Eicosanoid Release Research Articles

Failure of hCG/LH receptors to stimulate the transmembrane effector adenylyl cyclase in human endometrium

The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively; n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p tions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC; however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.

The sterols isolated from Evening Primrose oil modulate the release of proinflammatory mediators

Evening Primrose oil is a natural product extracted by cold-pressed from Oenothera biennis L. seeds. The unsaponifiable matter of this oil is an important source of interesting minor compounds, like long-chain fatty alcohols, sterols and tocopherols. In the present study, sterols were isolated from the unsaponifiable matter of Evening Primrose oil, and the composition was identified and quantified by GC and GC–MS. The major components of sterols fraction were β-Sitosterol and campesterol. We investigated the ability of sterols from Evening Primrose oil to inhibit the release of different proinflammatory mediators in vitro by murine peritoneal macrophages stimulated with lipopolysaccharide. Sterols significantly and dose-dependently decreased nitric oxide production. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression. Sterols also reduced tumor necrosis factor-α, interleukine 1β and tromboxane B2. However, sterols did not reduce prostaglandin E2. The reduction of eicosanoid release was related to the inhibition of cyclooxygenase-2 expression. These results showed that sterols may have a protective effect on some mediators involved in inflammatory damage development, suggesting its potential value as a putative functional component of Evening Primrose oil.

Regulation of Cytosolic Phospholipase A2 Phosphorylation by Proteolytic Cleavage of Annexin A1 in Activated Mast Cells

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.

Fasitibant chloride, a kinin B2 receptor antagonist, and dexamethasone interact to inhibit carrageenan‐induced inflammatory arthritis in rats

Bradykinin, through the kinin B₂ receptor, is involved in inflammatory processes related to arthropathies. B₂ receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated. Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1β, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h. The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg⁹-[Leu⁸]-bradykinin (B₂ receptor, leukotriene, catecholamine and B₁ receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg⁹-[Leu⁸]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg⁹-[Leu⁸]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment. Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B₂ receptor, or through the indirect effects mediated by release of eicosanoids and cytokines.

Rapid induction of lipid mediators is a novel effector function of the inflammasome in vivo (117.24)

Abstract Detection of microbial products by host inflammasomes is critical for innate immune surveillance, while eicosanoids, signaling lipids that include the prostaglandins and leukotrienes, are rapid triggers of inflammation. Here we report for the first time a link between inflammasome activation and eicosanoid synthesis. Using a novel protein toxin, we show that systemic cytosolic delivery of bacterial flagellin in vivo leads to loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (<30 min) death in mice. This unexpected response is dependent on the inflammasome components NAIP5, NLRC4, and CASP1, but interestingly, is independent of the known inflammasome effector functions, namely IL-1β/-18 production and pyroptotic cell death. Instead, inflammasome activation in resident peritoneal macrophages results, within minutes, in the release of prostaglandins and other eicosanoids that induce vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to the pathological effects of systemic inflammasome activation. Compared to other macrophage populations, resident peritoneal macrophages are specifically primed for inflammasome-dependent production of eicosanoids by high expression of Cox1 and other eicosanoid biosynthetic enzymes. Thus, our results identify eicosanoid production as a novel cell type-specific effector function of the inflammasome with dramatic physiological consequences in vivo.

Exposure of alveolar macrophages to polybrominated diphenyl ethers suppresses the release of pro-inflammatory products in vitro

