Inhibition of synovial LTB4 production by a specific leukotriene biosynthesis inhibitor,MK-0591, in a rabbit model of joint inflammation

The effect of a leukotriene biosynthesis inhibitor (MK-0591) on LTB₄ synthesis was examined in a rabbit model of joint inflammation. Intra-articular (ia) injection of human recombinant interleukin-1β (rlL-1β, 50–400 ng/joint) resulted in dose-dependent infiltration of leukocytes into the synovium but produced only background levels of LTB₄ over a time course of 14–16 h, suggesting that the leukocytes were not activated with respect to LTB₄ metabolism. The influx of leukocytes in the synovial space was not inhibited by MK-0591. Injection of A23187 (100 nmol/joint, ia) in addition to rlL-1β elicited significant LTB₄ release in the synovial fluid. MK-0591 given iv 30 min before A23187 significantly inhibited the release of LTB₄ (ED₅₀ = 0.3 mg/kg), without affecting the number of leukocytes in the synovium. In rabbit whole blood challenged with A23187 in vitro, MK-0591 also potently inhibited LTB₄ synthesis (IC₅₀ = 126 ± 29 nM). The in vivo inhibitory action of MK-0591 was in good agreement with the plasma levels of the compound (0.62 μM in the group treated with MK-0591 at 3 mg/kg) and its biochemical efficacy in vitro (100% inhibition of LTB₄ synthesis). The effect of MK-0591 was comparable to that of another leukotriene biosynthesis inhibitor, MK-886. Indomethacin at dose up to 3 mg/kg iv did not affect the production of LTB₄ in the joint. The present study demonstrates that MK-0591 inhibits LTB₄ biosynthesis with high potency in the rabbit synovium, a microenvironment rich in protein and leukocytes. It would be of interest to determine whether such an action would be beneficial in the management of rheumatoid arthritis or other inflammatory conditions in humans. ©1993 Wiley-Liss, Inc.     

Pharmacological intervention with 5-lipoxygenase: new insights and novel compounds

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs) that exert a large number of different biological activities mediated by specific G-protein-coupled receptors. LTB₄ is a typical pro-inflammatory mediator that recruits and activates leukocytes, whereas the cysteinyl-containing LTC₄, D4 and E₄ cause vascular permeability and smooth muscle contraction. Recent studies have implicated LTs and also other 5-LO products in bone metabolism, and the cardiovascular system, as well as in proliferation and (tumour) cell survival. Therefore, pharmacological intervention with 5-LO product synthesis represents a reasonable strategy for the treatment of a number of disease states, including allergic and inflammatory disorders, atherosclerosis and other cardiovascular diseases, osteoporosis and certain types of cancer. This review summarises the pharmacological concepts in 5-LO inhibition and focuses on novel pharmacological approaches in the devel-opment of drugs designed to intervene with dis-eases related to 5-LO prod-ucts.     

Inhibition of the arachidonic acid cascade by norathyriol via blockade of cyclooxygenase and lipoxygenase activity in neutrophils

Recent studies have suggested that dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LO) may be more beneficial in the treatment of inflammatory diseases in which platelet-leukocyte interaction dominates the underlying inflammatory process, than inhibitors of COX or LO alone. In this study, we examined oxygenated xanthones, shown previously to inhibit platelet and neutrophil activation, with respect to the potency of COX inhibition. 1,3,6,7-Tetrahydroxyxanthone (norathyriol) was the most potent. Norathyriol suppressed thromboxane B₂ (TXB₂) and leukotriene B₄ (LTB₄) formation in calcium ionophore (A23187)- and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils. Norathyriol was 3–4 times more active against LTB₄ formation than against TXB₂ formation (IC₅₀ about 2.8 vs. 10 µM, respectively). Norathyriol also inhibited prostaglandin D₂ (PGD₂) formation in A23187-stimulated rat mast cells (IC₅₀ 3.0±1.2 µM) and in arachidonic acid (AA)-activated mast cell lysate. Norathyriol was a more effective inhibitor of 5-LO activity than of COX, as shown also by analyses of enzyme activities in a cell-free system, of COX and 5-LO metabolic capacity in neutrophils and of ex vivo TXB₂ and LTB₄ formation in A23187-stimulated neutrophils. Moreover, norathyriol inhibited COX-2 and 12-LO with IC₅₀ values (19.6±1.5 and 1.2±0.1 µM, respectively) similar to those required for the inhibition of COX-1 and 5-LO (16.2±1.5 and 1.8±0.4 µM, respectively). Inhibition of 15-LO by norathyriol was slightly less active. Norathyriol had no effect on A23187-induced AA release from neutrophils and did not affect phospholipase A₂ (PLA₂) activity in a cell-free system. These results indicate that norathyriol inhibits the formation of PGs and LTs in neutrophils probably through direct blockade of COX and 5-LO activities. Norathyriol, a single molecule with multiple targets, might provide a potential therapeutic benefit in the treatment of inflammatory diseases.     

