Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis

Background

Blockade of Prostaglandin (PG) E₂ production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE₂ promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway.

Methodology/Principal Findings

Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE₂ production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE₂ production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice.

Conclusion

Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE₂ mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.     

Oxidative stress-mediated epidermal growth factor receptor activation regulates PM2.5-induced over-secretion of pro-inflammatory mediators from human bronchial epithelial cells

BackgroundExposure to PM_2.5 has been associated with increased morbidity and mortality of lung diseases although the underlying mechanisms have not been fully uncovered. Airway inflammation is a critical event in the pathogenesis of lung diseases. This study aimed to examine the role of oxidative stress and epidermal growth factor receptor (EGFR) in PM_2.5-induced pro-inflammatory response in a human bronchial epithelial cell line, BEAS-2B.MethodsBEAS-2B cells were exposed to 0, 20, 50, 100 and 150 μg/ml of PM_2.5. Secretion of pro-inflammatory mediators including interleukin-6 (IL-6), IL-8 and IL-1β was determined using enzyme linked immunosorbent assay. Levels of intracellular reactive oxygen species (ROS) were determined using flow cytometry. Phosphorylation of the EGFR was examined with immunoblotting.ResultsPM_2.5 exposure increased the secretion of IL-6, IL-8, and IL-1β in a concentration-dependent fashion. Moreover, exposure to PM_2.5 elevated intracellular levels of ROS, and phosphorylation of the EGFR (Y1068). Pretreatment of BEAS-2B cells with either an antioxidant or a specific EGFR inhibitor significantly reduced PM_2.5-induced IL-6, IL-8 and IL-1β secretion, implying that both oxidative stress and EGFR activation were involved in PM_2.5-induced pro-inflammatory response. Furthermore, pre-treatment of BEAS-2B cells with an antioxidant significantly blunted PM_2.5-induced EGFR activation, suggesting that oxidative stress was required for PM_2.5-induced EGFR activation.ConclusionPM_2.5 exposure induces pro-inflammatory response in human bronchial epithelial cells through oxidative stress-mediated EGFR activation.     

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background

Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms.

Methods

The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13).

Results

In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter.

Conclusions

This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD.     

ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli

Activation of the NADPH oxidase homolog dual oxidase 1 (DUOX1) within the airway epithelium represents a key mechanism of innate airway host defense, through enhanced production of H₂O₂, which mediates cellular signaling pathways that regulate the production of various inflammatory mediators. Production of the CXC chemokine interleukin (IL)-8/CXCL8 forms a common epithelial response to many diverse stimuli, including bacterial and viral triggers, environmental oxidants, and other biological mediators, suggesting the potential involvement of a common signaling pathway that may involve DUOX1-dependent H₂O₂ production. Following previous reports showing that DUOX1 is activated by extracellular ATP and purinergic receptor stimulation, this study demonstrates that airway epithelial IL-8 production in response to several bacterial stimuli involves ATP release and DUOX1 activation. ATP-mediated DUOX1 activation resulted in the activation of ERK1/2 and NF-κB pathways, which was associated with epidermal growth factor receptor (EGFR) ligand shedding by ADAM17 (a disintegrin and metalloproteinase-17). Although ATP-mediated ADAM17 activation and IL-8 release were not prevented by extracellular H₂O₂ scavenging by catalase, these responses were attenuated by intracellular scavengers of H₂O₂ or related oxidants, suggesting an intracellular redox signaling mechanism. Both ADAM17 activation and IL-8 release were suppressed by inhibitors of EGFR/ERK1/2 signaling, which can regulate ADAM17 activity by serine/threonine phosphorylation. Collectively, our results indicate that ATP-mediated DUOX1 activation represents a common response mechanism to several environmental stimuli, involving H₂O₂-dependent EGFR/ERK activation, ADAM17 activation, and EGFR ligand shedding, leading to amplified epithelial EGFR activation and IL-8 production.     

Demethylallosamidin, a chitinase inhibitor, suppresses airway inflammation and hyperresponsiveness

Acidic mammalian chitinase is upregulated in response to allergen exposure in the lung. We investigated the effects of chitinase inhibitors, allosamidin (Allo) and demethylallosamidin (Dma), on asthmatic responses. Mice were subjected to IL-13 instillation into the airways or to ovalbumin sensitization plus exposure with or without treatment of Allo or Dma. Airway hyperresponsiveness (AHR) and inflammation were evaluated. Allo and Dma attenuated airway eosinophilia and the upregulation of eotaxin after IL-13 instillation, while Dma, but not Allo, suppressed AHR in IL-13-induced asthma. Allo or Dma suppressed the elevated chitinase activity in BAL fluids after IL-13 to similar levels. The bronchoprotective PGE₂ levels in BAL fluids were elevated after IL-13 instillation. Allo, but not Dma, suppressed the overproduction of PGE₂ and the expression of COX-2 and PGE synthase-1 induced by IL-13. In ovalbumin-induced asthma, Dma suppressed AHR more strongly than Allo. These findings suggest that Dma attenuates asthmatic responses induced by IL-13 without affecting PGE₂ synthesis. Dma may have potential as therapeutic agents for asthma.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.     

