Leukotriene Production Research Articles

5‐Lipoxygenase and anandamide hydrolase (FAAH) mediate the antitumor activity of cannabidiol, a non‐psychoactive cannabinoid

It has been recently reported that cannabidiol (CBD), a non-psychoactive cannabinoid, is able to kill glioma cells, both in vivo and in vitro, independently of cannabinoid receptor stimulation. However, the underlying biochemical mechanisms were not clarified. In the present study, we performed biochemical analysis of the effect of CBD both in vivo, by using glioma tumor tissues excised from nude mice, and in vitro, by using U87 glioma cells. In vivo exposure of tumor tissues to CBD significantly decreased the activity and content of 5-lipoxygenase (LOX, by approximately 40%), and of its end product leukotriene B4 ( approximately 25%). In contrast cyclooxygenase (COX)-2 activity and content, and the amount of its end product prostaglandin E2, were not affected by CBD. In addition, in vivo treatment with CBD markedly stimulated ( approximately 175%) the activity of fatty acid amide hydrolase (FAAH), the main anandamide-degrading enzyme, while decreasing anandamide content ( approximately 30%) and binding to CB1 cannabinoid receptors ( approximately 25%). In vitro pre-treatment of U87 glioma cells with MK-886, a specific 5-LOX inhibitor, significantly enhanced the antimitotic effect of CBD, whereas the pre-treatment with indomethacin (pan-COX inhibitor) or celecoxib (COX-2 inhibitor), did not alter CBD effect. The study of the endocannabinoid system revealed that CBD was able to induce a concentration-dependent increase of FAAH activity in U87 cells. Moreover, a significantly reduced growth rate was observed in FAAH-over-expressing U87 cells, compared to wild-type controls. In conclusion, the present investigation indicates that CBD exerts its antitumoral effects through modulation of the LOX pathway and of the endocannabinoid system, suggesting a possible interaction of these routes in the control of tumor growth.

Inflammatory responses after endothelin B (ET B) receptor activation in human monocytes: New evidence for beneficial anti-inflammatory potency of ET B-receptor antagonism

Endothelin (ET) stimulates potent ET A/ET B receptors important in the pathogenesis of pulmonary arterial hypertension (PAH) and fibrosis. Though therapy with ET-receptor antagonists is well established uncertainty exists whether selective ET A or dual ET A/ET B-receptor antagonism is superior in PAH. The objective of this study was to further elucidate the pro-inflammatory effects of ET-1 on ET B receptors in cultured human monocytes (10 5/20 h) compared with non-specific stimulation with LPS in vitro and to define the antagonizing effects of bosentan, a dual ET A/ET B-receptor antagonist, on inflammatory mediator production. We further hypothesized that ET B-receptor antagonism reduces the requirement of PGE 2 to control inflammatory mediator production. Activation of the monocyte ET B subtype by ET (1 ng/ml) concentration-dependently stimulated TNF- α (744%) >PGE 2 (570%) > IL-1 β (112%) and had no effect on 5-lipoxygenase metabolism. Compared with ET a different profile of IL-1 β >TNF- α >PGE 2 was induced by LPS. ET B-receptor antagonism attenuated ET- and LPS-responses in monocytes, in particular of TNF- α and PGE 2 to a similar extend (40%) that were only demonstrable following LPS at therapeutic plasma concentrations of bosentan and had no effect on IL-1 β. Inhibition of ET B receptors in LPS-stimulated monocytes by bosentan was responded with suppression of PGE 2 and increased production of leukotrienes indicating strong effects in the cyclooxygenase pathway that is known to control cellular ET transcription. These data suggest an important signaling pathway between ET-induced cytokine production following ET B-receptor activation with no further control of ET transcription by PGE 2 required following ET B receptor antagonism. Therefore, in states of inflammation increased ET B-receptor expression and activation mediated by elevated ET concentrations may be an underestimated mechanism, which warrants the application of combined ET A/ET B-receptor antagonists.