Effect of norathyriol,isolated from Tripterospermum lanceolatum,on A 23 187-induced pleurisy and analgesia in mice

A 23 187-induced pleurisy in the mouse was demonstrated in this study. The protein leakage, leukocyte accumulation, LTB₄ and PGE₂ production in the pleural cavity of mice were increased by A 23 187 in a dose-dependent manner. At 7.5 nmole A 23 187 intrapleural injection, the protein level peaked at 0.5–2 h, PMN leukocytes accumulation peaked at 3–4 h, and LTB₄ and PGE₂ production peaked at 0.5–1 h. In this in vivo model we investigated the anti-inflammatory effect of norathyriol, isolated from Tripterospermum lanceolatum. A 23 187-induced protein leakage was reduced by norathyriol (ID₅₀ was about 30.6 mg/kg i.p.), indomethacin and BW 755 C. A 23 187-induced PMN leukocytes accumulation was suppressed by norathyriol (ID₅₀ was about 16.8 mg/kg, i.p.) and BW 755 C, while enhanced by indomethacin. Like BW 755 C, norathyriol reduced both LTB₄ and PGE₂ production (ID₅₀ was about 18.6and 29.1 mg/kg i.p., respectively), while indomethacin reduced PGE₂ but not LTB₄ generation. We also demonstrated the analgesic effect of norathyriol on the acetic acid-induced writhing response. Acetic acid-induced writhing response was depressed by norathyriol (ID₅₀ was about 27.9 mg/kg i.p.), indomethacin and ibuprofen. These results suggest that norathyriol, like BW 755 C, might be a dual, yet weak, cyclooxygenase and lipoxygenase pathway blocker. The inhibitory effect of norathyriol on the A 23 187-induced pleurisy and acetic acid-induced writhing response in mice is proposed to be dependent on the reduction of eicosanoids mediators formation in the inflammatory site. Correspondence to: J.-P. Wang at the above address     

857-860Anti-inflammatory Effect of Magnolol,Isolated from Magnolia officinalis,on A23187-induced Pleurisy in Mice

In the present study, A23187-induced pleurisy in mice was used to investigate the anti-inflammatory effect of magnolol, a phenolic compound isolated from Chinese medicine Hou p'u (cortex of Magnolia officinalis). A23187-induced protein leakage was reduced by magnolol (10mgkg^?1, i.p.), indomethacin (10mgkg^?1, i.p.) and BW755C (30mgkg^?1, i.p.). A23187-induced polymorphonuclear (PMN) leucocyte infiltration in the pleural cavity was suppressed by magnolol and BW755C, while enhanced by indomethacin. Like BW755C, magnolol reduced both prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) levels in the pleural fluid of A23187-induced pleurisy, while indomethacin reduced PGE₂ but increased LTB₄ formation. In the rat isolated peripheral neutrophil suspension, magnolol (3.7 μM) and BW755C (10 μM) also suppressed the A23187-induced thromboxane B₂ (TXB₂) and LTB₄ formation. These results suggest that magnolol, like BW755C, might be a dual cyclo-oxygenase and lipoxygenase inhibitor. The inhibitory effect of magnolol on the A23187-induced pleurisy is proposed to be, at least partly, dependent on the reduction of the formation of eicosanoids mediators in the inflammatory site.     

4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B₄ (LTB₄) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT₁ (cysLT₁) receptor antagonist, REV-5901 as well as a BLT₁ receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB₄ and cysLT (LTC₄ and LTD₄) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB₄ and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.     

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB₄, a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB₄ was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025–25 ng/mL for LTB₄ and 0.1–25 ng/mL for 5-HETE. The EIA method has a validated range covering 0.025–4 ng/mL. When both assays were applied to analyze LTB₄ from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r = 0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB₄ and 5-HETE measured by LC/MS/MS after ionophore stimulation (r = 0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.     

