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Roles of prostaglandins and leukotrienes in acute inflammation caused by bacterial infection

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Abstract

Prostanoids, including prostaglandins and thromboxanes, are generated by the phospholipase A(2)/cyclooxygenase pathway, and leukotrienes are generated by the 5-lipoxygenase pathway from arachidonic acid. At physiological concentrations, vasodilator prostaglandins enhance the vascular permeability effects of histamine and bradykinin, and leukotrienes are important mediators of leukocyte accumulation during acute inflammation. On the other hand, prostaglandin metabolites such as cyclopentenone prostaglandins contribute to the resolution of acute inflammation through inhibition of nuclear factor-chiB activation. Thus, arachidonic acid oxygenation products mediate diverse effects that induce and resolve acute inflammation caused by bacterial infection. Curr Opin Infect Dis 14:257-263. (C) 2001 Lippincott Williams & Wilkins.

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... EETs are metabolized to dihydroxyepoxyeicosatrienoic acid (DHETE) by soluble epoxide hydrolase. Each of these pathways yields multiple products, some of which act as intracellular messengers (47, 94) and others that leave the cell and act as ligands on G protein-coupled membrane receptors (26,55,94). The array of specific synthase enzymes for each of the PGs and leukotrienes that is present in a given cell varies and determines which PGs or leukotrienes are produced in that cell. ...
... However, the focus has been shifting to include fatty acids as we learn more about the functions of fatty acids beyond their role in energy metabolism. Fatty acids have been identified as ligands for members of the steroid superfamily of intracellular receptors (37,47,94) as well as for membrane GPCRs (43). Fatty acids have also been implicated in the accumulation of ceramide by stimulating de novo synthesis (31) or by increasing neutral sphingomyelinase activity (41). ...
... Since COX and LOX mediate inflammatory pathways, the reduced efficiency of these enzymes with-3 fatty acids as substrates allows-3 fatty acids to have an anti-inflammatory effect when they have been incorporated into cell membranes (37). As mentioned above, fatty acids have been shown to act as ligands at both intracellular (37,47,94) and membrane receptors (43) and can increase the concentration of ceramide in cell membranes (31,41). Whether the ability of-3 fatty acids to regulate the activity of these pathways differs from that of-6 fatty acids has not been thoroughly examined, but it is expected that their effects will differ. ...
Article
Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction. However, lipids serve a variety of roles in signal transduction. They act as ligands that activate signal transduction pathways as well as mediators of signaling pathways, and lipids are the substrates of lipid kinases and lipid phosphatases. Cell membranes are the source of the lipids involved in signal transduction, but membranes also constitute lipid barriers that must be traversed by signal transduction pathways. The purpose of this review is to explore the magnitude and diversity of the roles of the cell membrane and lipids in signal transduction and to highlight the interrelatedness of families of lipid mediators in signal transduction.
... Prostaglandins are also reported to inhibit macrophage production of NO and TNF-a [47,48]. Thus, COX inhibition, with reduction of the abnormally elevated PG production, would be expected to enhance the release of TNF-a and NO from the LPSstimulated macrophages from the malnourished mice. ...
... PGE 2 and PGI 2 increase intracellular cAMP levels, but decrease the level of the opposing cyclic nucleotide cGMP [65]. Conversely, LTB 4 and the cysLTs increase the level of cGMP [8] and inhibit adenyl cyclase, the enzyme that produces cAMP [48]. cGMP promotes the turnover of phosphatidylinositol whereas cAMP inhibits this process [10]. ...
... Prostanoids are also potent regulators of the vasculature (Yoshikai, 2001;Bos et al., 2004;Hata & Breyer, 2004;Yuhki et al., 2004). Administration of PGE 1 , PGE 2 or PGF 2a into the testis has direct inhibitory effects on testicular blood flow, by increasing testicular venous pressure and reducing testicular arterial pressure (Free & Jaffe, 1972). ...
... Clinical trials with celecoxib have not uncovered overt effects on normal fertility or testicular function but more subtle effects, such as effects on the testicular vasculature, may have been overlooked. Moreover, as prostanoids have complex effects on various aspects of the inflammatory response (Yoshikai, 2001;Hata & Breyer, 2004), interfering with endogenous COX-2 levels might either diminish or exacerbate the effects of inflammation on the testis. This would have important implications for men receiving chronic anti-inflammatory treatment, particularly those men with pre-existing fertility problems. ...
Article
Celecoxib (Celebrex), an inhibitor of cyclooxygenase-2 (COX-2; prostaglandin-endoperoxide synthase 2; EC 1.14.99.1), is widely used in the treatment of chronic inflammation and pain. COX-2 is constitutively expressed in the testis, where it is responsible for prostaglandin production, so inhibition of this enzyme should have effects on testicular function. The effects of administering celecoxib (oral with feed, 0.15% w/w) for 5 weeks on normal testis function and the response to low dose (0.1 mg/kg body weight) or high dose (5.0 mg/kg) lipopolysaccharide (LPS) were examined in adult male rats. Celecoxib caused a 60% reduction in testicular interstitial fluid (IF) prostaglandin E(2) (PGE(2)) concentrations, accompanied by a compensatory increase in COX-2 mRNA expression. Celecoxib increased IF volume by 30%, but had no effect on testis weight, testis morphology or serum testosterone levels. In the celecoxib-fed rats, the dose-dependent inhibitory effects of LPS on testis weight, IF volume and serum testosterone levels were significantly diminished. However, celecoxib had no effect on COX-2 protein levels or LPS-induced expression of the inflammatory mediators interleukin-1beta, tumour necrosis factor-alpha or inducible nitric-oxide synthase. A similar lack of inhibition of LPS-induced cytokine expression by another COX-2 inhibitor, NS-398, was observed in vitro. These data indicate that celecoxib reduces intratesticular activity of COX-2 (as indicated by PGE(2) levels) and inhibits IF formation in the testis, but has no appreciable effect on steroidogenesis or spermatogenesis, at least in the short term. Celecoxib does not appear to alter the ability of the testis to mount an inflammatory response but opposes the deleterious effects of inflammation on IF formation and testosterone production. These results indicate significant roles for products of the COX-2 pathway in testicular vascular control and steroidogenesis, which may have implications for men with marginal fertility taking celecoxib for extended periods, but also highlight the potential of this drug to ameliorate testicular damage caused by systemic or local inflammation.
... Występujące tutaj leukocyty wytwarzają wolne rodniki i jony nadtlenkowe [28]. Granulocyty aktywują uwalnianie prostaglandyny (PGE 2 ) [27,148] potęgującej działanie takich mediatorów jak bradykinina, histamina i serotonina [144]. ...
... Gromadzące się leukocyty i makrofagi w następstwie chemotaktycznego działania składowych dopełniacza C3a i C5a są pobudzane do fagocytozy i uwalniania wolnych rodników [28]. Granulocyty aktywują uwalnianie prostaglandyny-PGE 2 , która potęguje działanie niektórych związków, takich jak: bradykinina, histamina i serotonina [27,148]. ...
The intensity of an inflammatory response in a tissue or an organ is dependent on the efficiency of the organism's homeostatic mechanisms, which restrict the extent of the reaction. The type of factor inducing a inflammatory response and its strength have significant influence on the dynamics of an inflammatory reaction. The prompt eradication of an inflammatory factor and its biologically adverse effects attest to the efficacious adaptive mechanisms of the organism. The inflammatory response expresses biochemical, hematological, and immunological responses at the local or systemic level.
... Cell mobility, e.g., migration, adhesion, diapedesis, chemotaxis, and humoral immune response, is often observed [7]. In other cases, there are inflammatory mediators locally occurring in humor (e.g., histamine, CRP protein, complement proteins, interleukins, prostacyclins, prostaglandins, thromboxane) [8][9][10]. The homeostatic response is also characteristic and the symptoms are platelet aggregation, blood clotting, and disseminated intravascular coagulation (DIC) [11,12]. ...
... A lot of neutrophils, eosinophiles, and thrombocytes appear in the fourth and fifth hour of the inflammatory reaction [19,21,22]. The leukocytes produce free radicals and superoxide ions [20,[23][24][25], and granulocytes activate the release of prostaglandins (e.g., PGE 2 ) [10,14,19], which accelerate the effects of bradykinin, histamine, and serotonin [9,[26][27][28]. Migrating granulocytes in the inflammatory focus cause the local necrobiotic changes, and they damage the intrafollicular and intralobular septum in the lungs because of the presence of elastase in neutrophils [4,6,21,22,28]. ...
Article
Full-text available
Proper functioning of homeostatic mechanisms is characteristic for every healthy organism and enables adapting to environmental changes. These complicated systematic reactions can neutralize the harmful stress factors leading to various inflammatory reactions. The aim of this study was to determine dynamic changes in the inflammatory reaction after single 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) administration of 5 μg/kg body weight into rats with experimentally induced pleuritis. These changes were observed by monitoring the hematological blood parameters during inflammation. The obtained results proved that dioxins contribute to various changes in the character of the inflammatory response. TCDD administration before pleuritis initiation caused an increase of lymphocytes and significant decrease of the number of neutrophils during inflammation. The current study proved that administration of low TCDD dose (seven times lower than used in other studies) can cause thymus, spleen, or lymphatic gland atrophy. This finding indicates the toxic influence of small TCDD dose especially on the immune system.