Human CMV infection induces 5-lipoxygenase expression and leukotriene B4 production in vascular smooth muscle cells

Leukotrienes (LTs) are powerful proinflammatory lipid mediators that may play a central role in cardiovascular diseases, including arteriosclerosis, myocardial infarction, and stroke. Owing to restricted expression of 5-lipoxygenase (5-LO), the enzyme required for their synthesis, LTs are almost exclusively produced by myeloid cells. Here, we report that human cytomegalovirus (HCMV) infection of human vascular smooth muscle cells (SMCs) increases 5-LO mRNA levels by up to 170-fold in a dose- and time-dependent manner. Infected cells expressed 5-LO protein, as shown by immunohistochemistry, enabling them to synthesize bioactive LTB4. HCMV-infected vascular SMCs expressing 5-LO protein were readily detected in tissue samples from CMV-infected patients with inflammatory bowel disease or AIDS. Thus, pathogen-induced LT production in HCMV-infected tissues may contribute to local inflammation, consistent with the ability of HCMV to control cellular and immunological functions. HCMV-induced LT biosynthesis in SMCs offers a molecular mechanism to explain HCMV-induced pathogenesis in inflammatory diseases.

Expression of Enzymes Involved in the Prostanoid Metabolism by Cortical Astrocytes after LPS-induced Inflammation

Neuroinflammatory processes are a common epiphenomenon for a number of neurological and neurodegenerative diseases. Besides microglia, astrocytes are implicated in brain inflammation in response to harmful stimuli and pathological processes. Bacterial endotoxins can induce the synthesis and release of proinflammatory mediators, i.e., cytokines and chemokines, by astroglia. In this study, we have investigated the effect of lipopolysaccharide (LPS) treatment on the expression of enzymes of prostanoid synthesis and degradation in cultured mouse cortical astrocytes using an Affymetrix Gene Chip array, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and an enzyme-immunosorbent assay. LPS treatment induced an upregulation of enzymes responsible for prostaglandin E2 synthesis, a downregulation of enzymes that catalyzes prostaglandin E2 (PGE2) degradation and production of proinflammatory leukotrienes. Changes in enzyme expression were accompanied by a highly significant increase in extracellular PGE2. Our data demonstrate that astrocytes are directly involved in the complex regulation of proinflammatory prostanoids in the CNS under pathological processes, thus being of potential interest as targets for therapeutical interventions. Further studies are required to unravel the different roles and interactions between astroglia and other cells of the brain-intrinsic innate immune system during inflammation.

Anti-inflammatory Activity of Pectolinarigenin and Pectolinarin Isolated from Cirsium chanroenicum

In order to identify the active anti-inflammatory ingredient(s) in Cirsium chanroenicum (Compositae), its methanol extract and several solvent fractions were prepared; the methanol extract and the ethylacetate fraction inhibited cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE2) and 5-lipoxygenase (5-LOX)-mediated leukotriene (LT) production in lipopolysaccharide-treated RAW 264.7 cells and A23187-treated rat basophilic leukemia (RBL-1) cells, respectively. Further bioactivity-guided fractionation of the ethylacetate fraction using column chromatography led to the isolation of pectolinarigenin (5,7-dihydroxy-4',6-dimethoxyflavone), along with pectolinarin [pectolinarigenin 7-rhamnosyl-(1-->6)-glucoside]. Pectolinarigenin strongly inhibited COX-2-mediated PGE2 and 5-LOX-mediated LT production at >1 microM, indicating that it is a dual inhibitor of COX-2/5-LOX. However, pectolinarigenin did not affect COX-2 expression or nuclear transcription factor (NF-kappaB) activation. In addition, in vivo studies demonstrated that oral administration of these two compounds at 20-100 mg/kg resulted in similar inhibitory activities against several animal models of inflammation/allergy: arachidonic acid-induced mouse ear edema, carrageenan-induced mouse paw edema and passive cutaneous anaphylaxis. All of these results suggest that pectolinarigenin and pectolinarin possess anti-inflammatory activity and that they may inhibit eicosanoid formation in inflammatory lesions. These activities certainly contribute to the anti-inflammatory mechanism of C. chanroenicum.