Regulation of interleukin-8 via an airway epithelial signaling cascade

Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-alpha mediates IL-8 production. Third, we show that tumor necrosis factor-alpha-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-alpha into soluble TGF-alpha, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-alpha-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.     

Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway

Background

Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.

Methods and results

Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE₂ and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling.

Conclusion

SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.

     

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites

Leukotriene D₄ (LTD₄)-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D₂-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD₄ and PGD₂ were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD₄-induced bronchoconstriction and thromboxane (TX) A₂ release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT₁ receptor activation by LTD₄ was established by measurements of immunoreactive 6-keto PGF_1α (a stable metabolite of PGI₂) and PGE₂. In contrast, TP receptor-mediated bronchoconstriction by PGD₂ was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE₂ was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD₂ nor U-46619 actively released PGE₂, but continuous production of PGE₂ and PGI₂ was established, and the response to PGD₂ was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD₄ and PGD₂ in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD₄ was amplified predominantly by secondarily released TXA₂, that induced by PGD₂ was attenuated by bronchoprotective PGE₂ and PGI₂, presumably tonically produced in the airways.     

The free radical scavenging and anti-inflammatory activities of gallate-chitooligosaccharides in human lung epithelial A549 cells

In this study, gallic acid-conjugated chitooligosaccharides (gallate-COS) was evaluated for its enhancing capabilities in free radical scavenging and anti-inflammatory activities in human lung epithelial A549 cells. It was found that gallate-COS possesses significantly high DPPH radical scavenging activity and protective effect against H₂O₂-induced DNA damage. Likewise, gallate-COS decreased the production of intracellular reactive oxygen species in H₂O₂-stimulated A549 cells. Moreover, the suppressive effects of gallate-COS on COX-2 expression and PGE₂ production from LPS-stimulated A549 cells were evidenced. Finally, the productions of cytokines including IL-8 and TNF-α were also inhibited by gallate-COS in a dose-dependent manner. Collectively, these results suggest that gallate-COS could be used as a functional ingredient with potent antioxidant and anti-inflammatory benefits.     

Induction of cyclooxygenase-2 expression in human tracheal smooth muscle cells by interleukin-1β: Involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-κB

Interleukin-1 (IL-1) has been recognized as a potent stimulus for the synthesis of prostaglandin (PG), which has been implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1-induced cyclooxygenase (COX) expression and PGE₂ synthesis via activation of p42/p44 and p38 mitogen-activated protein kinases (MAPKs) in human tracheal smooth muscle cells (HTSMCs) are not completely understood. We found that IL-1 increased COX-2 expression and PGE₂ synthesis in time- and concentration-dependent manners. Both specific phosphatidylcholine-phospholipase C inhibitor (D609) and protein kinase C inhibitor (GF109203X) attenuated IL-1-induced responses in HTSMCs. IL-1-induced COX-2 expression and PGE₂ synthesis were also inhibited by an inhibitor of MEK1/2 (PD98059) and inhibitors of p38 MAPK (SB203580 and SB202190), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the transient activation of p42/p44 and p38 MAPKs induced by IL-1. Furthermore, IL-1-induced activation of nuclear factor-B (NF-B) was inversely correlated with the degradation of IB- in HTSMCs. IL-1-induced COX-2 expression and PGE₂ synthesis were inhibited by the NF-B inhibitor pyrrolidinedithiocarbamate. These findings suggest that the expression of COX-2 is correlated with the release of PGE₂ from IL-1-challenged HTSMCs, which is mediated, at least in part, through p42/p44 and p38 MAPKs and NF-B signaling pathways in HTSMCs.     

Ozone-induced pulmonary inflammation and epithelial proliferation are partially mediated by PAF

Ozone(O₃) exposure stimulates airwayinflammation and epithelial sloughing in a number of species, includingmice. Platelet-activating factor (PAF) is a lipid mediator released byactivated mast cells, macrophages, and epithelial cells and causespulmonary inflammation and hyperpermeability. We hypothesized that theactivation of PAF receptors is central to the development ofinflammation and epithelial injury induced by acuteO₃ exposure in mice. To test thishypothesis, O₃-susceptibleC57BL/6J mice were treated with a PAF-receptor antagonist, UK-74505, orvehicle either before or immediately after 3-h exposure toO₃ (2 parts/million) or filtered air. Bronchoalveolar lavage (BAL) fluids were collected 6 and 24 hafter exposure. Differential cell counts and protein content of thelavage were used as indicators of inflammation in the airways. O₃-induced epithelial injury wasassessed by light microscopy, and DNA synthesis in epithelium ofterminal bronchioles was estimated by using abromodeoxyuridine-labeling index. Intercellular adhesion molecule 1 (ICAM-1) expression was also examined in the lung by immunohistochemical localization.O₃ caused significant increases inpolymorphonuclear leukocytes and protein in the BAL fluid, increasedpulmonary epithelial proliferation, and increased epithelial expressionof ICAM-1 compared with air-exposed, vehicle-treated control mice.Relative to O₃-exposed,vehicle-treated control mice, UK-74505 before exposure significantly(P < 0.05) decreased BAL protein,polymorphonuclear leukocytes, and epithelial cells. O₃-induced inflammation wassimilarly attenuated in mice treated with UK-74505 after exposure.These experiments thus support the hypothesis thatO₃-induced airways inflammationand epithelial damage in mice are partially mediated by activation ofPAF receptors, possibly through modulation of ICAM-1 expression.