Inhalation of chemical pollutants has been associated with a reduced immune response in humans. Inhalation of dust is a major route of exposure for one endocrine-disrupting chemical and suspected xenoestrogen, polybrominated diphenyl ethers (PBDEs); however, the impact of PBDEs on immune function is unclear. The objective of this study was to investigate the action of PBDEs on cytokine and eicosanoid release by alveolar macrophages and determine whether the effects are mediated via the estrogen receptor. The production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, IL-10 and prostaglandin E(2) (PGE(2)) by porcine alveolar macrophages exposed to different concentrations of the pentabrominated diphenyl ether mixture, DE-71, were measured; cells were also exposed to varying concentrations of 17β-estradiol and the selective estrogen receptor-modulating agent, tamoxifen. Cells exposed to PBDEs released significantly less pro-inflammatory cytokines (TNF-α and IL-6) and PGE(2) compared with controls; IL-1β and IL-10 were not detected in the culture medium. Cells exposed to 17β-estradiol released significantly less TNF-α compared with controls, an effect that was reversed by the addition of tamoxifen; tamoxifen had no effect on the inhibition of TNF-α release by PBDEs. Although the suppression of TNF-α with DE-71 was similar to that of estrogen, the inhibitory effects of DE-71 were not found to be dependent on the estrogen receptor. Findings of this study suggest that chronic exposure to PBDEs suppressed innate immunity in vitro. Whether the immunosuppressant effects of PBDEs occur in vivo, remains to be determined.

Activation of Mitochondrial Calcium-independent Phospholipase A2γ (iPLA2γ) by Divalent Cations Mediating Arachidonate Release and Production of Downstream Eicosanoids

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

Infrapatellar fat pad from osteoarthritis patients displays a distinctive eicosanoid release profile compared to healthy donors

Infrapatellar fat pad from osteoarthritis patients displays a distinctive eicosanoid release profile compared to healthy donors

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

Effects of Antihistamines on Leukotriene and Cytokine Release from Dispersed Nasal Polyp Cells

In this study the effects of antihistamines on the release of eicosanoids and the pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha) were compared. Enzymatically dispersed cells from human nasal polyps served as an in vitro model of chronic respiratory mucosal inflammation. Nasal polyp cells (2 x 10(5)/ml) were sensitized with human IgE pre-incubated azelastine (CAS 58581-89-8), terfenadine (CAS 50679-08-8), levocabastine (CAS 79516-68-0) or cetirizine (CAS 83881-51-0), and stimulated with anti-human immunoglobulin E (IgE). Thromboxane B2 (TBX2) and leukotriene C4 (LTC4) were measured by radioimmunoassay (RIA), TNF alpha by enzyme-linked immunosorbent assay (ELISA). Data represent mean values of % inhibition estimated from the untreated positive control or mean IC50 (n = 5). Azelastine and terfenadine inhibited TNF alpha release with IC50 values of 6.2 mumol/l and 4.3 mumol/l, respectively. Terfenadine reduced TXB2 release by 37 +/- 15%, and LTC4 release was decreased by azelastine and terfenadine very potently by 86% and 100%, respectively. Azelastine shows anti-inflammatory properties in therapeutically relevant concentrations as assessed by its ability to reduce TNF alpha release as well as its ability to inhibit LTC4 production in allergically stimulated human nasal polyp cells.

TRP-channel-specific cutaneous eicosanoid release patterns

Analyzing mechanisms and key players in peripheral nociception nonneuronal skin cells are getting more and more into focus. Herein we investigated the functional expression of TRPV1 and TRPA1 in human keratinocytes and fibroblasts and assessed proinflammatory lipid mediator release upon their stimulation as well as sensory effects after topical application, combining in vitro and in vivo approaches. In vitro, the expression of functional TRPV1 and TRPA1 channels on fibroblasts and keratinocytes was confirmed via immunofluorescence, qualitative real time (RT) polymerase chain reaction, and cellular Ca 2+ influx measurements. Additionally, the agonists allyl isothiocyanate (TRPA1) and capsaicin (TRPV1) induce a differential secretion pattern of the eicosanoids PGE 2 and LTB 4 in human dermal fibroblasts and keratinocytes, which was also detectable in vivo, analyzing suction blister fluid at various times after short-term topical application. Capsaicin provoked the release of LTB 4 at 2 and 24 hours. In contrast, PGE 2 levels were reduced upon stimulation. Allyl isothiocyanate, however, increased PGE 2 levels only at 24 hours, but did not alter LTB 4 levels. In parallel, heat pain thresholds were reduced by both agents after short-term topical application, but only AITC provoked a long-lasting local erythema. In conclusion, the agonist-induced activation of nociceptors by TRPA1 and TRPV1 elicits painful sensations, whereas nonneuronal tissue cells respond with differential release of inflammatory mediators, thus influencing local vasodilatation and neuronal sensitization. These results have implications for the application of transient receptor potential antagonists to improve inflammatory skin conditions and pain management.