Nigella sativa seeds: Folklore treatment in modern day medicine

<i>Nigella sativa</i> seeds: Folklore treatment in modern day medicine

The Role of PPARgamma Receptors and Leukotriene B(4) Receptors in Mediating the Effects of LY293111 in Pancreatic Cancer.

Pancreatic cancer is a devastating disease in which current therapies are inadequate. Separate lines of research have identified the 5-lipoxygenase/leukotriene B4 receptor pathway and the PPARγ pathway as potential targets for prevention or treatment of this disease. LY293111 was originally designed as a potent leukotriene B4 receptor antagonist for treatment of inflammatory conditions. LY293111 was also known to have inhibitory effects on 5-lipoxygenase, which is upstream of the production of leukotrienes. LY293111 was shown to have potent anticancer effects in pancreatic cancer and several other solid malignancies, where it caused cell cycle arrest and marked apoptosis. Subsequently, it came to light that LY293111 exhibited PPARγ agonist activity in addition to its effects on the 5-lipoxygenase pathway. This raises the question of which of the two targets is of greatest importance with regard to the anticancer effects of this agent. The evidence to date is not conclusive, but suggests that the effects of LY293111 may be mediated by both LTB4 receptors and PPARγ.

Development of Synthetic Modulators of PPARs: Current Challenges and Future Opportunities

The cloning of the mouse PPAR alpha gene in 1990 by Issemann and Green [1] stimulated intense interest in this family of nuclear receptors, and research efforts over the next decade established important roles for the PPAR isotypes in glucose and lipoprotein metabolism, inflammation, and atherosclerosis. Though the fibrates (PPAR-α agonists) had been used for the treatment of dyslipidemia for nearly 40 years, the discovery of the insulin-sensitizing effects of PPAR-γ agonists brought about the development and commercialization of the thiazolidinedione (TZD) class of oral anti-hyperglycemic medications: troglitazone, approved by the FDA in 1997, and pioglitazone and rosiglitazone, both approved by the FDA in 1999. The withdrawal of troglitazone from the market in 2000 was an early indicator of the potential safety issues of PPAR drugs. Even so, the clinical use of fibrates and TZDs has allowed for a better understanding of the safety profiles and safety issues of PPAR-α and PPAR-γ agonists [2, 3]. At the beginning of this decade, many pharmaceutical companies had development programs focused on delivering “new and improved” PPAR agonists to the market. For example, several programs were developing PPAR-α/γ dual agonists (i.e., glitazars) for the treatment of type 2 diabetes. Although preliminary data from various PPAR-α/γ dual agonist research programs was promising, nearly all of these research programs were discontinued due to safety issues identified during clinical testing and/or during preclinical testing [4–6]. More recently, the highly publicized (and controversial) meta-analysis of rosiglitazone reported by Nissen and Wolski [7] called into question the safety of the TZDs and prompted changes to the labels of both rosiglitazone and pioglitazone. And yet, the promise of the therapeutic potential of PPAR drugs remains. A PubMed search using the term “peroxisome proliferator-activated receptor” yielded 1578 manuscripts (161 of which were review articles) between January 1 and December 1, 2008. In addition to the roles of the PPAR isotypes in lipid/lipoprotein/glucose metabolism, additional roles in diverse physiological processes and disease states are currently being investigated. As additional functions are identified; the PPARs will continue to be important molecular targets for identifying ligands (drugs) with potential applications to reproduction and fertility, normal development; function of the reproductive, gastrointestinal, respiratory, and central nervous systems; skin biology and wound healing; and cell cycle control and cancer. Reports highlighting both challenges and opportunities in PPAR drug development are included in this special issue. For example, edema, weight gain, and a reduction in bone mass (particularly in women) are challenges limiting the clinical utility of the currently marketed TZDs. The renal and vascular mechanisms of TZD-induced fluid retention are reviewed by Yang and Soodvilai, and the recent clinical data describing the effects of TZDs on bone are reviewed by Schwartz. In contrast, several reports highlight opportunities in PPAR drug development. Deeg and Tan compare the effects of rosiglitazone and pioglitazone on lipids, lipoproteins, and apolipoproteins as reported in head-to-head, randomized clinical studies. Two papers describe the effects of PPARs/PPAR ligands on immune/inflammatory responses; Fernandez reviews the roles of the PPARs in modulating the immune/inflammatory response in atherosclerosis, while Yamashita reviews the receptor-independent effects of PPAR-α and PPAR-γ ligands on cysteinyl leukotriene production in mast cells as it relates to the development of potential anti-asthma medications. Technological and methodological approaches that may prove useful in the identification and assessment of new PPAR drugs are also reported. Clarke et al. describe an approach used to determine the species differences in plasma protein binding to MBX-102, a novel PPAR-γ agonist currently in Phase 2 clinical development, and the corresponding differences in PPAR-γ activation across species. Cho et al. review the role of PPARs in metabolic disorders as well as various strategies and technologies used in the identification and assessment of PPAR drugs. Finally, Miyachi and Hashimoto describe the synthesis and SAR of subtype-specific PPAR agonists derived from a single 3,4-disubstituted phenylpropanoic acid “versatile template” scaffold, and Higgins and Mantzoros review the development and safety profile of INT-131, a potent non-TZD selective PPAR modulator (SPPARM) currently in Phase 2 clinical development. In conclusion, though the full therapeutic potential of PPARs has yet to be realized, and serious safety issues are associated with the currently marketed PPAR drugs (PPAR-α and PPAR-γ), there remains intense interest in exploring new physiological roles of the PPARs and in the identification of new and improved PPAR agonists drugs. Jane A. Pinaire Anne Reifel Miller Francine M. Gregoire