The generation and metabolism of leukotrienes in the ionophore-stimulated blood of normal and asthmatic subjects

The generation and metabolism of leukotrienes (LTs) 1341 C₄, D4, and E₄ were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB₄ and LTC₄ in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB₄, which are significantly higher in the AA blood than in N blood. Incubations of ³H-LTB₄ in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB₄ and 20-COON-LTB₄.LTC₄ is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p < 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC₄ elicits significantly greater amounts of LTD₄, and consistently higher levels of LTE₄, in the AA blood. Parallel incubations of 3H-LTC₄ in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB₄ and LTC₄ in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB₄ and LTC₄ generation and was equipotent in N and AA blood.     

Saucerneol F,a New Lignan Isolated from Saururus chinensis,Attenuates Degranulation via Phospholipase Cγ 1 Inhibition and Eicosanoid Generation by Suppressing MAP Kinases in Mast Cells

During our on-going studies to identify bioactive compounds in medicinal herbs, we found that saucerneol F (SF), a naturally occurring sesquilignan isolated from Saururus chinensis (S. chinensis), showed in vitro anti-inflammatory activity. In this study, we examined the effects of SF on the generation of 5-lipoxygenase (5-LO) dependent leukotriene C₄ (LTC₄), cyclooxygenase-2 (COX-2) dependent prostaglandin D₂ (PGD₂), and on phospholipase Cγ1 (PLCγ1)-mediated degranulation in SCF-induced mouse bone marrow-derived mast cells (BMMCs). SF inhibited eicosanoid (PGD₂ and LTC₄) generation and degranulation dose-dependently. To identify the molecular mechanisms underlying the inhibition of eicosanoid generation and degranulation by SF, we examined the effects of SF on the phosphorylation of PLCγ1, intracellular Ca²⁺ influx, the translocation of cytosolic phospholipase A₂ (cPLA₂) and 5-LO, and on the phosphorylation of MAP kinases (MAPKs). SF was found to reduce intracellular Ca²⁺ influx by inhibiting PLCγ1 phosphorylation and suppressing the nuclear translocations of cPLA₂ and 5-LO via the phosphorylations of MAPKs, including extracellular signal-regulated protein kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Taken together, these results suggest that SF may be useful for regulating mast cell-mediated inflammatory responses by inhibiting degranulation and eicosanoid generation.     

Effects of Phosgene Exposure on Lung Arachidonic Acid Metabolism

Abstract

Phosgene can acylate macromolecules and may react with and affect enzymes involved in arachidonic acid metabolism. We examined the effects of an acute phosgene exposure in vivo and in vitro on lung arachidonic acid metabolism. Fischer 344 rats were exposed either to air or to phosgene (0.05–7.0 ppm) for 4 h and the lungs were lavaged at 0, 4, 20, and 44 h postexposure. Leukotriene B, (LTBJ, peptide leuko-trienes C₄ D₄ and E₄ (LTC₄/D/E₄, and prostaglandin E₂ (PCE₂) were measured in la-vage fluid by radioimmunoassay Phosgene exposure in vivo (0.7–7.0 ppm for 4 b) produced significant decreases in concentrations of PGE, (maximal decrease of 74%), LTB, (maximal decrease of 59%), and LTC₄/D₄/E₄ (maximal decrease of 97%) measured in rat lavage fluid immediately postexposure. Associated with this decrease in eicosa-noid production was a decrease in the number of alveolar macrophages and an increased number of neutrophils recovered in the lavage fluid of phosgene-exposed rats. Lung lavage eicosanoid concentrations in rats exposed to either 0.7 or 0.25 ppm phosgene returned to or exceeded air control values by 44 h postexposure. However, rats exposed to 0.5 or 7.0 ppm phosgene had a persistent decrease in the concentration of PGE, and LTC₄/D₄/E₄ at44h after the exposure. Phosgene exposure in vitro (7.0 ppm for 4 h) of rat or human alveolar macrophages induced a 56% decrease in the production of LTB₄ in the rat macrophages and a 31% nonsignificant decrease in the formation of LTB₄ in human alveolar macrophages. Production of PCE, and the LTC₄/D₄/E₄ by the rat macrophages, and PCE₂ by the human macrophages, was not different from air-exposed culture values. These data suggested that phosgene exposure altered arachidonic acid metabolism in rat lungs after in vivo exposure and in rat and human alveolar macrophages after exposure in vitro. These changes in lung mac-rophage arachidonic acid metabolism may be involved in some of the lung responses to phosgene exposure.     