Role of gastrointestinal inflammations in the development and treatment of depression

Recent studies have revealed that inflammation, among other factors, may be involved in the pathogenesis of depression. One line of studies has shown that depression is frequently associated with manifest gastrointestinal inflammations and autoimmune diseases as well as with cardiovascular diseases, neurodegenerative diseases, type 2-diabetes and also cancer, in which chronic low-grade inflammation is a significant contributing factor. Thus depression may be a neuropsychiatric manifestation of a chronic inflammatory syndrome. Another line of studies has shown that the primary cause of inflammation may be the dysfunction of the "gut-brain axis". Although, this is a bidirectional mechanism, life style factors may primarily affect the symbiosis between host mucous membrane and the microbiota. Local inflammation through the release of cytokines, neuropeptides and eicosanoids may also influence the function of the brain and of other organs. Role of metabolic burst due to inflammation represents a new aspect in both pathophysiology and treatment of the depression. Finally, an increasing number of clinical studies have shown that treating gastrointestinal inflammations with probiotics, vitamin B, D and omega 3 fatty acids, through attenuating proinflammatory stimuli to brain, may also improve depression symptoms and quality of life. All these findings justify an assumption that treating gastrointestinal inflammations may improve the efficacy of the currently used treatment modalities of depression and related diseases. However, further studies are certainly needed to confirm these findings.

An inflammatory pathway to apnea and autonomic dysregulation

Infection in infancy may dramatically aggravate an underlying cardiorespiratory dysfunction during a susceptible postnatal period. Children with immature brainstem respiratory control, as well as infants, may have periodic irregular breathing with potential detrimental apneas that are increased during sleep as well as during infectious episodes. Data now indicate that the proinflammatory cytokine interleukin (IL)-1β impairs respiration during infection via prostaglandin E2 (PGE(2)) and that infection, with associated eicosanoid release, is one of the main causes of respiratory disorders in preterm infants. Moreover, brainstem microsomal prostaglandin E synthase-1 (mPGES-1) is rapidly activated during transient hypoxia. An inflammatory mediated activation of the mPGES-1 pathway, e.g., by viral or bacterial infection, rapidly induces release of PGE(2) in the vicinity of brainstem cardio-respiratory-related centers. This will depress the autonomic control networks, including the central drive to breathe. Hypoxia may then further reduce the activity of vital brainstem centers and the ability to autoresuscitate. This might have fatal consequences in vulnerable infants during a susceptible time frame. Here the evidence from human, animal and molecular studies to support this hypothesis is reviewed and how the pathogenesis of apnea and the response to hypoxia is associated with inflammatory pathways is discussed.

A targeted lipidomics approach to the study of eicosanoid release in synovial joints

IntroductionArticular tissues are capable of producing a range of eicosanoid mediators, each of which has individual biological effects and may be affected by anti-inflammatory treatment. We set out to develop and evaluate a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) approach for the simultaneous analysis of multiple eicosanoid lipid mediators in equine synovial fluid (SF), and to illustrate its use for investigation of the in vivo effects of non-steroidal anti-inflammatory drug (NSAID) treatment.MethodsSynovial fluid samples were obtained from normal joints of 6 adult horses at baseline (0 hr) and at 8, 24 and 168 hours after experimental induction of transient acute synovitis, with horses treated once daily with oral NSAID (meloxicam, 0.6 mg/kg) or placebo. Following solid-phase extraction, SF lipid mediator quantitation was based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis, and results were compared between disease states using linear discriminant analysis (LDA) and analysis of variance (ANOVA) with multiple comparisons corrections.ResultsOf a total of 23 mediators targeted, 14 could be reliably identified and quantified in SF samples based on detection of characteristic fragment ions at retention times similar to those of commercial standards. LDA analysis of baseline, 8, 24 and 168 hour synovial fluid samples revealed a separation of these groups into discrete clusters, reflecting dynamic changes in eicosanoid release over the course of synovitis. Prostaglandin (PG) E2 was significantly lower in NSAID vs. placebo treated samples at all time points; PGE1, 11-hydroxyeicosatetraenoic acid (11-HETE) and 13,14-dihydro-15keto PGF2α were reduced at 8 and 24 hours by NSAID treatment; while 15-HETE, 6-keto PGF1α, PGF2α, 13,14-dihydro-15keto PGE2 and thromboxane B2 (TXB2) were reduced at the 8 hour time point only. An interesting pattern was seen for Leukotriene B4 (LTB4), NSAID treatment causing an initial increase at 8 hours, but a significant reduction by 168 hours.ConclusionsThe described method allows a comprehensive analysis of synovial fluid eicosanoid profiles. Eicosanoid release in inflamed joints as well as differences between NSAID treated and placebo treated individuals are not limited to PGE2 or to the early inflammatory phase.