PPARα/γ‐Independent Effects of PPARα/γ Ligands on Cysteinyl Leukotriene Production in Mast Cells

Peroxisome proliferator-activated receptor (PPAR) α ligands (Wy-14,643, and fenofibrate) and PPARγ ligands (troglitazone and ciglitazone) inhibit antigen-induced cysteinyl leukotriene production in immunoglobulin E-treated mast cells. The inhibitory effect of these ligands on cysteinyl leukotriene production is quite strong and is almost equivalent to that of the anti-asthma compound zileuton. To develop new aspects for anti-asthma drugs the pharmacological target of these compounds should be clarified. Experiments with bone-marrow-derived mast cells from PPARα knockout mice and pharmacological inhibitors of PPARγ suggest that the inhibitory effects of these ligands are independent of PPARs α and γ. The mechanisms of the PPAR-independent inhibition by these agents on cysteinyl leukotriene production are discussed in this review.

The potential link between atherosclerosis and the 5-lipoxygenase pathway: investigational agents with new implications for the cardiovascular field

The 5-lipoxygenase pathway is responsible for the production of leukotrienes – inflammatory lipid mediators that have a role in innate immunity, but that can also have pathological effects in inflammatory diseases. Recently, a potential link between leukotriene production and atherosclerosis has been proposed. The expression of leukotriene biosynthetic enzymes and leukotriene receptors has been identified in coronary and carotid atherosclerotic plaques, and the levels of biosynthetic enzymes have been correlated with the clinical symptoms of unstable plaques. Genetic variants in 5-lipoxygenase pathway genes have also been associated with a relative risk of developing myocardial infarction and stroke. On the basis of these discoveries, antileukotriene compounds are now being evaluated for the treatment of cardiovascular disease. Several tool compounds have been shown to limit the progression of lesion development in preclinical models of atherosclerosis, and three compounds, including two drugs previously developed for asthma, are undergoing clinical trials in patients with acute coronary syndromes.