... Prostaglandins (PG) are a class of lipid hormones responsible for a wide range of functions within the body. PGs are synthesized from arachidonic acid that is released from the cell membrane by phospholipase A2 and then modified by the cyclooxygenase enzymes (COX1 and COX2) to enter the PG pathway (Figure 1) [1,2]. COX1 is constitutively active, whereas COX2 is induced under inflammatory conditions [2]. ...
... The involvement of COX1 and COX2 in regulating inflammation is evidenced by the increased cardiovascular risk associated with the inhibition of COX2 [4], and the increased susceptibility to colitis in mice lacking these two enzymes [5]. Two waves of COX2 activity have been identified: the first (early) activity is associated with the pro-inflammatory response, whereas the second wave mediates the resolution of inflammation [6], and is associated with high levels of PGD 2 and 15-deoxy-D 12,14 -PGJ 2 (hereafter referred to as 15d-PGJ 2 ) [1,6]. 15d-PGJ 2 has recently been identified as an anti-inflammatory PG. ...
Article
Full-text available
15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory downstream product of the cyclooxygenase enzymes. It has been implicated to play a protective role in a variety of inflammatory mediated diseases, including rheumatoid arthritis, neural damage, and myocardial infarctions. Here we show that 15d-PGJ2 also plays a role in Salmonella infection. Salmonella enterica Typhimurium is a Gram-negative facultative intracellular pathogen that is able to survive and replicate inside phagocytic immune cells, allowing for bacterial dissemination to systemic sites. Salmonella species cause a wide range of morbidity and mortality due to gastroenteritis and typhoid fever. Previously we have shown that in mouse models of typhoid fever, Salmonella infection causes a major perturbation in the prostaglandin pathway. Specifically, we saw that 15d-PGJ2 production was significantly increased in both liver and feces. In this work we show that 15d-PGJ2 production is also significantly increased in macrophages infected with Salmonella. Furthermore, we show that the addition of 15d-PGJ2 to Salmonella infected RAW264.7, J774, and bone marrow derived macrophages is sufficient to significantly reduce bacterial colonization. We also show evidence that 15d-PGJ2 is reducing bacterial uptake by macrophages. 15d-PGJ2 reduces the inflammatory response of these infected macrophages, as evidenced by a reduction in the production of cytokines and reactive nitrogen species. The inflammatory response of the macrophage is important for full Salmonella virulence, as it can give the bacteria cues for virulence. The reduction in bacterial colonization is independent of the expression of Salmonella virulence genes SPI1 and SPI2, and is independent of the 15d-PGJ2 ligand PPAR-γ. 15d-PGJ2 also causes an increase in ERK1/2 phosphorylation in infected macrophages. In conclusion, we show here that 15d-PGJ2 mediates the outcome of bacterial infection, a previously unidentified role for this prostaglandin.
... The eicosanoids, a group of lipid mediators such as prostaglandins, thromboxane, leukotrienes, and lipoxins, are involved in diverse processes of the immune and inflammatory responses and in many physiological functions (3,13,28). The first step in eicosanoid production in response to bacterial products or inflammatory cytokines is the release of arachidonic acid (AA) from the cell membrane by the family of phospholipases A 2 (PLA 2 ). ...
... In addition, it serves as a potent vasodilator and has been implicated in the induction of inflammatory symptoms, such as edema, pain, and fever. Although studies have shown that PGE 2 promotes inflammation, there is growing evidence that it also exerts strong immunosuppressive effects, including shifting of the T-helper (Th) cell response from Th1 to Th2 cytokine production and inhibition of leukocyte chemotaxis and phagocytosis (1,10,13,22,28). ...
Article
Full-text available
Rifampin, a potent antimicrobial agent, is a major drug in the treatment of tuberculosis. There is evidence that rifampin also serves as an immunomodulator. Based on findings that arachidonic acid and its metabolites are involved in the pathogenesis of Mycobacterium tuberculosis infections, we investigated whether rifampin affects prostaglandin E2 (PGE2) production in human alveolar epithelial cells stimulated with interleukin-1. Rifampin caused a dose–dependent inhibition of PGE2 production. At doses of 100, 50, and 25 g/ml it inhibited PGE2 production by 75%, 59% and 45%, respectively (p<0.001). Regarding the mechanism involved, rifampin caused a time- and dose-dependent inhibition of arachidonic acid release from the alveolar cells. At doses of 100, 50, 25 and 10 g/ml it significantly inhibited the release of arachidonic acid by 93%, 64%, 58%, and 35%, respectively (p<0.001). Rifampin did not affect the phosphorylation of cytosolic phospholipase A2 or the expression of cyclooxygenase 2. The inhibition of PGE2, and presumably other arachidonic acid products, probably contributes to the efficacy of rifampin in the treatment of tuberculosis, and may explain some of its adverse effects.
... The prostaglandins (PGs) are a family of lipid-soluble metabolites of arachidonic acid that act as autocrine and paracrine signaling molecules throughout the body. These molecules are particularly important in the regulation of vascular responses, apoptosis, and inflammation [1]. They also regulate several key reproductive processes in the female, including ovulation, fertilization, and parturition [2], but their roles in the male reproductive system, especially in the adult, are very poorly defined. ...
... Apart from the immunoregulatory actions of the PGs, expression of PTGS2 and production of PGs by numerous cell types in the testis suggest an overall, ongoing role in controlling the development, steroidogenic activity, and inflammatory functions of the Sertoli and Leydig cells [10,[13][14][15][64][65][66]. Since PG production regulates changes in vascular permeability and blood flow in other tissues, most notably during inflammation [1], endogenous PG production by the testis also may be involved in maintaining fluid balance and vascular function. Balanced against this, the limited data so far available have not provided any direct evidence for this possibility [17,18]. ...
Article
Prostaglandins (PGs), particularly PGE(2), have been implicated in the control of testicular steroidogenesis, spermatogenesis, and local immunity. However, virtually nothing is known about the expression or activity of the prostaglandin-endoperoxide synthases (PTGSs; also referred to as the cyclooxygenases), the specific rate-limiting enzymes responsible for PG production, in the adult testis. This activity was investigated in rats under normal conditions and during lipopolysaccharide-induced inflammation using quantitative real-time PCR, in situ hybridization, Western blotting, and PGE(2) measurements by ELISA. The mRNA for both the "constitutive" Ptgs1 and the "inducible" Ptgs2 forms was detected in multiple testicular cell types. Testicular Ptgs2 expression was substantially higher than that of Ptgs1, and testicular production of PGE(2) in vitro was found to be suppressed by a specific PTGS2 inhibitor (NS-398), but not by an inhibitor of PTGS1. Further investigation indicated that 1) PGE(2) production in the adult testis is attributable to constitutive expression of PTGS2 by somatic (Leydig cells and Sertoli cells) and spermatogenic cells; 2) testicular macrophages constitutively produce relatively low levels of PTGS2 and PGE(2) but are the only cell type to respond significantly to an inflammatory stimulus by increasing production of PGE(2); and 3) testicular PTGS2 expression and intratesticular PGE(2) levels are only marginally affected by acute inflammation. These data point toward a previously unanticipated maintenance role for the "inducible" PTGS2 enzyme in normal testicular function, as well as an anomalous response of testicular PTGS2 to inflammatory stimuli. Both observations are consistent with the reduced capacity of the testis to initiate and support inflammatory reactions.
... The eicosanoids, a group of lipid mediators such as prostaglandins, thromboxane, leukotrienes, and lipoxins, are involved in diverse processes of the immune and inflammatory responses and in many physiological functions (3,13,28). The first step in eicosanoid production in response to bacterial products or inflammatory cytokines is the release of arachidonic acid (AA) from the cell membrane by the family of phospholipases A 2 (PLA 2 ). ...
... In addition, it serves as a potent vasodilator and has been implicated in the induction of inflammatory symptoms, such as edema, pain, and fever. Although studies have shown that PGE 2 promotes inflammation, there is growing evidence that it also exerts strong immunosuppressive effects, including shifting of the T-helper (Th) cell response from Th1 to Th2 cytokine production and inhibition of leukocyte chemotaxis and phagocytosis (1,10,13,22,28). ...
Article
Full-text available
Rifampin, a potent antimicrobial agent, is a major drug in the treatment of tuberculosis. There is evidence that rifampin also serves as an immunomodulator. Based on findings that arachidonic acid and its metabolites are involved in the pathogeneses of Mycobacterium tuberculosis infections, we investigated whether rifampin affects prostaglandin E2 (PGE2) production in human alveolar epithelial cells stimulated with interleukin-1β. Rifampin caused a dose-dependent inhibition of PGE2 production. At doses of 100, 50, and 25 μg/ml, it inhibited PGE2 production by 75%, 59%, and 45%, respectively (P < 0.001). Regarding the mechanism involved, rifampin caused a time- and dose-dependent inhibition of arachidonic acid release from the alveolar cells. At doses of 100, 50, 25, and 10 μg/ml, it significantly inhibited the release of arachidonic acid by 93%, 64%, 58%, and 35%, respectively (P < 0.001). Rifampin did not affect the phosphorylation of cytosolic phospholipase A2 or the expression of cyclooxygenase-2. The inhibition of PGE2, and presumably other arachidonic acid products, probably contributes to the efficacy of rifampin in the treatment of tuberculosis and may explain some of its adverse effects.