     

The Moraxella catarrhalis-induced pro-inflammatory immune response is enhanced by the activation of the epidermal growth factor receptor in human pulmonary epithelial cells

Background

Chronic lower airway inflammation is considered to be a major cause of pathogenesis and disease progression in chronic obstructive pulmonary disease (COPD). Moraxella catarrhalis is a COPD-associated pathogen causing exacerbations and bacterial colonization in the lower airways of patients, which may contribute to chronic inflammation. Increasing evidence suggests that the epidermal growth factor receptor (EGFR) modulates inflammatory processes in the human airways. The goal of this study was to investigate the role of EGFR in the M. catarrhalis-induced pro-inflammatory immune response in airway epithelial cells.

Methods

The effects of inhibition and gene silencing of EGFR on M. catarrhalis-dependent pro-inflammatory cytokine expression in human primary bronchial epithelial cells (NHBEs), as well as the pulmonary epithelial cell lines BEAS-2B and A549 were analyzed. We also assessed the involvement of EGFR-dependent ERK and NF-κB signaling pathways.

Results

The M. catarrhalis-induced pro-inflammatory immune response depends, at least in part, on the phosphorylation and activation of the EGF receptor. Interaction of M. catarrhalis with EGFR increases the secretion of pro-inflammatory cytokines, which is mediated via ERK and NF-κB activation.

Conclusion

The interaction between M. catarrhalis and EGFR increases airway inflammation caused by this pathogen. Our data suggest that the inhibition of EGFR signaling in COPD could be an interesting target for reducing M. catarrhalis-induced airway inflammation.     

Tiludronate inhibits interleukin-6 synthesis in osteoblasts: Inhibition of phospholipase D activation in MC3T3-E1 cells

In previous studies, we have reported that PGF_2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF_2α and PGE₁ induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF_2α- and PGE₁-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF_2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE₁ or (Bu)₂cAMP was hardly affected by tiludronate. The choline formation induced by PGF_2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF_2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF_2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252–259, 1998. © 1998 Wiley-Liss, Inc.     

Murine lung injury caused by Leptospira interrogans glycolipoprotein,a specific Na/K-ATPase inhibitor

Background

Leptospiral glycolipoprotein (GLP) is a potent and specific Na/K-ATPase inhibitor. Severe pulmonary form of leptospirosis is characterized by edema, inflammation and intra-alveolar hemorrhage having a dismal prognosis. Resolution of edema and inflammation determines the outcome of lung injury. Na/K-ATPase activity is responsible for edema clearance. This enzyme works as a cell receptor that triggers activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therefore, injection of GLP into lungs induces injury by triggering inflammation.

Methods

We injected GLP and ouabain, into mice lungs and compared their effects. Bronchoalveolar lavage fluid (BALF) was collected for cell and lipid body counting and measurement of protein and lipid mediators (PGE₂ and LTB₄). The levels of the IL-6, TNFα, IL-1B and MIP-1α were also quantified. Lung images illustrate the injury and whole-body plethysmography was performed to assay lung function. We used Toll-like receptor 4 (TLR4) knockout mice to evaluate leptospiral GLP-induced lung injury. Na/K-ATPase activity was determined in lung cells by nonradioactive rubidium incorporation. We analyzed MAPK p38 activation in lung and in epithelial and endothelial cells.

Results

Leptospiral GLP and ouabain induced lung edema, cell migration and activation, production of lipid mediators and cytokines and hemorrhage. They induced lung function alterations and inhibited rubidium incorporation. Using TLR4 knockout mice, we showed that the GLP action was not dependent on TLR4 activation. GLP activated of p38 and enhanced cytokine production in cell cultures which was reversed by a selective p38 inhibitor.

Conclusions

GLP and ouabain induced lung injury, as evidenced by increased lung inflammation and hemorrhage. To our knowledge, this is the first report showing GLP induces lung injury. GLP and ouabain are Na/K-ATPase targets, triggering intracellular signaling pathways. We showed p38 activation by GLP-induced lung injury, which was may be linked to Na/K-ATPase inhibition. Lung inflammation induced by GLP was not dependent on TLR4 activation.     

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.