Honeybee venom secretory phospholipase A2 induces leukotriene production but not histamine release from human basophils

The role of basophils in an anaphylactic response is well recognized but is usually masked by mast cells, which contain similar mediators for the induction of generalized vasodilatation and laryngeal constriction. The rapid onset of systemic anaphylactic symptoms, particularly in insect stings and ingested food, suggest that basophils, a circulating pool of cells containing histamine and other potent mediators such as leukotrienes, may be more involved in systemic anaphylaxis than originally thought. We wished to examine if secretory phospholipase A2, a systemic allergen found in honey bee venom (HBV-sPLA2) may activate basophils directly leading to rapid systemic mediator release. Basophils were isolated from human blood and stimulated with increasing concentrations of HBV-sPLA2. We found that physiological concentrations of HBV-sPLA2 induce rapid leukotriene C4 production from purified human basophils within 5 min, while interleukin (IL)-4 expression and production was induced at later time-points. Histamine release was not induced, signifying that HBV-sPLA2 did not induce generalized degranulation. Surface expression of CD63, CD69 and CD11b were up-regulated following HBV-sPLA2 treatment. Stimulation of basophils with anti-immunoglobulin E (IgE) following treatment with HBV-sPLA2 did not induce more leukotriene release. To investigate the mechanism of leukotriene production, 9-12 octadecadiynioc acid, a cyclooxygenase-1 (COX-1) and 15-lipoxygenase inhibitor, was used and this abrogated leukotriene production. These results indicate that HBV-sPLA2 can directly activate human basophils in vitro to induce leukotriene production.

Counterpoint: Lung Impedance Measurements are not More Useful than Simpler Measurements of Lung Function in Animal Models of Pulmonary Disease

Counterpoint: Lung Impedance Measurements are not More Useful than Simpler Measurements of Lung Function in Animal Models of Pulmonary Disease

γ‐Tocopherol prevents airway eosinophilia and mucous cell hyperplasia in experimentally induced allergic rhinitis and asthma

Traditional therapies for asthma and allergic rhinitis (AR) such as corticosteroids and antihistamines are not without limitations and side effects. The use of complementary and alternative approaches to treat allergic airways disease, including the use of herbal and dietary supplements, is increasing but their efficacy and safety are relatively understudied. Previously, we have demonstrated that gamma-tocopherol (gammaT), the primary form of dietary vitamin E, is more effective than alpha-tocopherol, the primary form found in supplements and tissue, in reducing systemic inflammation induced by non-immunogenic stimuli. We used allergic Brown Norway rats to test the hypothesis that a dietary supplement with gammaT would protect from adverse nasal and pulmonary responses to airway allergen provocation. Ovalbumin (OVA)-sensitized Brown Norway rats were treated orally with gammaT before intranasal provocation with OVA. Twenty-four hours after two challenges, histopathological changes in the nose, sinus and pulmonary airways were compared with gene expression and cytokine production in bronchoalveolar lavage fluid and plasma. We found that acute dosing for 4 days with gammaT was sufficient to provide broad protection from inflammatory cell recruitment and epithelial cell alterations induced by allergen challenge. Eosinophil infiltration into airspaces and tissues of the lung, nose, sinus and nasolacrimal duct was blocked in allergic rats treated with gammaT. Pulmonary production of soluble mediators PGE(2), LTB(4) and cysteinyl leukotrienes, and nasal expression of IL-4, -5, -13 and IFN-gamma were also inhibited by gammaT. Mucous cell metaplasia, the increase in the number of goblet cells and amounts of intraepithelial mucus storage, was induced by allergen in both pulmonary and nasal airways and decreased by treatment with gammaT. Acute treatment with gammaT inhibits important inflammatory pathways that underlie the pathogenesis of both AR and asthma. Supplementation with gammaT may be a novel complementary therapy for allergic airways disease.