... The PPARγ antagonist, the synthetic compound, 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907) that is bonded with PPARγ, but has no capability to stimulate the transcriptional action of PPARγ, rather antagonised PPARγ agonised to trigger transcriptional and adipogenic actions of this receptor (Wright et al., 2000;Yoshikai, 2001;Funk, 2001;Croasdell et al., 2015). ...
... Prostaglandins (PGs) are produced from arachidonic acid which is released from cell membrane by phospholipase A2 and then altered by the cyclooxygenase enzymes (COX-1 and COX-2) to enter the PG pathway (Yoshikai, 2001;Funk, 2001;Choi et al., 2005;Chien et al., 2013). Funk, (2001) revealed that COX-1 is constitutively active, while COX-2 is induced under inflammatory situations. ...
Article
Full-text available
Sepsis is the overwhelming inflammatory response to infection, especially bacterial infection and associated bacterial products. It has major healthcare impacts, affecting an estimated 19-30 million persons/year worldwide with a mortality of 30-70%. Despite intense research, no specific therapy has been established for sepsis and in addition to the high mortality, the associated economic costs are very high. For example, recent data shows that the annual cost of patients with sepsis is more than $20 billion in the USA, and £2.5 billion in the UK. Therefore, novel targets and new therapies for sepsis are required which will have an important impact on both mortality and economic benefits. Recent work has demonstrated that the phopsholipid modifying enzyme, LPCAT, has a role in the regulation of inflammatory responses to bacterial infections. However, the mechanism of action in this regard is not well understood. This project aimed to identify the role of LPCAT-2 in inflammatory response to infections. This project has utilized the RAW264.7 murine macrophage cell line as an experimental model and LPS or Pam3CSK4 as infectious stimuli to investigate the role of overexpressing LPCAT-2 as well as silencing the over-expressed LPCAT-2 using siRNA technique. RAW264.7 cells transiently or stably transfected with the LPCAT-2 gene were used to study the role of LPCAT-2 in the inflammatory responses of macrophages. LPCAT-2 was successfully over-expressed in RAW264.7 cells and the overexpression was successfully confirmed with real time polymerase chain reaction (RT-PCR) and western blotting. The overexpression of LPCAT-2 significantly upregulated the pro-inflammatory cytokines TNF-α and IL-6, at both gene expression, and protein level, while the anti-inflammatory cytokine, IL-10, was down regulated in these cells. Moreover, overexpression of LPCAT-2 significantly decreased the expression of TLR4, peroxisome proliferator-activated receptors –gamma (PPARγ) and CD206 (a marker of M2 macrophages) while it significantly increased CD14, TLR2, COX-2 and iNOS (M1 markers). LPCAT-2 gene expression was also increased when PPARγ was blocked with the selective (PPAR-γ) antagonist T0070907. Importantly, silencing the transiently over-expressed murine LPCAT-2 resulted in a significant reduction in TNF-alpha and a significant increase in IL-10 gene expression. Both the transient and stably transfected RAW264.7 cells have been used to study the role of LPCAT-2 in regulating inflammatory responses in macrophages. The results have significantly added to knowledge of the role of LPCAT-2 in the inflammatory response and will aid in the development of novel therapies for inflammatory conditions such as sepsis.
... The PPARγ antagonist, the synthetic compound, 2-chloro-5-nitro-N-4-pyridinylbenzamide (T0070907) that is bonded with PPARγ, but has no capability to stimulate the transcriptional action of PPARγ, rather antagonised PPARγ agonised to trigger transcriptional and adipogenic actions of this receptor (Wright et al., 2000;Yoshikai, 2001;Funk, 2001;Croasdell et al., 2015). ...
... Prostaglandins (PGs) are produced from arachidonic acid which is released from cell membrane by phospholipase A2 and then altered by the cyclooxygenase enzymes (COX-1 and COX-2) to enter the PG pathway (Yoshikai, 2001;Funk, 2001;Choi et al., 2005;Chien et al., 2013). Funk, (2001) revealed that COX-1 is constitutively active, while COX-2 is induced under inflammatory situations. ...
Thesis
Full-text available
Sepsis is the overwhelming inflammatory response to infection, especially bacterial infection and associated bacterial products. It has major healthcare impacts, affecting an estimated 19-30 million persons/year worldwide with a mortality of 30-70%. Despite intense research, no specific therapy has been established for sepsis and in addition to the high mortality, the associated economic costs are very high. For example, recent data shows that the annual cost of patients with sepsis is more than $20 billion in the USA, and £2.5 billion in the UK. Therefore, novel targets and new therapies for sepsis are required which will have an important impact on both mortality and economic benefits. Recent work has demonstrated that the phopsholipid modifying enzyme, LPCAT, has a role in the regulation of inflammatory responses to bacterial infections. However, the mechanism of action in this regard is not well understood. This project aimed to identify the role of LPCAT-2 in inflammatory response to infections. This project has utilized the RAW264.7 murine macrophage cell line as an experimental model and LPS or Pam3CSK4 as infectious stimuli to investigate the role of overexpressing LPCAT-2 as well as silencing the over-expressed LPCAT-2 using siRNA technique. RAW264.7 cells transiently or stably transfected with the LPCAT-2 gene were used to study the role of LPCAT-2 in the inflammatory responses of macrophages. LPCAT-2 was successfully over-expressed in RAW264.7 cells and the overexpression was successfully confirmed with real time polymerase chain reaction (RT-PCR) and western blotting. The overexpression of LPCAT-2 significantly upregulated the pro-inflammatory cytokines TNF-α and IL-6, at both gene expression, and protein level, while the anti-inflammatory cytokine, IL-10, was down regulated in these cells. Moreover, overexpression of LPCAT-2 significantly decreased the expression of TLR4, peroxisome proliferator-activated receptors –gamma (PPARγ) and CD206 (a marker of M2 macrophages) while it significantly increased CD14, TLR2, COX-2 and iNOS (M1 markers). LPCAT-2 gene expression was also increased when PPARγ was blocked with the selective (PPAR-γ) antagonist T0070907. Importantly, silencing the transiently over-expressed murine LPCAT-2 resulted in a significant reduction in TNF-alpha and a significant increase in IL-10 gene expression. Both the transient and stably transfected RAW264.7 cells have been used to study the role of LPCAT-2 in regulating inflammatory responses in macrophages. The results have significantly added to knowledge of the role of LPCAT-2 in the inflammatory response and will aid in the development of novel therapies for inflammatory conditions such as sepsis.
... Arachidonic acid, one of the major products of PLA 2 metabolism, has been implicated in the modulation of ion-channel conductance, the release of neurotransmitters and neuritogenesis (Kim and Clapham, 1989;Hwang et al., 1990;Miller et al., 1992). In addition, arachidonic acid is primarily involved in the production of mediators of the inflammatory response, namely leukotrienes, prostaglandins and tromboxanes, generated by the activity of 5-lipoxygenase and cyclooxygenase (Musser and Kreft, 1992;Yoshikai, 2001). ...
Article
Full-text available
Phospholipase A(2) (PLA(2)) is a key enzyme in cerebral phospholipid metabolism. Preliminary post-mortem studies have shown that PLA(2) activity is decreased in frontal and parietal areas of the AD brain, which is in accordance with recent (31)P-Magnetic Resonance Spectroscopy evidence of reduced phospholipid turnover in the pre-frontal cortex of moderately demented AD patients. Such abnormality may also be observed in peripheral cells, and reduced PLA(2) activity in platelet membranes of AD patients, and correlates with the severity of dementia. In rat hippocampal slices, PLA(2) has been implicated in mechanisms of synaptic plasticity. In adult rats, the stereotaxic injection of PLA(2) inhibitors in the CA1 area of hippocampus impaired, in a dose-dependent manner, the formation of short- and long-term memory. Additionally, such inhibition resulted in a reduction of the fluidity of hippocampal membranes. In primary cultures of cortical and hippocampal neurons, the inhibition of PLA(2) precluded neurite outgrowth, and the sustained inhibition of the enzyme in mature cultures lead to loss of viability. Taken together, these findings reinforce the involvement of PLA(2) enzymes in neurodevelopment and neurodegeneration processes, and further suggest that reduced PLA(2) activity, probably reducing membrane phospholipids breakdown, may contribute to the memory impairment in AD.
... The prostanoid PGE2 increases blood flow, vascular permeability, and edema. It mediates fever generation and interacts with various pathways of the innate immune system in a concentration-dependent manner (37). In our model of E. coli meningitis, PGE2 and TNF-␣ peak concentrations were reached 2 h after onset of ceftriaxone treatment, reflecting the liberation of proinflammatory compounds such as endotoxin by bacterial lysis. ...
Article
Mortality and long-term sequelae rates are high among adults and children with acute bacterial meningitis. Adjunctive treatment with dexamethasone has been shown to reduce systemic complications in bacterial meningitis patients, but corticosteroid treatment may have detrimental effects on hippocampal function. We evaluated the effect of dexamethasone treatment in addition to antibiotic therapy in a rabbit model of Escherichia coli meningitis. A moderate anti-inflammatory effect of dexamethasone could be demonstrated with respect to the inflammatory mediator prostaglandin E2, whereas no significant effect of dexamethasone on tumor necrosis factor-alpha, cerebrospinal fluid pleocytosis, protein, lactate, indicators of global neuronal damage, or blood gas analysis was found. Dexamethasone, however, increased the rate of apoptotic neurons in the granular layer of the hippocampal dentate gyrus. In view of the proapoptotic effect of adjunctive dexamethasone on hippocampal neuronal cells in animal models of Gram-positive and Gram-negative meningitis, the application of dexamethasone should be considered carefully in those forms of bacterial meningitis for which no evidence-based data of beneficial effect in humans are available, such as neonatal meningitis, bacillary Gram-negative meningitis or nosocomial forms of meningitis (e.g. following neurosurgery).
