD animals. CLA feeding reduced interstitial inflammation (P < 0.001), fibrosis (P = 0.03), and renal PGE2 release (P = 0.02). Cystic change and oxidized low-density lipoprotein (LDL) staining did not change significantly. CLA feeding produced increased renal and hepatic CLA isomers. Hepatic, but not renal, LA proportion was reduced on the CLA diet. The renal proportion of the PGE2 precursor, arachidonic acid (AA), was not changed by diet, but hepatic AA proportion increased significantly with CLA feeding (P= 0.009). CLA reduces renal production of PGE2, without reduced availability of the precursor fatty acid, AA. Short-term feeding of CLA to Han:SPRD-cy rats also has significant renal anti-inflammatory and antifibrotic effects. As inflammation and fibrosis are important components of the progression of chronic renal injury, CLA may be a useful agent in dietary amelioration of renal disease.

Osteopontin-Deficient Bone Cells Are Defective in Their Ability to Produce NO in Response to Pulsatile Fluid Flow

Osteopontin (OPN) is a noncollagenous component of bone matrix. It mediates cell attachment and activates signal transduction pathways. In this work, bone cells, cultured from fragments of long bones derived from wild-type and OPN−/− (“knock-out”) mice, were exposed to pulsatile fluid flow (PFF) over a 60-min period. The medium was assayed periodically for nitric oxide (NO) and prostaglandin E2 (PGE2) release. OPN+/+ cells exhibited a peak of NO production 5–10 min after the onset of PFF, decreasing to a stable plateau at 15 min; much less NO was produced by the OPN−/− cells. PFF resulted in reduced PGE2 release by both cell types, although the reduction was less for the OPN−/− cells in the 15–30 min window. Both cell types exhibited a similar enhancement of cyclooxygenase2 mRNA levels 60 min after initiation of PFF. These results suggest that bone cells require OPN to respond fully to PFF as assessed by increased NO and reduced PGE2 synthesis.

Gadolinium blocks rat Kupffer cell calcium channels: relevance to calcium-dependent prostaglandin E2 synthesis and septic mortality.

Hepatic Kupffer cells (KC), the major tissue macrophage population, produce the septic response mediators, tumor necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2), and have been shown to internalize gadolinium chloride (GD), a rare earth metal of the lanthanide series. Because GD pretreatment of rats has been shown to inhibit the mortality of sepsis, we studied the secretory response to lipopolysaccharide (LPS) by KC isolated from rats injected with either saline or GD (7 mg/kg, intravenously) on the 2 days before KC isolation. Using culture conditions modified to reflect the intrasinusoidal milieu of arginine (RPMI-1640 media with 10 or 100 micromol/L arginine), KC from GD-treated rats responded to LPS (0. 0025 microg/mL) with significantly (P <.01) reduced PGE2 release. In contrast, TNF-alpha release by treated KC was significantly (P <.05) enhanced, consistent with the loss of PGE2 autocoid inhibition of TNF-alpha. Calcium flux is an early signaling event in eicosanoid synthesis, and GD is known to block calcium channels. Therefore, KC were loaded with fura-2-AM to study the effect of GD on KC calcium flux. GD prevented ionomycin and platelet-activating factor (PAF)-mediated [Ca++]i increase and calcium-dependent PGE2 synthesis, while GD did not affect PGE2 synthesis when protein kinase C (PKC) was directly activated with tetradecanoylphorbolacetate (TPA). The inhibition of calcium flux and calcium-dependent PGE2 synthesis in the major cell of the monocytic phagocytic system by GD may explain the previously reported ability of this lanthanide to prevent the mortality of endotoxemia.

Opposing Effects of Indomethacin and Nordihydroguaiaretic Acid on Macrophage Function and Tumor Growth

The effects of indomethacin (INDO) and nordihydroguaiaretic acid (NDGA) were compared on macrophage function and tumor growth. Intraperitoneal injections (5 mg/kg/day, 6, 8 or 9 days) of INDO in C57BL/6 mice stimulated the cytotoxicity of peritoneal macrophages and inhibited growth of subcutaneous B16 melanoma, whereas NDGA injections suppressed macrophage cytotoxicity, increased macrophage prostaglandin‐E2 (PGE2) release, and enhanced the tumor growth. Further, pretreatment of macrophages in vitro with the INDO group serum increased cytostasis against B16 tumor cells, while the use of NDGA group serum reduced it. Incubation of tumor‐bearer's macrophages in vitro with INDO (10‐6M and 10‐7M) or 10‐6M NDGA for 4 h reduced PGE2 release, but 10 ‐7M NDGA markedly enhanced the PGE2 release. Our data indicate that INDO and NDGA are able to modulate the macrophage function directly, and produce opposing effects on macrophage function and tumor growth. Inhibitory actions of INDO and NDGA on the cyclooxygenase or lipoxygenase pathway of arachidonic acid metabolism appear to contribute to their effects.

Effect of atrial natriuretic factor on renal prostaglandin E2 release in the anaesthetized dog.