Novel leukotriene biosynthesis inhibitors (2012-2016) as anti-inflammatory agents

Introduction: Leukotrienes (LTs) are lipid mediators produced from arachidonic acid with a broad variety of bioactivities in allergy and inflammation. The biosynthesis of LTs mainly involves 5-lipoxygenase (5-LO) and its 5-lipoxygenase-activating protein (FLAP), LTA₄ hydrolase and LTC₄ synthase that all may represent potential targets for LT biosynthesis inhibitors.

Areas covered: We introduce the LT biosynthetic pathway and its cellular regulation, the diverse biological actions of LTs and their receptors, and we briefly describe the pharmacological strategies for suppression of LT formation as well as the classes of current LT biosynthesis inhibitors. The main focus is placed on the comprehensive discussion of recently reported inhibitors of 5-LO, FLAP, LTA₄ hydrolase and LTC₄ synthase, based on literature search (PubMed and Thomson Innovation Patents Searches), covering 2012–2016.

Expert opinion: Although many new series of 5-LO inhibitors have been presented without patenting, essentially by academia, novel FLAP inhibitors (many patented) are most advanced in clinical development and are apparently the focus of pharmaceutical companies. Only few novel inhibitors of LTA₄ hydrolase and LTC₄ synthase were reported. Major issues in the development of LT synthesis inhibitors are related to loss of potency in biological relevant environment, poor pharmacokinetics, lack of oral efficacy, and side effects.     

Curcumin Inhibits the Activation of Immunoglobulin E-Mediated Mast Cells and Passive Systemic Anaphylaxis in Mice by Reducing Serum Eicosanoid and Histamine Levels

Curcumin is naturally occurring polyphenolic compound found in turmeric and has many pharmacological activities. The present study was undertaken to evaluate anti-allergic inflammatory activity of curcumin, and to investigate its inhibitory mechanisms in immunoglobulin E (IgE)/Ag-induced mouse bone marrow-derived mast cells (BMMCs) and in a mouse model of IgE/Ag-mediated passive systemic anaphylaxis (PSA). Curcumin inhibited cyclooxygenase-2 (COX-2) dependent prostaglandin D₂ (PGD₂) and 5-lipoxygenase (5-LO) dependent leukotriene C₄ (LTC₄) generation dose-dependently in BMMCs. To probe the mechanism involved, we assessed the effects of curcumin on the phosphorylation of Syk and its downstream signal molecules. Curcumin inhibited intracellular Ca²⁺ influx via phospholipase Cγ1 (PLCγ1) activation and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-κB (NF-κB) pathway. Furthermore, the oral administration of curcumin significantly attenuated IgE/Ag-induced PSA, as determined by serum LTC₄, PGD₂, and histamine levels. Taken together, this study shows that curcumin offers a basis for drug development for the treatment of allergic inflammatory diseases.     

黄芩苷的抗炎机理

目的：探讨黄芩苷抗炎作用机理。方法：以大鼠腹腔白细胞为实验材料,分别采用高效液相色谱法（HPLC）,放射免疫分析法（RIA）和荧光分光光度法测定白细胞内白三烯（LTs）、环一磷酸腺苷(cAMP)和细胞内游离钙水平。结果：黄芩苷可显著抑制白细胞内白三烯B₄、白三烯C₄的生物合成,其IC₅₀值分别为0.48和3.15 μmol.L^-1;　黄芩苷还可显著抑制fMLP激发的白细胞内Ca²⁺升高,并促进细胞内cAMP水平提高。结论：黄芩苷显著影响白细胞的多种功能,提示此功能与其抗炎作用机理有关。     