... Abnormalities in lipid composition in all mutants manifested itself by increased levels of lysophospholipids in the membrane fraction and of arachidonic acid in the raft and myelin fractions. Arachidonic acid is produced by phospholipase A 2 and serves as a precursor for eicosanoids, that is, prostaglandins, thromboxanes, and leukotrienes, all of which have been implicated in immune system functions as vasodilators and potent mediators of vascular inflammation (Campbell, 2000;Yoshikai, 2001;Zurier, 1993). Interestingly, arachidonic acid has also been implicated in modulating synaptic efficacy as a retrograde messenger ( Bazan et al., 1997;Bolshakov and Siegelbaum, 1995;Cunha and Ribeiro, 1999). ...
Article
The present study was carried out to compare different myelin-compromised mouse mutants with regard to myelin morphology in relation to axon-, lipid-, and immunopathology as a function of age. Mouse mutants deficient in the myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) display subtle and severe myelin pathologies in the central nervous system (CNS), respectively. Animals doubly deficient in MAG and the neural cell adhesion molecule (NCAM) show defects similar to those present in MAG single mutants while mice deficient in MAG and the nonreceptor type tyrosine kinase Fyn are severely hypomyelinated, in addition to the MAG-specific myelin abnormalities. These mutant mice showed distinct myelin pathologies in different regions of the central nervous system and generally displayed a decrease in axonal integrity with age. Myelin pathology did not correlate locally with axon transection and with an involvement of the immune system as seen by numbers of CD3-positive lymphocytes and MAC-3-positive macrophages. Interestingly, the degree of these cellular abnormalities also did not correlate with abnormalities in levels of phospholipids, arachidonic acid, cholesterol, and apolipoprotein E (apoE). Moreover, these changes in lipid metabolism, including immune system-related arachidonic acid, preceded cellular pathology. The combined observations point to differences, but also similarities in the relation of myelin, axon, and immunopathology with genotype, and to a common aggravation of the phenotype with age.
... 19 Similarly, the generation of lipid mediators (such as prostaglandins and leukotrienes), which are crucially important as potent regulators of immune responses, is altered by dietary lipids. 20,21 In the present study, we investigated the effect of several dietary lipids, such as FO, OO and hydrogenated coconut oil (HCO), on the circulating concentrations of IL-12p70, IL-4, prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) in the serum from mice experimentally infected with a virulent strain of L. monocytogenes , a ubiquitous Gram-positive facultative intracellular bacterial pathogen. Here, an initial reduction of IL-12p70 production by the administration of FO or HCO diets might be a critical factor involved in increased host susceptibility to L. monocytogenes infection. ...
Article
The direct examination of the effects that fish oil diets (composed of long-chain n-3 polyunsaturated fatty acids) exert on immune system function indicates a reduction of host natural resistance to infectious diseases mainly because of a suppression of immune function generated by the fatty acids contained in this diet. Here, we evaluated the concentration of IL-12, IL-4, prostaglandin E2 and leukotriene B4 in the serum from BALB/c mice receiving four different diets. Each group was fed a diet that differed only in the source of fat: a low-fat diet (2.5% by weight), an olive oil diet (20% by weight), a fish oil diet (20% by weight) or a hydrogenated coconut oil diet (20% by weight). Mice were fed for 4 weeks and then infected with the intracellular pathogen Listeria monocytogenes. An initial reduction in the Th1-type response as a result of a decrease in IL-12p70 secretion, an inefficient action of IL-4 (Th2-type response) and no modification of pro-inflammatory lipid-mediator production could be, at least in part, the key events responsible for the inadequate elimination of L. monocytogenes from the spleens of mice fed a fish oil diet. Furthermore, our results suggest that the type of dietary lipids may affect the circulating concentration of IL-12p70 and IL-4, leading to a modulation in the protective cellular immune response to L. monocytogenes infection.
... Thromboxanes were first discovered in platelets, whereas prostaglandins were named after their initial detection in the seminal fluid (Hamberg and Samuelsson, 1966; Hamberg et al., 1975). Later research has shown that virtually all mammalian tissues contain these important molecules and that they play a major role in the host defense (Vane, 1976; Samuelsson, 1987; Yoshikai, 2001). The nomenclature of prostanoids reflects two important structural features. ...
Article
The discovery of endogenous molecules involved in counterregulation of inflammatory responses that may lead to tissue injury provides an opportunity to explore new therapeutic approaches based on manipulation of new pathways. Natural counterregulatory pathways may reduce the possibility of unwanted toxic side-effects. Lipoxins are trihydroxytetraene-containing eicosanoids that are generated within the vascular lumen during platelet-leukocyte interactions and at mucosal surfaces via leukocyte-epithelial cell interactions. During cell-cell interactions, transcellular biosynthetic pathways are the major lipoxin biosynthetic routes, and thus, in humans, lipoxins are formed in vivo during multicellular responses, such as inflammation and asthma. This branch of the eicosanoid cascade generates specific tetraene-containing products that serve as "stop signals" for neutrophils that regulate key steps in leukocyte trafficking and prevent neutrophil-mediated tissue injury. These novel anti-inflammatory lipid mediators also appear to facilitate the resolution of the acute inflammatory response. In this review, recent findings and new concepts pertaining to the generation of lipoxins and their impact on the resolution of acute inflammation, and organ protection from leukocyte-mediated injury, are presented. The parallels and possible associations with periodontal diseases are discussed.
... Interestingly, the presence of proinflammatory cytokines IL-1β, IFN-γ and TNF-α, but not nitric oxide, during infection exacerbates the effects of Rickettsia on endothelial permeability, further suggesting that the changes in the barrier properties of vascular endothelium are most likely due to a combinatorial effect of the presence of intracellular Rickettsiae and immune responses of the host cell [102]. Another possibility in this regard is the potential contributions of prostaglandins, which are secreted as a result of increased expression of COX-2 and are also known to cause increased vascular permeability and edema [75,103]. ...
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Pathogenic Rickettsia species are Gram-negative, obligate intracellular bacteria responsible for the spotted fever and typhus groups of diseases around the world. It is now well established that a majority of sequelae associated with human rickettsioses are the outcome of the pathogen's affinity for endothelium lining the blood vessels, the consequences of which are vascular inflammation, insult to vascular integrity and compromised vascular permeability, collectively termed 'Rickettsial vasculitis'. Signaling mechanisms leading to transcriptional activation of target cells in response to Rickettsial adhesion and/or invasion, differential activation of host-cell signaling due to infection with spotted fever versus typhus subgroups of Rickettsiae, and their contributions to the host's immune responses and determination of cell fate are the major subtopics of this review. Also included is a succinct analysis of established in vivo models and their use for understanding Rickettsial interactions with host cells and pathogenesis of vasculotropic rickettsioses. Continued progress in these important but relatively under-explored areas of bacterial pathogenesis research should further highlight unique aspects of Rickettsial interactions with host cells, elucidate the biological basis of endothelial tropism and reveal novel chemotherapeutic and vaccination strategies for debilitating Rickettsial diseases.
... Leukotriene B 4 (LTB 4 ) 3 is a polyunsaturated fatty acid derived from the oxygenation of arachidonic acid. Leukotrienes represent a large family of lipidic molecules whose precursor, arachidonic acid, is the substrate of many biologically active molecules such as prostaglandins and thromboxanes (11), some of which have been used in the clinic for many years. The major sources of LTB 4 are neutrophils and macrophages (12,13). ...
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Human CMV is often associated with transplant rejection and opportunistic infections such as pneumonia in immunosuppressed patients. Current anti-CMV therapies, although effective, show relatively high toxicity, which seriously limits their long-term use. In this study, we provide evidence that leukotriene B(4) (LTB(4)) plays an important role in the fight against murine CMV (MCMV) infection in vivo. Intravenous administration of 50 and 500 ng/kg/day of LTB(4) to mice infected with a lethal dose of MCMV significantly increases their survival (50 and 70%, respectively), compared with the placebo-treated group (10% of survival). In mice infected with a sublethal dose of MCMV and treated daily with 50 ng/kg/day of LTB(4), the salivary gland viral loads were found to be reduced by 66% compared with the control group. Furthermore, using an allogeneic bone marrow transplantation mouse model, the frequency of MCMV reactivation from latently infected mice was much lower (38%) in LTB(4) (500 ng/kg)-treated mice than in the placebo-treated group (78%). Finally, in experiments using 5-lipoxygenase-deficient mice, MCMV viral loads in salivary glands were found to be higher in animals unable to produce leukotrienes than in the control groups, supporting a role of endogenous 5-lipoxygenase products, possibly LTB(4), in host defense against CMV infection.