Experiments were undertaken in two groups of barbiturate anaesthetized dogs to examine whether atrial natriuretic factor (ANF) exerts an effect on renal release of prostaglandin E2 (PGE2). In the first group, intravenous infusion of ANF (50 ng min-1 kg-1 body wt) reduced basal PGE2 release from 4.4 +/- 0.8 pmol min-1 to 1.8 +/- 0.7 pmol min-1. In the second group, intrarenal infusion of an alpha 1-adrenoceptor agonist, phenylephrine (2.5-6.75 micrograms min-1), raised PGE2 release from 2.7 +/- 0.5 pmol min-1 to 7.5 +/- 1.3 pmol min-1. During continuous alpha 1-adrenergic stimulation, intravenous infusion of ANF (100 ng min-1 kg-1 body wt) reduced PGE2 release to 3.5 +/- 1.0 pmol min-1. These results demonstrate that ANF reduces basal and alpha 1-adrenergic stimulated renal PGE2 release.

Chloroquine Attenuates Hemorrhagic Shock—Induced Immunosuppression and Decreases Susceptibility to Sepsis

Hemorrhagic shock causes a severe suppression of cellular immunity and an increased susceptibility to sepsis that may be due to increased release of prostaglandin E2 by macrophages. Since chloroquine inhibits the secretion of prostaglandin E2 by macrophages in vitro, the effects of chloroquine administration in vivo following hemorrhagic shock on macrophage prostaglandin E2 secretion and on depressed cellular immunity were examined. Inbred C3H/HeN male mice, aged 6 to 8 weeks, were bled to a mean blood pressure of 35 mm Hg, which was maintained for 60 minutes, and adequately, resuscitated. Mice then received intramuscular injections of either saline (vehicle) or chloroquine (10 mg/kg of body weight). Prostaglandin E2 in macrophage supernatants (radioimmunoassay) concanavalin A-dependent splenocyte proliferation, and interleukin 2 in splenocyte supernatants (CTLL 20 interleukin 2-dependent proliferation) were determined 2 or 24 hours later. Hemorrhage caused a significant decrease of splenocyte proliferation (47%) and interleukin 2 release (49%) at 24 hours, while prostaglandin E2 secretion from macrophages was elevated at 2 hours. Chloroquine treatment attenuated depression of splenocyte functions and reduced prostaglandin E2 release. Furthermore, chloroquine treatment decreased the mortality of septic mice after hemorrhage to levels comparable with those of sham-operated mice. Thus, chloroquine may be a useful adjunct in the clinical setting for the treatment of shock-induced immunodepression and increased susceptibility to sepsis following hemorrhage.

Further evidence that accelerated muscle protein breakdown during sepsis is not mediated by prostaglandin E2.

Prostaglandin E2 (PGE2) reportedly increases protein break-down in skeletal muscle. The role of PGE2 for accelerated muscle proteolysis during sepsis, however, is controversial. In this study, the effect of the prostaglandin synthesis inhibitor indomethacin on PGE2 release and protein breakdown in skeletal muscle from nonseptic and septic rats was evaluated. Sepsis was induced in male Sprague-Dawley rats (40-60 g) by cecal ligation and puncture (CLP). After 16 hours the extensor digitorum longus (EDL) and soleus (SOL) muscles were dissected with intact tendons and incubated in an oxygenated medium, and the release of tyrosine (protein breakdown) and PGE2 into the incubation medium was determined. Paired muscles were incubated in the absence or presence of indomethacin (3 mumol/L or 6 mumol/L). In some experiments the effect of indomethacin was investigated in the presence of different concentrations of insulin (1, 10, or 100 mU/mL) since previous reports suggested an interaction between insulin and prostaglandins on protein turnover in skeletal muscle. In other experiments muscles were incubated in a flaccid or stretched state, which is known to influence the metabolic response to different substances. Protein breakdown rate was 0.210 +/- 0.013 and 0.492 +/- 0.025 mumol Tyr/g X 2 hours in EDL from nonseptic and septic rats, respectively (p less than 0.01). The corresponding values for SOL were 0.480 +/- 0.037 and 0.712 +/- 0.039 mumol Tyr/g X 2 hours (p less than 0.01). Addition of indomethacin to the incubation medium reduced PGE2 release from 29.1 +/- 3.1 to 6.8 +/- 0.7 ng/g X 2 hours in nonseptic SOL and from 50.6 +/- 10.4 to 5.6 +/- 0.7 ng/g X 2 hours in septic SOL. Protein breakdown rate in SOL and EDL from sham-operated or septic rats was unaffected by indomethacin, both when muscles were incubated in a flaccid or stretched state, and when they were incubated in the presence or absence of insulin. The present results do not suggest a role of PGE2 for accelerated muscle proteolysis in the present experimental septic model.

慢性関節リウマチ患者関節液中マクロファージ，好中球のプロスタグランディン産生能

Macrophages in synovial fluid of patients with rheumatoid arthritis (RASF-Mφ) released prostaglandin E2 (PGE2) when they were stimulated by lipopolysaccharide (LPS), aggregated-IgG (Agg-IgG) and opsonized-Zymosan (Zymosan) . Monocytes in peripheral blood of patients with rheumatoid arthritis (RA-Mo) and normal subjects (healthy-Mo) similarly released PGE2 when they were stimulated by LPS, Agg-IgG and Zymosan. Healthy-Mo released more amount of PGE2 than RA-Mo. The reduced PGE2 release of RA-Mo might be due to the effect of nonsteroidal antiinflammatory drugs taken by RA patients. Polymorphonuclear leukocytes (PMN), in synovial fluid and peripheral blood of patients with rheumatoid arthritis along with PMN of normal subjects, released little PGE2.