Abstract 365: Genetic Variants in ALOX5 and ALOX5AP are Associated with Reduced Response to Aspirin in African American Families with Premature Coronary Artery Disease

Background: ALOX5 (arachidonate 5-lipoxygenase) and ALOX5AP (arachidonate 5-lipoxygenase activating protein) are involved in the production of leukotrienes, which may interact with cyclo-oxygenase to alter the formation of thromboxane. Reduced suppression of thromboxane production by aspirin (ASA) was associated with worse cardiovascular outcomes in the HOPE study. We therefore examined whether genetic variants in ALOX5 or ALOX5AP could modify the effect of ASA on thromboxane production. Methods: We measured urinary 11-dehydro-thromboxane B2 (UTX) levels after 2 weeks of ASA, 81 mg/day, in 1546 healthy individuals from 2-generational families with premature coronary heart disease (CHD) (n=573 African Americans from 120 families; n = 973 whites from 202 families). We genotyped 18 SNPs in ALOX5 and 10 SNPs in ALOX5AP with about 4 kb density. FBAT software was used to determine whether any SNPs were associated with the upper quartile of UTX (the least ASA suppression). The analysis was adjusted for known risk factors such as smoking, high blood pressure, sex, age, obesity, fibrinogen, diabetes and cholesterol. Results: We obtained significant associations between SNPs in both genes and the upper quartile of UTX in African Americans, but not in whites (Table ). The SNPs were not in linkage disequilibrium except for rs7896431 and rs901681 (r 2 =0.80) in African Americans. Conclusion: Both the ALOX5 and ALOX5AP genes have polymorphisms associated in African Americans with less suppression of UTX by ASA. The response to ASA may be affected by an interaction between the leukotriene and thromboxane pathways. Association of SNPs and the upper quartile of UTX

Maternal fish oil supplementation in pregnancy modifies neonatal leukotriene production by cord-blood-derived neutrophils

Fish oil supplementation has been shown to reduce neutrophil production of inflammatory LTB4 (leukotriene B4) in adults. The present study is the first to examine the effects on neonatal neutrophil function following supplementation in pregnancy. Pregnant women with allergic disease (n=98) were randomized to receive either fish oil [3.7 g of n-3 long-chain PUFAs (polyunsaturated fatty acids)/day] or a placebo supplement for the final 20 weeks of pregnancy. Leukotriene production by neonatal neutrophils was measured after stimulation with the calcium ionophore A23187. This was examined in relation to supplementation, cell membrane fatty acid levels and mononuclear cytokine production. Neutrophil LTB4 production was significantly reduced in neonates whose mothers had received fish oil in pregnancy. This was most evident for isomer 2 of LTB4 (P=0.031), although this was also observed for total LTB4 (P=0.051) and isomer 1 (P=0.088). There was also a trend for lower production of other PUFA metabolites, namely 5-HETE (5-hydroxyeicosatetraenoic acid; P=0.054) in the fish oil group. Accordingly, LTB4 levels were inversely related to membrane n-3 PUFA levels. Less inflammatory products (LTB5) were only produced at very low levels, although there was a trend for higher levels of this metabolite in the fish oil group. Consistent with this, LTB5 levels were positively correlated with n-3 PUFA membrane levels, particularly EPA (eicosapentanoic acid) and negatively correlated with n-6 PUFAs. Neonates with lower neutrophil LTB4 production also had lower production of pro-inflammatory IL (interleukin)-6 responses (r=0.35, P=0.005) and regulatory IL-10 responses (r=0.37, P=0.003) by LPS (lipopolysaccharide)-stimulated neonatal mononuclear cells. In conclusion, maternal dietary changes can modify neonatal neutrophil function. This has implications for the early immune programming, which can be influenced by the inflammatory milieu of local tissues during initial antigen encounter. It also provides evidence of another pathway through which long-chain PUFAs status can influence early immune development.