... However, the role of toxins in leukocyte recruitment process in vitro remained to be elucidated. Lipid mediators derived from arachidonic acid oxygenation are known for mediate diverse effects in acute inflammation, as well as endogenous regulators of inflammatory process (Larsen and Henson, 1982;Yoshikai, 2001). Leukotriene B4 induces an increase in the expression of LFA-1, MAC-1 and a2 integrin in the leukocytes and ICAM-1 (CD54) on the surface of the endothelial cells (Seo and Smith, 2001). ...
Article
Thalassophryne maculosa fish envenomation is characterized by severe pain, dizziness, fever, edema and necrosis. Here, the dynamic of cellular influx, activation status of phagocytic cells, and inflammatory modulator production in the acute inflammatory response to T. maculosa venom was studied using an experimental model. Leukocyte counting was performed (2 h to 21 days) after venom injection in BALB/c mice footpads. Our results showed an uncommon leukocyte migration kinetic after venom injection, with early mononuclear cell recruitment followed by elevated and delayed neutrophil influx. The pattern of chemokine expression is consistent with the delay in neutrophil recruitment to the footpad: T. maculosa venom stimulated an early production of IL-1beta, IL-6, and MCP-1, but was unable to induce an effective early TNF-alpha and KC release. Complementary to these observations, we detected a marked increase in soluble KC and TNF-alpha in footpad at 7 days post-venom injection when a prominent influx of neutrophils was also detected. In addition, we demonstrated that bone marrow-derived macrophages and dendritic cells were strongly stimulated by the venom, showing up-regulated ability to capture FITC-dextran. Thus, the reduced levels of KC and TNF-alpha in footpad of mice concomitant with a defective accumulation of neutrophils at earlier times provide an important clue to uncovering the mechanism by which T. maculosa venom regulates neutrophil movement.
... OmCI binds directly to C5 and thus inhibits cleavage into anaphylatoxin C5a and C5b, a subunit of the membrane attack complex (MAC; C5b-9) and prevents MAC-mediated lysis and destruction of red blood cells in paroxysmal nocturnal hemoglobinuria (PNH) and tissue destruction in various other complement-mediated inflammatory and autoimmune diseases ( Hepburn et al., 2007;Fredslund et al., 2008). In addition, OmCI captures the inflammatory mediator leukotriene B4, a potent chemotactic agent and activator of neutrophils (Yoshikai, 2001). A recombinant form of OmCI (known as Coversin and rEV576) has shown efficacy in numerous animal models of complement-mediated diseases and successfully accomplished a phase Ia clinical trial. ...
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Ticks are obligatory blood-feeding ectoparasites, causing blood loss and skin damage in their hosts. In addition, ticks also transmit a number of various pathogenic microorganisms that cause serious diseases in humans and animals. Ticks evolved a wide array of salivary bioactive compounds that, upon injection into the host skin, inhibit or modulate host reactions such as hemostasis, inflammation and wound healing. Modulation of the tick attachment site in the host skin involves mainly molecules which affect physiological processes orchestrated by cytokines, chemokines and growth factors. Suppressing host defense reactions is crucial for tick survival and reproduction. Furthermore, pharmacologically active compounds in tick saliva have a promising therapeutic potential for treatment of some human diseases connected with disorders in hemostasis and immune system. These disorders are often associated to alterations in signaling pathways and dysregulation or overexpression of specific cytokines which, in turn, affect mechanisms of angiogenesis, cell motility and cytoskeletal regulation. Moreover, tick salivary molecules were found to exert cytotoxic and cytolytic effects on various tumor cells and have anti-angiogenic properties. Elucidation of the mode of action of tick bioactive molecules on the regulation of cell processes in their mammalian hosts could provide new tools for understanding the complex changes leading to immune disorders and cancer. Tick bioactive molecules may also be exploited as new pharmacological inhibitors of the signaling pathways of cytokines and thus help alleviate patient discomfort and increase patient survival. We review the current knowledge about tick salivary peptides and proteins that have been identified and functionally characterized in in vitro and/or in vivo models and their therapeutic perspective.
... intrOduÇãO O processo inflamatório é um mecanismo fisiopatológico útil, em resposta a diversos tipos de doenças ou lesões, envolvendo interações complexas entre células do sistema imunológico (circulantes e residentes) e tecido vascular (células endoteliais e do músculo liso; TEDGUI e MALLAT, 2001), com objetivo de eliminar do organismo a causa inicial da lesão e suas consequências (MEADOR et al., 2009). O processo inflamatório agudo refere-se à resposta que começa de maneira abrupta e precoce, sendo caracterizada por três eventos principais, mediados por moléculas solúveis ou por células do sistema imune: a) vasodilatação e diminuição da velocidade do fluxo sanguíneo local, sinalizando calor e rubor; b) permeabilidade vascular aumentada, ambos os eventos, conduzindo ao extravasamento de proteínas, assim como o recrutamento de leucócitos para o espaço extravascular e, subsequente, formação de edema (ou tumor) e, c) liberação de uma variedade de agentes pró-inflamatórios também capazes de produzir dor ou hiperalgesia (AL-NAEMI e BALDWIN, 1999;TROWBRIDGE e EMLING, 1996;WEBSTER e GALLEY, 2003;WOOLF, 2011;YOSHIKAI, 2001). ...
... Further, LA activates prostaglandin and leukotriene production [56], which can act as pro-inflammatory molecules. Prostaglandins are produced by the phospholipase A 2 / cyclooxygenase (COX) pathway, and leukotrienes are produced by the 5-lipoxygenese (LOX) pathway from AA [57]. PGE 2 is one of the most abundant prostaglandins, and is associated with inflammation [58,59]. ...
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Background/aims: The omega 6 fatty acid (FA) linoleic acid (LA) is required for embryonic development; however, omega 6 FAs can alter cellular metabolism via inflammation or modulation of mitochondrial function. Fetal LA is obtained from the maternal diet, and FAs are transported to the fetus via placental FA transporters (FATPs) and binding proteins (FABPs), but specific proteins responsible for LA transport in placental trophoblasts are unknown. Dietary LA consumption is increasing, but the effect of elevated LA on trophoblast function is not clear. Methods: Swan71 trophoblasts were exposed to physiological and supraphysiological concentrations of LA for 24 hours. Quantification of mRNA was determined using real time PCR, and protein concentration was determined by Western blot analysis. Cell viability, citrate synthase activity and mitochondrial respiration were determined. Results: Exposure to 300 and 500 μM LA increased FATP1 and FATP4 mRNA expression. 500 μM LA increased FATP1 and FATP4 protein expression. Exposure to 500 μM increased FABP5 mRNA expression, while exposure to 100 to 500 μM LA decreased FABP3 mRNA expression. 300 and 500 μM LA decreased FABP3 protein expression. Cell viability was decreased by exposure to LA (100 to 1000 μM). Citrate synthase activity and routine mitochondrial respiration were significantly decreased by exposure to 300 and 500 μM LA, and maximal respiration and spare respiratory capacity were decreased by exposure to 100 to 500 μM LA. 300 and 500 μM LA increased reactive oxygen species generation in human trophoblasts. Moreover, exposure to 300 and 500 μM LA decreased IL-6 secretion. Exposure to 500 μM LA increased IL-8, NF-κB and PPAR-γ mRNA expression, but decreased NF-κB protein expression. 300 μM LA decreased IL-8 protein expression. Further, exposure to 100 to 500 μM LA increased prostaglandin E2 and leukotriene B₄ release. Conclusion: Exposure to LA decreases cell viability, alters mRNA expression of FA transport related proteins, mitochondrial respiration and function, and inflammatory responses in trophoblasts. These findings may have implications on placental function when women consume high levels of LA.
... According to one of the inflammatory pathways, PLA 2 acts on the membrane phosopholipids and mediates the formation of acarhidonic acid which is further utilized by other enzymes such as cyclooxygenase and lipoxygenase, triggering the production of prostaglandins and leukotrines, respectively. Prostaglandins and leukotrines are also strong inflammatory mediators (9,10). Hence, inhibition of any of these enzymes is considered to be an efficient way to control inflammation (11)(12)(13). ...
... Based on our studies, interfering with the synthesis and/or function of the eicosanoid chemoattractant HXA 3 represents a potentially compelling mechanistic target for developing and exploring a novel class of pharmaceutical compounds with significant untapped therapeutic potential for alleviating PMN-mediated mucosal surface injury during lung disease. Eicosanoid subsets, including leukotrienes and PGs, have been exploited as therapeutic targets to alleviate overzealous inflammation (65). Multiple leukotriene inhibitors are available for treatment of allergies and asthma, including the 5-LO inhibitor zileuton (66). ...
Article
A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.
... Prostaglandins are a large, varied family of fatty acids, a number of which, such as prostaglandin E 2 , are early markers of inflammation. These prostaglandins are produced by activated macrophages during infection and increase symptoms of sepsis and sys-temic inflammation, such as vascular permeability and edema, as well as stimulating other immune cells (64). Prostaglandins are synthesized by cyclooxygenase (COX) enzymes, COX-1 and COX-2, which can be inhibited by pharmaceuticals in order to prevent the production of inflammatory prostaglandins. ...
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Inflammation is the body's first line of defence against infection or injury, responding to challenges by activating innate and adaptive responses. Microbes have evolved a diverse range of strategies to avoid triggering inflammatory responses. However, some pathogens such as the Influenza virus and the Gram negative bacterium Francisella tularensis, do trigger life-threatening cytokine storms in the host which can result in significant pathology and ultimately death. For these diseases, it has been proposed that down-regulating inflammatory immune responses may improve outcome. We review some of the current candidates for treatment of cytokine storms which may prove useful in the clinic in the future, and compare them to more traditional therapeutic candidates that target the pathogen rather than the host response.