大鼠角叉菜胶胸膜炎渗出白细胞磷脂酶D活性的变化

目的　观察在整体炎症过程中炎症白细胞磷脂酶D(PLD)活性的变化和炎症的关系。方法　采用大鼠角叉菜胶胸膜炎模型 ,以渗出液量和细胞数及渗出液中髓过氧化物酶活性 (中性粒细胞脱颗粒指标 )作为炎症程度。用酶偶联比色法测定白细胞PLD活性。结果　正常大鼠外周血白细胞PLD活性极低 ,为 ( 0 14± 0 0 3) μmol·g-1·min-1。致炎后各时间点胸膜腔渗出白细胞的PLD活性明显升高 ,分别可达 40～ 6 0倍 ,并在 3h达峰值 ,明显早于炎症高峰 ( 12h)。不同剂量 ( 5 0 0 μg和 10 0 0 μg)角叉菜胶可引起致炎 12h明显不同程度的炎症 ,但渗出白细胞PLD活性两者差别不大。低剂量吲哚美辛 ( 2mg·kg-1,ip)和地塞米松 ( 0 1mg·kg-1,ip)均明显抑制致炎 6h大鼠胸膜腔的渗出 ,但渗出白细胞PLD活性与对照组相比差别无显著性。结论　大鼠角叉菜胶性胸膜炎白细胞PLD活性显著升高 ,提示PLD活性升高在该炎症模型中是原发性表现 ,低剂量吲哚美辛和地塞米松的抗炎机制与PLD无关     

Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments

Leukotriene B₄ (LTB₄) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A₄ (LTA₄) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB₄. As a result, pharmacological strategies to modulate the synthesis of LTB₄ (inhibition of PLA2, 5-LO or LTA₄ hydrolase) or the effects of LTB₄ itself (antagonism of LTB₄ receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB₄, namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.     

Mechanisms for anti-inflammatory effects of 1-[15(S)-hydroxyeicosapentaenoyl] lysophosphatidylcholine, administered intraperitoneally, in zymosan A-induced peritonitis

BACKGROUND AND PURPOSE

Lysophosphatidylcholines (lysoPCs) with polyunsaturated acyl chains are known to exert anti-inflammatory actions. 15-Lipoxygeanation is crucial for anti-inflammatory action of polyunsaturated acylated lysoPCs. Here, the anti-inflammatory actions of 1-(15-hydroxyeicosapentaenoyl)-lysoPC (15-HEPE-lysoPC) and its derivatives were examined in a mechanistic analysis.

EXPERIMENTAL APPROACH

Anti-inflammatory actions of 15-HEPE-lysoPC in zymosan A-induced peritonitis of mice were examined by measuring plasma leakage and leucocyte infiltration, and determining levels of lipid mediators or cytokines.

KEY RESULTS

When each lysoPC, administered i.v., was assessed for its ability to suppress zymosan A-induced plasma leakage, 15-HEPE-lysoPC was found to be more potent than 1-(15-hydroperoxyeicosapentaenoyl)-lysoPC or 1-eicosapentaenoyl-lysoPC. Separately, i.p. administration of 15-HEPE-lysoPC markedly inhibited plasma leakage, in contrast to 15-HEPE, which had only a small effect. 15-HEPE-lysoPC also decreased leucocyte infiltration. Moreover, it reduced the formation of LTC₄ and LTB₄, 5-lipoxygenation products, as well as the levels of pro-inflammatory cytokines. The time-course study indicated that 15-HEPE-lysoPC might participate in both the early inflammatory phase and resolution phase. Additionally, 15-HEPE-lysoPC administration caused a partial suppression of LTC₄-induced plasma leakage and LTB₄-induced leucocyte infiltration. In the metabolism study, peritoneal exudate was shown to contain lysoPC-hydrolysing activity, crucial for anti-inflammatory activity, and a system capable of generating lipoxin A from 15-hydroxy eicosanoid precursor.

CONCLUSIONS AND IMPLICATIONS

15-HEPE-lysoPC, a precursor for 15-HEPE in target cells, induced anti-inflammatory actions by inhibiting the formation of pro-inflammatory leukotrienes and cytokines, and by enhancing the formation of lipoxin A. 15-HEPE-lysoPC might be one of many potent anti-inflammatory lipids in vivo.     