Pathways Regulating Cytosolic Phospholipase A2 Activation and Eicosanoid Production in Macrophages by Candida albicans

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A2 (cPLA2α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the β-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88−/− but not Dectin-1−/− macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA2α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA2α phosphorylation, and COX2 expression in response to particulate β-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA2α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both β-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA2α and eicosanoid production.

Hypercapnic acidosis in ventilator-induced lung injury

Permissive hypercapnia is established in lung injury management. Therapeutic hypercapnia causes benefit or harm, depending on the context. Ventilator-associated lung injury has a wide spectrum of candidate mechanisms, affording multiple opportunities for intervention such as hypercapnia to exert benefit or harm. To confirm (1) that hypercapnia attenuates in vivo ventilator-induced lung injury (VILI); (2) biological plausibility of such protection (e.g., dose-response, time series, inflammatory profile); and (3) that the associated biochemical events are consistently beneficial. A mouse model of VILI was established in vivo. Injurious ventilation was established, hypercapnia applied and markers of inflammation measured. Lung injury was quantified by gas exchange, elastance, microvascular leak, histology and levels of cytokines and eicosanoids, cyclooxygenase and tissue nitrotyrosine. Injurious ventilation caused significant lung injury (mechanics, microvascular leak, histology) and release of inflammatory cytokines, chemokines and eicosanoids. Hypercapnia attenuated these responses, with dose-response and time-dependent effects. No adverse effects of hypercapnia were observed in controls. Hypercapnia suppressed the transcription (mRNA) and translation (protein) of the major inducible prostanoid-generating enzyme (COX-2), but the effects on the downstream eicosanoids were modest. However, hypercapnia significantly increased lung tissue nitrotyrosine-at PaCO(2) levels that were protective. Hypercapnia provided consistent and biologically plausible in vivo protection against VILI, but elevated lung tissue levels of nitro-tyrosine as previously described in sepsis. Clinicians and those designing clinical trials need to be aware of the potential for detrimental effects when using hypercapnia in order to balance benefits versus harm with this approach.

Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2

Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.

Cyclic guanosine monophosphate dependent pathway contributes to human mast cell inhibitory actions of the nitric oxide donor, diethylamine NONOate

We have previously demonstrated that exogenous nitric oxide (NO) inhibited anti-IgE-mediated histamine release from human cultured mast cells. In the current study, we further investigated if syntheses of eicosanoids and cytokines were also suppressed by NO donors and evaluated if activation of soluble guanylyl cyclase (sGC) was an underlying mechanism. The effects of the NO donor diethylamine NONOate (DEA/NO) on IgE-dependent syntheses of eicosanoids (prostaglandin D 2 and cysteinyl leukotrienes) and cytokines (tumor necrosis factor-α and interleukin-8) from buffy coat derived human cultured mast cells were examined. The effects of sGC related agents on human mast cell activation were studied by measuring histamine release. DEA/NO (10 − 7 –10 − 4 M) dose-dependently inhibited anti-IgE induced release of histamine, eicosanoids and cytokines. It could also significantly increase intracellular cyclic guanosine monophosphate (cGMP) but reduce anti-IgE induced activation of ERK1/2, JNK1/2 and NF-κB. The inhibition of anti-IgE induced histamine release by DEA/NO was reversed by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 − 7 M) and the cGMP-dependent protein kinase (PKG) inhibitor, Rp-8-(4-Chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS, 10 − 5 M). The current study confirmed the inhibitory action of exogenous NO on immunological activation of human mast cells. We also provided evidence for the first time that the activation of the sGC–cGMP–PKG pathways together with the suppression of phosphorylation of MAPKs and NF-κB contributed to the mast cell modulating action of NO in human.