... Thus, the overall reduction in LTB4 levels shown in Fig. 2 (lower panel) may be due to both complement inhibition and direct binding to LTB4. OmCI has the potential to reduce the migration of neutrophils, as LTB4 promotes neutrophil chemotaxis, increases adherence to capillary walls, and is a potent inducer of chemokinesis, neutrophil infiltration, and degranulation (55). Furthermore, this might have been reflected in the histopathological lung changes, with the presence of less swollen septa and fewer foci filled with inflammatory cells in the OmCI-treated group compared with positive control group. ...
Article
Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.
... This process was determined by Shibata et al. using an achiral-phase high-performance liquid chromatography method in 2002 [8]. Studies indicate that both PGD2 and 15d-PGJ2 levels increase in the later phases of COX2 activity, which mediates the resolution of inflammation, whereas early COX2 activity has been associated with a proinflammatory response [9,10]. After its discovery, the role of 15d-PGJ2 in moderating inflammation has been extensively studied. ...
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Objective. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) reduces inflammation and has been identified as an anti-inflammatory prostaglandin in numerous animal models. In this study, we investigated both effects of 15d-PGJ2 and its protection mechanism in concanavalin A- (ConA-) induced autoimmune hepatitis in mice. Materials and Methods. In vivo, Balb/C mice were injected with ConA (25 mg/kg) to induce acute autoimmune hepatitis, and 15d-PGJ2 (10 μg or 25 μg) was administered 1 h before the ConA injection. The histological grade, proinflammatory cytokine levels, and NF-κB and PPARγ activity were determined 6, 12, and 24 h after the ConA injection. In vitro, LO2 cells and RAW264.7 cells were pretreated with 15d-PGJ2 (2 μM) 1 h before the stimulation with ConA (30 μg/mL). The NF-κB and PPARγ activity were determined 30 min after the ConA administration. Results. Pretreatment with 15d-PGJ2 reduced the pathological effects of ConA-induced autoimmune hepatitis and significantly reduced the levels of cytokines after injection. 15d-PGJ2 activated PPARγ, blocked the degradation of IκBα, and inhibited the translocation of NF-κB into the nucleus. Conclusion. These results indicate that 15d-PGJ2 protects against ConA-induced autoimmune hepatitis by reducing proinflammatory cytokines. This reduction in inflammation may correlate with the activation of PPARγ and the reduction in NF-κB activity.
... Pain perception is also known as nociception, and is therefore an individual perception alert or sensation of pain (Tracey and Mantyh 2007). Pain is directly linked to inflammatory processes, and it is important to highlight that inflammatory processes can be associated with bacterial infections (Yoshikai 2001;Chiu et al. 2013). ...
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Bioactive phytocompounds are studied by several bioactivities demonstrated, as their cytotoxic effects. The aim of this work was to evaluate the phytochemical profile, the toxic effect using the Drosophila melanogaster animal model and the anti-inflammatory and antimicrobial effect of the Alternanthera brasiliana (EEAB) ethanol extract. The phytochemical profile was performed using HPLC. The cytotoxic effect was evaluated in vivo using D. melanogaster. The anti-inflammatory effect was determined by neurogenic and antiedematogenic assays, and the antimicrobial activity was assayed using a microdilution method to determine the minimum inhibitory concentration (MIC) of the EEAB alone and in association with antibiotics. The main compound identified on the EEAB was luteolin (1.93%). Its cytotoxic effect was demonstrated after 24 h in the concentrations of 10, 20 and 40 mg/mL. The extract demonstrated an antiedematogenic effect, with a reduction of the edema between 35.57 and 64.17%. The MIC of the extract was ≥1.024 μg/mL, thus being considered clinically irrelevant. However, when the EEAB was associated with gentamicin, a synergism against all bacterial strains assayed was observed: Staphylococcus aureus (SA10), Escherichia coli (EC06) and Pseudomonas aeruginosa (PA24). Due to these results, the EEAB demonstrated a low toxicity in vivo and anti-inflammatory and synergistic activities. These are promising results, mainly against microbial pathogens, and the compounds identified can be a source of carbon backbones for the discovery and creation of new drugs.
... arachidonic acid through cyclooxygenase pathway. The arachidonic acid oxygenation products mediate diverse effects that induce acute inflammation caused by bacterial infection [16]. The present work designed to synthesize antibacterial compounds which possess anti-inflammatory potential additionally. ...
... Leukotrienes are important multifunctional mediators of inflammation and promote neutrophil chemotaxis and adherence to capillary walls [48]. LTB4 is expressed on leucocytes after myocardial IRI [36], gets elevated in plasma in the course of myocardial infarction [42] and has been shown to be able to discriminate between cardiac and non-cardiac chest pain [26]. ...
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Inhibition of complement factor 5 (C5) reduced myocardial infarction in animal studies, while no benefit was found in clinical studies. Due to lack of cross-reactivity of clinically used C5 antibodies, different inhibitors were used in animal and clinical studies. Coversin (Ornithodoros moubata complement inhibitor, OmCI) blocks C5 cleavage and binds leukotriene B4 in humans and pigs. We hypothesized that inhibition of C5 before reperfusion will decrease infarct size and improve ventricular function in a porcine model of myocardial infarction. In pigs (Sus scrofa), the left anterior descending coronary artery was occluded (40 min) and reperfused (240 min). Coversin or placebo was infused 20 min after occlusion and throughout reperfusion in 16 blindly randomized pigs. Coversin significantly reduced myocardial infarction in the area at risk by 39% (p = 0.03, triphenyl tetrazolium chloride staining) and by 19% (p = 0.02) using magnetic resonance imaging. The methods correlated significantly (R = 0.92, p < 0.01). Tissue Doppler echocardiography showed increased systolic displacement (31%, p < 0.01) and increased systolic velocity (29%, p = 0.01) in coversin treated pigs. Interleukin-1β in myocardial microdialysis fluid was significantly reduced (31%, p < 0.05) and tissue E-selectin expression was significantly reduced (p = 0.01) in the non-infarcted area at risk by coversin treatment. Coversin ablated plasma C5 activation throughout the reperfusion period and decreased myocardial C5b-9 deposition, while neither plasma nor myocardial LTB4 were significantly reduced. Coversin substantially reduced the size of infarction, improved ventricular function, and attenuated interleukin-1β and E-selectin in this porcine model by inhibiting C5. We conclude that inhibition of C5 in myocardial infarction should be reconsidered.
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Unlabelled: Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome of S. aureus USA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H₁₂₁Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genes farR and farE, for regulator and effector of fatty acid resistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression of farE was strongly induced by arachidonic and linoleic acids in an farR-dependent manner. Second, an H₁₂₁Y substitution in FarR resulted in increased expression of farE and was alone sufficient to promote increased resistance of S. aureus to linoleic acid. Third, inactivation of farE resulted in a significant reduction in the inducible resistance of S. aureus to the bactericidal activity of 100 μM linoleic acid, increased accumulation of [(14)C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen. Importance: Staphylococcus aureus colonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts, S. aureus must have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of human innate defense mechanisms. We have identified a novel pair of genes, farR and farE, that constitute a dedicated regulator and effector of S. aureus resistance to linoleic and arachidonic acids, which are major fatty acids in human membrane phospholipid. Expression of farE, which encodes an efflux pump, is induced in an farR-dependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess.
Article
Oxylipins comprise a family of oxygenated fatty acid-derived signaling molecules that initiate critical biological activities in animals, plants, and fungi. Mammalian oxylipins, including the prostaglandins (PGs), mediate many immune and inflammation responses in animals. PG production by pathogenic microbes is theorized to play a role in pathogenesis. We have genetically characterized three Aspergillus genes, ppoA, ppoB, and ppoC, encoding fatty acid oxygenases similar in sequence to specific mammalian prostaglandin synthases, the cyclooxygenases. Enzyme-linked immunosorbent assay analysis showed that production of PG species is decreased in both Aspergillus nidulans and A. fumigatus ppo mutants, implicating Ppo activity in generating PGs. The A. fumigatus triple-ppo-silenced mutant was hypervirulent in the invasive pulmonary aspergillosis murine model system and showed increased tolerance to H2O2 stress relative to that of the wild type. We propose that Ppo products, PG, and/or other oxylipins may serve as activators of mammalian immune responses contributing to enhanced resistance to opportunistic fungi and as factors that modulate fungal development contributing to resistance to host defenses.