The suppressive effects of YM-58483/BTP-2, a store-operated Ca 2+ entry blocker, on inflammatory mediator release in vitro and airway responses in vivo

YM-58483/BTP-2, 4-methyl-4′-[3,5- bis(trifluoromethyl)-1H-pyrazol-1- yl]-1,2,3-thiadiazole-5-carboxanilide, blocks the store-operated Ca 2+ entry (SOCE) that mediates the activation of non-excitable cells. This study investigated the pharmacological profile and therapeutic potential of YM-58483 as anti-asthma drug. YM-58483 inhibited DNP antigen-induced histamine release from and leukotrienes (LTs) production in IgE-primed RBL-2H3 cells, a rat basophilic leukemia cell line, with IC 50 values of 460 and 310 nM, respectively. Prednisolone did not inhibit either of these responses. YM-58483 also inhibited phytohemagglutinin-P (PHA)-stimulated IL-5 and IL-13 production in human peripheral blood cells with IC 50 values of 125 and 148 nM, respectively, which is approximately 5 times less potent than prednisolone. YM-58483 (30 mg/kg, p.o.) significantly suppressed ovalbumin (OVA)-induced bronchoconstriction in OVA-sensitized guinea pigs, whereas prednisolone did not. YM-58483 (3–30 mg/kg, p.o.) and prednisolone (100 mg/kg, p.o.) both significantly and completely suppressed airway hyperresponsiveness (AHR) caused by OVA exposure. Since YM-58483 inhibits two major characteristic symptoms of bronchial asthma, namely bronchoconstriction and AHR via the suppression of inflammatory mediator and cytokine production, SOCE inhibition is a potential approach for treatment.

Role of platelet activating factor in pathogenesis of acute respiratory distress syndrome

Role of platelet activating factor in pathogenesis of acute respiratory distress syndrome

Increased interleukin‐10 production and Th2 skewing in the absence of 5‐lipoxygenase

Eicosanoids (prostaglandins and leukotrienes) are important mediators of inflammatory responses. These lipid mediators may also regulate the production of peptide mediators of the immune system. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on interleukin (IL)-10 production. IL-10 is a key regulator of immune and inflammatory responses, and previous studies have suggested that prostaglandins effect their immunosuppressive functions in part by stimulation of IL-10 production. We therefore investigated whether leukotriene production would have a similar role in regulation of IL-10 production. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased IL-10 production with a concomitant decrease in the production of pro-inflammatory cytokines, including tumour necrosis factor (TNF)-alpha and IL-12. Moreover, T-cell cytokine production in the absence of 5-LO-derived leukotrienes results in increased IL-4 production and decreased interferon (IFN)-gamma production. This may be in part secondary to increased IL-10 production and its effects on dendritic cell function resulting in altered T-cell differentiation. These findings indicate that, in addition to the central role leukotrienes play in the acute inflammatory response, endogenous leukotrienes are also important regulators of inflammatory cytokine production, via regulation of IL-10 production and in vivo differentiation of T cells.

A Medicinal Extract ofScutellaria baicalensisandAcacia catechuActs as a Dual Inhibitor of Cyclooxygenase and 5-Lipoxygenase to Reduce Inflammation

A mixed extract containing two naturally occurring flavonoids, baicalin from Scutellaria baicalensis and catechin from Acacia catechu, was tested for cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibition via enzyme, cellular, and in vivo models. The 50% inhibitory concentration for inhibition of both ovine COX-1 and COX-2 peroxidase enzyme activities was 15 microg/mL, while the mixed extract showed a value for potato 5-LOX enzyme activity of 25 microg/mL. Prostaglandin E2 generation was inhibited by the mixed extract in human osteosarcoma cells expressing COX-2, while leukotriene production was inhibited in both human cell lines, immortalized THP-1 monocyte and HT-29 colorectal adenocarcinoma. In an arachidonic acid-induced mouse ear swelling model, the extract decreased edema in a dose-dependent manner. When arachidonic acid was injected directly into the intra-articular space of mouse ankle joints, the mixed extract abated the swelling and restored function in a rotary drum walking model. These results suggest that this natural, flavonoid mixture acts via "dual inhibition" of COX and LOX enzymes to reduce production of pro-inflammatory eicosanoids and attenuate edema in an in vivo model of inflammation.

Biosynthesis and metabolism of leukotrienes

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.