Effect of an LTB4 Aerosol Exposure on Pulmonary Function,Cell Populations,and Mediators in the Lungs of Rhesus Monkeys

Abstract

Six adult rhesus monkeys (5–7 kg) were anesthetized with sodium pentobarbital, 30 mg/kg iv, intubated, and exposed for 4 h, once per week, to air (baseline), normal saline, an inactive isomer of leukotriene B₄ (LTB₄), and LTB₄. Anesthesia was maintained with sodium pentobarbital, S mg/kg/h iv. Pulmonary function was monitored for a 30-min baseline period and during the 4-h exposure. After each exposure, the upper and lower airways were lavaged. The upper airway lavage was performed by inclining the animal head down, inserting a small catheter through the endotracheal (ET) tube to the carina, then instilling 15 ml normal saline through the catheter and collecting the fluid as it came out the ET tube. The lower airway lavage was performed with a fiberoptic bronchoscope placed at the level of generation 5–7 of a lower lobe. Two 10-ml aliquots of saline were instilled and gently suctioned via syringe. Cell counts and differentials were performed. Supernatant was analyzed for levels of LTB₄, LTC₄ and TxB₂. After the LTB₄ exposure, the animals were killed and the lungs were removed for histopathology. There were no significant changes in pulmonary function or cell populations from the upper airways after any treatment. LTB₄ levels were significantly increased over baseline only in the upper air-ways after the LTB₄ aerosol exposure. The lower airways showed a significant increase in total white cells due to a dramatic (1100%) increase in neutrophils after LTB₄ treatment only. The pulmonary tissue response was characterized by a multifocal accumulation of neutrophils in alveoli, neutrophilic infiltration of bronchiolar wall smooth muscle, and a slight accumulation of cell and mucous debris within small bronchi. It was concluded that, in these monkeys, an LTB₄ aerosol causes neutrophils to accumulate in the lungs without evidence of inflammation or altered pulmonary function.The LTB₄ and the 12-epi,6-trans-LTB₄ were synthesized and kindly supplied by D. R. Dobson and Dr. S. R. Baker, respectively, Eli Lilly and Company.     

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B₄ (LTB₄) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB₄ production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB₄. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB₄, subsequent MMP-9 production and plaque rupture.     

Pinusolide Isolated from <i>Biota orientalis</i> Inhibits 5-Lipoxygenase Dependent Leukotriene C₄ Generation by Blocking c-Jun N-Terminal Kinase Pathway in Mast Cells

Pinusolide, an herbal medicine isolated from Biota orientalis L. (B. orientalis), inhibited 5-lipoxygenase (5-LO)-dependent leukotriene C4 (LTC4) generation in immunoglobulin E (IgE)/Ag-induced bone marrow-derived mast cells (BMMCs) in a concentration-dependent manner. To clarify the action mechanism of pinusolide on the inhibition of LTC4 generation, we examined the effect of pinusolide on phosphorylation of cytosolic phospholipase A2 (cPLA2), as well as translocation phospho-cPLA2 and 5-LO to nucleus. Inhibition of LTC4 generation by pinusolide was accompanied by a decrease in cPLA2 phosphorylation which occurred via a decrease in intracellular Ca²⁺ influx and blocking the c-Jun N-terminal kinase (JNK) pathways. However, pinusolide had no effect on extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinas phosphorylation. Taken together, the present results suggest that potent inhibition of 5-LO dependent LTC4 generation by pinusolide requires both suppression of calcium influx and JNK phosphorylation.     

Treatment in vivo with Ph CL28A alters prostaglandin E2, prostacyclin and leukotriene C4 metabolism in rat isolated lung

We have studied the effects of a potent inhibitor of prostaglandin (PG) catabolism, Ph CL28A, given in vivo, on PG metabolism in rat perfused lungs, isolated at different times after treatment. Two doses of Ph CL28A were used, 10 and 30 mg/kg, and lungs isolated at 1, 2 and 4h after a single injection (i.p.). The catabolism of exogenous PGE₂ was decreased for up to 2h after injection. Synthesis of PGI₂ using exogenous arachidonic acid, was increased by treatment with Ph CL28A. Stimulation of the turnover of endogenous arachidonic acid with the calcium ionophore A23187 led to the synthesis of LTC₄ as well as PGI₂. Treatment in vivo with Ph CL28A, decreased the output of LTC₄ but increased that of PGI₂. However in the presence of indomethacin, the perfused lung did not form any PGI₂ and the output of LTC₄ was still inhibited by Ph CL28A. We conclude that the inhibition of LT formation in lung by Ph CL28A was not due to the increased production of PGI₂. These two properties, inhibition of LT synthesis and potentiation of PGI₂ formation, may synergize in vivo to give anti-inflammatory activity.