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Die Phospholipid-Hydroperoxid Glutathion-Peroxidase (phGPx) ist ein monomeres Selenoprotein, welches innerhalb der Familie der Glutathion-Peroxidasen aufgrund seiner breiten Substratspezifität und der Fähigkeit Proteinthiole zu modifizieren eine Sonderstellung einnimmt. Vom Gen der phGPx werden nach heutigem Kenntnisstand drei verschiedene Protein-Isoformen gebildet. Die mitochondriale Isoform enthält am N-Terminus ein mitochondriales Insertionssignal und wird bevorzugt im Testis exprimiert. Von einem im Leserahmen stromabwärts liegenden Startkodon wird die kürzere, ubiquitär exprimierte zytosolische Isoform synthetisiert. Eine dritte phGPx-Isoform besitzt eine N-terminale nukleäre Lokalisationssequenz (kodiert von einem alternativen Exon 1) und wird vornehmlich in den Kernen post-meiotischer Zellen der Spermatogenese gefunden. Aufgabe dieser Arbeit war es, die molekularen Mechanismen zu untersuchen, die am Zustandekommen des vielfältigen Expressionsmusters der phGPx-Isoformen beteiligt sind. Im ersten Teil der Arbeit wurden transkriptionelle Regulationsmechanismen der phGPx-Expression untersucht. Im proximalen Promotorbereich (-100 bp – +228 bp) des phGPx-Gens wurden unter in vitro (Supershift-Assay) und in vivo (Chromatin-Immunopräzipitation) Bedingungen die Transkriptionsfaktoren Sp1 und NF-Y identifiziert, die an drei GC-reiche Motive beziehungsweise zwei inverse CCAAT-Boxen binden. Darüber hinaus konnten in kompetetiven Gelshift-Assays im proximalen Promotorbereich zwei Bindungssequenzen identifiziert werden, die von Faktoren der Smad-Familie gebunden werden. Funktionelle in vitro Promotorstudien mit mutierten Promotorkonstrukten zeigten, dass die Mutagenesen der Sp1- und NF-Y Bindestellen einen starken Einfluss auf die Reportergenaktivität hatten. Im zweiten Teil der Arbeit wurden durch Untersuchungen von Protein-RNA-Interaktionen post-transkriptionelle Mechanismen der Expressionsregulation studiert. Mit Hilfe des in vivo Ansatzes des Hefe Drei-Hybrid Systems wurde der Guanin-reiche Sequenz bindende Faktor 1 (GRSF1) identifiziert, der in der 5’-untranslatierte Region der mitochondrialen phGPx-mRNA bindet. In RNA Gelshift-Assays wurde die Spezifität dieser Interaktion bestätigt und näher charakterisiert. Schließlich wurden für GRSF1 und die phGPx Expressionsprofile in murinen Gewebe erstellt sowie die zeitabhängige Expression beider Proteine während der Embryogenese verfolgt. Die auffällig ähnlichen Expressionsmuster lassen ähnliche Regulationsmechanismen vermuten. Die in dieser Arbeit identifizierten trans-regulatorischen Proteine Sp1, NF-Y, Smad und GRSF1 sollten an der differentiellen Expression der phGPx-Isoformen beteiligt sein. The Phospholipid Hydroperoxide Glutathione Peroxidase (phGPx) is a monomeric selenoprotein that is unique in the family of Glutathione Peroxidases due to its low substrate specificity and its ability to oxidise protein thiols. Three different isoforms are known to derive from one common gene. The mitochondrial Isoform contains an N-terminal mitochondrial insertion sequence and is preferentially expressed in postpubertal testis. The shorter, ubiquitously expressed, cytosolic isoform is expressed from an in-frame start codon. A third isoform contains an N-terminal nuclear localization signal coded for by an alternative exon 1 and is preferentially expressed in the nuclei of post-meiotic spermatides. The aim of the present study is to investigate the molecular mechanisms leading to the different isoforms and causing their tissue specific expression pattern. In the first part of this work transcriptional regulatory mechanisms will be analysed. Within the proximal promoter region (-100 to +228 bp) of the phGPx gene the transcription factors SP1 and NF-Y were identified to bind to three GC-boxes and two CCAAT-boxes respectively using in vitro methods (Supershift Assays) and in vivo methods (Chromatine immunoprecipitation). Moreover, performing competitive gel shift assays two binding elements for the smad family of transcription factors could be identified. Functional in vitro reporter gene assays provided evidence that the mutagenesis of the binding sequences for NF-Y and Sp1 has a strong impact on promoter activity. In the second part of this work post-transcriptional events in the expression regulation of the phGPx were analysed on the basis of protein/RNA interactions. Applying the in vivo approach of the yeast three hybrid system the Guanin-riche sequence binding factor 1 (GRSF1) could be identified binding to the 5’-untranslated region of the mitochondrial phGPx messenger. RNA mobility shift assays were performed to further characterize the specificity of this protein/RNA interaction. Eventually, the tissue distribution of GRSF1 and phGPx was studied in murine tissues and their expression kinetics were followed during murine embryogenesis. The obvious parallel expression kinetics for mitochondrial phGPx and GRSF1 suggest common regulatory mechanisms for these two genes. All the identified trans-regulatory elements are very likely to be involved in the differential expression regulation of the phGPx isoforms.
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"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maître ès sciences (M. Sc.) en sciences pharmaceutiques option pharmacologie." Thèse (M. Sc.)--Université de Montréal, 2004.
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"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maître ès sciences (M.Sc.) en sciences pharmaceutiques option pharmacologie" Thèse (M. Sc.)--Université de Montréal, 2004. Microfilm du manuscrit.
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Cyclooxygenase (COX), first purified in 1976 and cloned in 1988, is the key enzyme in the synthesis of prostaglandins (PGs) from arachidonic acid. In 1991, several laboratories identified a product from a second gene with COX activity and called it COX-2. However, COX-2 was inducible, and the inducing stimuli included pro-inflammatory cytokines and growth factors, implying a role for COX-2 in both inflammation and control of cell growth. The two isoforms of COX are almost identical in structure but have important differences in substrate and inhibitor selectivity and in their intracellular locations. Protective PGs, which preserve the integrity of the stomach lining and maintain normal renal function in a compromised kidney, are synthesized by COX-1. In addition to the induction of COX-2 in inflammatory lesions, it is present constitutively in the brain and spinal cord, where it may be involved in nerve transmission, particularly that for pain and fever. PGs made by COX-2 are also important in ovulation and in the birth process. The discovery of COX-2 has made possible the design of drugs that reduce inflammation without removing the protective PGs in the stomach and kidney made by COX-1. These highly selective COX-2 inhibitors may not only be anti-inflammatory but may also be active in colon cancer and Alzheimer's disease.
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The influence of prostaglandins on glial functions and, more specifically, on glial activation is not well understood. We report here that prostaglandin E(2) (PGE(2)), one of the major prostaglandins produced in the brain, acts as a potent and selective inhibitor of tumor necrosis factor alpha (TNF-alpha) production in lipopolysaccharide-stimulated primary microglia and the microglial cell line BV-2. The IC(50) for this effect is 1 nM, and 100 nM PGE(2) suppresses TNF-alpha production by >95%. More detailed studies of BV-2 cells show that PGE(2) also prevents the secretion of interleukin (IL)-6 but does not significantly modify lipopolysaccharide-stimulated expression of cyclooxygenase-2, pro-IL-1beta, or inducible nitric oxide synthase. PGE(2) appears to act primarily at the level of translation or protein stability, because TNF-alpha and IL-6 mRNA levels were only modestly decreased at high PGE(2) concentrations; concomitantly with this inhibition, PGE(2) up-regulated the levels of IL-1beta mRNA. The effects of PGE(2) could be largely mimicked by 8-bromo-cAMP, suggesting that, as in other cell types, PGE(2) action is mediated at least in part by a rise in intracellular cyclic AMP. However, the protein kinase A inhibitor H89 only partially reversed the inhibition of TNF-alpha production by PGE(2), implying that the PGE(2) effect in BV-2 cells is mediated through both protein kinase A-dependent and -independent pathways.
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Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet-activating factor (PAFR), and leukotriene B(4) (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a Galpha(i) inhibitor. These receptors were chosen to represent the spectrum of G(i) usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B(4), and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3-kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G(i), whereas BLTR and PAFR utilize both G(i) and a Ptx-insensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G(i)-mediated pathway.
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To investigate roles in intestinal inflammation for the 2 cyclooxygenase (COX) isoforms, we determined susceptibility to spontaneous and induced acute colitis in mice lacking either the COX-1 or COX-2 isoform. We treated wild-type, COX-1(-/-), COX-2(-/-), and heterozygous mice with dextran sodium sulfate (DSS) to provoke acute colonic inflammation, and we quantified tissue damage, prostaglandin (PG) E(2), and interleukin-1beta. No spontaneous gastrointestinal inflammation was detected in mice homozygous for either mutation, despite almost undetectable basal intestinal PGE(2) production in COX-1(-/-) mice. Both COX-1(-/-) and COX-2(-/-) mice showed increased susceptibility to a low-dose of DSS that caused mild colonic epithelial injury in wild-type mice. COX-2(-/-) mice were more susceptible than COX-1(-/-) mice, and selective pharmacologic blockade of COX-2 potentiated injury in COX-1(-/-) mice. At a high dose, DSS treatment was fatal to 50% of the animals in each mutant group, but all wild-type mice survived. DSS treatment increased PGE(2) intestinal secretion in all groups except COX-2(-/-) mice. These results demonstrate that COX-1 and COX-2 share a crucial role in the defense of the intestinal mucosa (with inducible COX-2 being perhaps more active during inflammation) and that neither isoform is essential in maintaining mucosal homeostasis in the absence of injurious stimuli.