Transglutaminase 2, a Novel Regulator of Eicosanoid Production in Asthma Revealed by Genome-Wide Expression Profiling of Distinct Asthma Phenotypes

BackgroundA frequent manifestation of asthma, exercise-induced bronchoconstriction (EIB), occurs in 30–50% of asthmatics and is characterized by increased release of inflammatory eicosanoids. The objective of this study was to identify genes differentially expressed in EIB and to understand the function of these genes in the biology of asthma.Methodology/Principal FindingsGenome-wide expression profiling of airway leukocytes and epithelial cells obtained by induced sputum was conducted in two groups of subjects with asthma with and without EIB (n = 7 per group), at baseline and following exercise challenge. Based on the results of the gene expression study, additional comparisons were made with a normal control group (n = 10). Localization studies were conducted on epithelial brushings and biopsies from an additional group of asthmatics with EIB (n = 3). Genes related to epithelial repair and mast cell infiltration including β-tryptase and carboxypeptidase A3 were upregulated by exercise challenge in the asthma group with EIB. A gene novel to asthma pathogenesis, transglutaminase 2 (TGM2), was the most differentially expressed at baseline between the groups. In vivo studies confirmed the increased expression of TGM2 in airway cells and airway lining fluid, and demonstrate that TGM2 is avidly expressed in the asthmatic airway epithelium. In vitro studies using recombinant human enzymes reveal that TGM2 augments the enzymatic activity of secreted phospholipase A2 (PLA2) group X (sPLA2-X), an enzyme recently implicated in asthma pathogenesis.Conclusions/SignificanceThis study found that TGM2, a mediator that is novel to asthma pathogenesis, is overexpressed in asthmatic airways and functions to increase sPLA2-X enzymatic activity. Since PLA2 serves as the first rate-limiting step leading to eicosanoid formation, these results suggest that TGM2 may be a key initiator of the airway inflammatory cascade in asthma.

Characterization and modulation of canine mast cell derived eicosanoids

Mast cells play an important role in both innate and acquired immunity as well as several pathological conditions including allergy, arthritis and neoplasia. They influence these processes by producing a variety of mediators including cytokines, chemokines and eicosanoids. Very little is currently known about the spectrum of inflammatory mediators, particularly eicosanoids (prostaglandins and leukotrienes), produced by canine mast cells. This is important since modulating mast cell derived eicosanoids may help in the treatment of autoimmune and inflammatory disorders. The purpose of this study was to investigate the spectrum of eicosanoids produced by normal canine mast cells and to evaluate the effects of cytokines and non-steroidal anti-inflammatory mediators (NSAIDS) on eicosanoid production and release. Canine bone marrow derived cultured mast cells (cBMCMCs) expressed COX-1, COX-2, and 5-LOX and synthesized and released PGD2, PGE2, LTB4, and LTC4 following activation by a variety of stimuli. The selective COX-2 NSAIDs carprofen (Rimadyl®) and deracoxib (Deramaxx®) inhibited PGD2 and PGE2 production but only slightly inhibited LTB4 and LTC4. The mixed COX-1/COX-2 inhibitor piroxicam blocked PGD2 and PGE2 production, but upregulated LTC4 following treatment while tepoxalin (Zubrin®), a pan COX/LOX inhibitor, markedly reduced the production of all eicosanoids. The LOX inhibitor nordihydroguaiaretic acid (NDGA) prevented LTB4/LTC4 release and BMBMC degranulation. Pre-incubation of cBMCMCs with IL-4 and SCF sensitized these cells to degranulation in response to substance P. In conclusion, canine BMCMCs produce an array of eicosanoids similar to those produced by mast cells from other species. Tepoxalin appeared to be the most effective NSAID for blocking eicosanoid production and thus may be useful for modulating mast cell mediated responses in dogs.