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The contractile and inflammatory actions of the cysteinyl leukotrienes (CysLTs), LTC(4), LTD(4), and LTE(4), are thought to be mediated through at least two distinct but related CysLT G protein-coupled receptors. The human CysLT(1) receptor has been recently cloned and characterized. We describe here the cloning and characterization of the second cysteinyl leukotriene receptor, CysLT(2), a 346-amino acid protein with 38% amino acid identity to the CysLT(1) receptor. The recombinant human CysLT(2) receptor was expressed in Xenopus oocytes and HEK293T cells and shown to couple to elevation of intracellular calcium when activated by LTC(4), LTD(4), or LTE(4). Analyses of radiolabeled LTD(4) binding to the recombinant CysLT(2) receptor demonstrated high affinity binding and a rank order of potency for competition of LTC(4) = LTD(4) LTE(4). In contrast to the dual CysLT(1)/CysLT(2) antagonist, BAY u9773, the CysLT(1) receptor-selective antagonists MK-571, montelukast (Singulair(TM)), zafirlukast (Accolate(TM)), and pranlukast (Onon(TM)) exhibited low potency in competition for LTD(4) binding and as antagonists of CysLT(2) receptor signaling. CysLT(2) receptor mRNA was detected in lung macrophages and airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, and brain, and the receptor gene was mapped to chromosome 13q14, a region linked to atopic asthma.
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Leukotrienes are derived from arachidonic acid and serve as mediators of inflammation and immediate hypersensitivity. Leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) act through G protein-coupled receptors LTB(4) receptor (BLTR) and Cys-LTR, respectively. To investigate the physiological role of BLTR, we produced mice with a targeted disruption of the BLTR gene. Mice deficient for BLTR (BLTR(-/-)) developed normally and had no apparent hematopoietic abnormalities. Peritoneal neutrophils from BLTR(-/-) mice displayed normal responses to the inflammatory mediators C5a and platelet-activating factor (PAF) but did not respond to LTB(4) for calcium mobilization or chemotaxis. Additionally, LTB(4) elicited peritoneal neutrophil influx in control but not in BLTR(-/-) mice. Thus, BLTR is the sole receptor for LTB(4)-induced inflammation in mice. Neutrophil influx in a peritonitis model and acute ear inflammation in response to arachidonic acid was significantly reduced in BLTR(-/-) mice. In mice, intravenous administration of PAF induces immediate lethal anaphylaxis. Surprisingly, female BLTR(-/-) mice displayed selective survival (6 of 9; P = 0.002) relative to male (1 of 11) mice of PAF-induced anaphylaxis. These results demonstrate the role of BLTR in leukotriene-mediated acute inflammation and an unexpected sex-related involvement in PAF-induced anaphylaxis.
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Leukotriene B(4) (LTB(4)) is a potent chemoattractant and activator of both granulocytes and macrophages. The actions of LTB(4) appear to be mediated by a specific G protein-coupled receptor (GPCR) BLT1, originally termed BLT (Yokomizo, T., T. Izumi, K. Chang, Y. Takuwa, and T. Shimizu. 1997. Nature. 387:620-624). Here, we report the molecular cloning of a novel GPCR for LTB(4), designated BLT2, which binds LTB(4) with a Kd value of 23 nM compared with 1.1 nM for BLT1, but still efficiently transduces intracellular signaling. BLT2 is highly homologous to BLT1, with an amino acid identity of 45.2%, and its open reading frame is located in the promoter region of the BLT1 gene. BLT2 is expressed ubiquitously, in contrast to BLT1, which is expressed predominantly in leukocytes. Chinese hamster ovary cells expressing BLT2 exhibit LTB(4)-induced chemotaxis, calcium mobilization, and pertussis toxin-insensitive inhibition of adenylyl cyclase. Several BLT1 antagonists, including U 75302, failed to inhibit LTB(4) binding to BLT2. Thus, BLT2 is a pharmacologically distinct receptor for LTB(4), and may mediate cellular functions in tissues other than leukocytes. BLT2 provides a novel target for antiinflammatory therapy and promises to expand our knowledge of LTB(4) function. The location of the gene suggests shared transcriptional regulation of these two receptors.
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Leukotriene B4 (LTB4) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB4 exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB4 in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB4 elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B4 receptor has been recently identified (LTB4-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB4-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB4-R1. Expression of LTB4-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB4-R2 cDNA displayed high affinity (K d = 0.17 nm) for [3H]LTB4. Radioligand competition assays revealed high affinities of the receptor for LTB4 and LTB5, and 20-hydroxy-LTB4, and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB4 receptor antagonists, 14,15-dehydro-LTB4, LTB4-3-aminopropylamide, and U-75302, had high affinity for LTB4-R1 but not for LTB4-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB4 functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB4 in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.
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Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.
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Leukotrienes are potent inflammatory mediators synthesized from arachidonic acid (AA) predominately by cells of myeloid origin. The synthesis of these lipids is believed to be dependent not only on the expression of the enzyme 5-lipoxygenase (5-LO), which catalyzes the first steps in the synthesis of leukotrienes, but also on expression of a nuclear membrane protein termed the 5-LO–activating protein (FLAP). To study the relationship of these two proteins in mediating the production of leukotrienes in vivo and to determine whether the membrane protein FLAP has additional functions in various inflammatory processes, we have generated a mouse line deficient in this protein. FLAP-deficient mice develop normally and are healthy. However, an array of assays comparing inflammatory reactions in FLAP-deficient mice and in normal controls revealed that FLAP plays a role in a subset of these reactions. Although examination of DTH and IgE-mediated passive anaphylaxis showed no difference between wild-type and FLAP-deficient animals, mice without FLAP possessed a blunted inflammatory response to topical AA and had increased resistance to platelet-activating factor–induced shock compared to controls. Also, edema associated with Zymosan A–induced peritonitis was markedly reduced in animals lacking FLAP. To determine whether these differences relate solely to a deficit in leukotriene production, or whether they reflect an additional role for FLAP in inflammation, we compared the FLAP-deficient mice to 5-LO–deficient animals. Evaluation of mice lacking FLAP and 5-LO indicated that production of leukotrienes during inflammatory responses is dependent upon the availability of FLAP and did not support additional functions for FLAP beyond its role in leukotriene production.
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Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.
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Arachidonic acid is released from membrane phospholipids upon cell stimulation (for example, by immune complexes and calcium ionophores) and converted to leukotrienes by a 5-lipoxygenase that also has leukotriene A4 synthetase activity. Leukotriene A4, an unstable epoxide, is hydrolyzed to leukotriene B4 or conjugated with glutathione to yield leukotriene C4 and its metabolites, leukotriene D4 and leukotriene E4. The leukotrienes participate in host defense reactions and pathophysiological conditions such as immediate hypersensitivity and inflammation. Recent studies also suggest a neuroendocrine role for leukotriene C4 in luteinizing hormone secretion. Lipoxins are formed by the action of 5- and 15-lipoxygenases on arachidonic acid. Lipoxin A causes contraction of guinea pig lung strips and dilation of the microvasculature. Both lipoxin A and B inhibit natural killer cell cytotoxicity. Thus, the multiple interaction of lipoxygenases generates compounds that can regulate specific cellular responses of importance in inflammation and immunity.
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Prostaglandins are formed from arachidonic acid by the action of cyclooxygenase (COX) and subsequent downstream synthetases. Recently, it has been found that there are two closely related forms of COX, which are now known as COX-1 and COX-2. Although both isoforms of this enzyme convert arachidonate to prostaglandins, there are significant differences in their distribution in the body and their roles in health and disease. The basis for these important differences lies in the genes for COX-1 and COX-2 and the regulation of these genes.
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Prostaglandin E series (PGEs) are known to protect against lipopolysaccharide (LPS)-induced liver injury by down-regulating the production of inflammatory cytokines. We show here a novel mechanism whereby prostaglandin E(1) protects mice against liver injury after Escherichia coli infection. Prostaglandin E(1) administration suppressed circulating interleukin 12 (IL-12) levels but increased the IL-10 production after E. coli challenge. Furthermore, prostaglandin E(1)-alpha-cyclodextrin (PGE(1)) shifted the Th1/Th2 balance of CD3(intermediate) IL-2Rbeta(+) T cells in the liver to a dominant Th2-like response. Neutralization of endogenous IL-4 by administration of anti-IL-4 monoclonal antibody (mAb) diminished the inhibitory effect of prostaglandin E(1) on liver injury after E. coli challenge. These results suggested that the Th2-like response of liver T cells may be at least partly involved in the mechanism whereby prostaglandin E(1) protects against E. coli-induced liver injury.
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T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARgamma mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPARgamma involvement in the regulation of T cell function. Here we show that PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14) prostaglandin J(2), but not PPARalpha agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPARgamma2-expressing, not the PPARgamma-lacking, subpopulation of transfected Jurkat cells. The activated PPARgamma physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPARgamma in T lymphocytes and could suggest a previously unrecognized clinical potential for PPARgamma ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.
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Prostaglandin J(2) (PGJ(2)) and its metabolites Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are naturally occurring derivatives of prostaglandin D(2) that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ(2) is a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor alpha, in a PPARgamma-dependent manner. We report here that 15d-PGJ(2) potently inhibits NF-kappaB-dependent transcription by two additional PPARgamma-independent mechanisms. Several lines of evidence suggest that 15d-PGJ(2) directly inhibits NF-kappaB-dependent gene expression through covalent modifications of critical cysteine residues in IkappaB kinase and the DNA-binding domains of NF-kappaB subunits. These mechanisms act in combination to inhibit transactivation of the NF-kappaB target gene cyclooxygenase 2. Direct inhibition of NF-kappaB signaling by 15d-PGJ(2) may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.
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Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.