Elevated Expressions of 15-Lipoxygenase and Lipoxin A4 in Children with Acute Poststreptococcal Glomerulonephritis

Anti-inflammatory effects of the 15-lipoxygenase (15-LO) derivatives lipoxin A₄ (LXA₄) and 15-S-hydroxyeicosatetraenoic acid (15-S-HETE) have been documented in many experimental models of acute inflammation. However, the expression levels of 15-LO and its products in human renal diseases remain unknown. This study investigated the expression levels of LXA₄, leukotriene B₄ (LTB₄), and 15-LO in leukocytes and glomeruli obtained from 22 children with acute poststreptococcal glomerulonephritis (APSGN), and determined the modulatory effects of both 15-S-HETE and LXA₄ on LTB₄ synthesis in leukocytes and LTB₄-evoked chemotaxis of polymorphonuclear leukocytes (PMNs) obtained from children during the first 3 days after onset of APSGN. Expression levels of both LXA₄ and 15-LO in leukocytes and glomeruli were up-regulated during the acute phase of disease, further peaking between days 10 and 14, and remained increased after 6 to 8 weeks of APSGN onset. In contrast, blood and urinary levels of LTB₄ as well as the number of glomerular PMNs peaked during the acute phase of disease and then decreased during the resolution phase. Administration of both 15-S-HETE and LXA₄ in vitro inhibited LTB₄-induced chemotaxis of PMNs and production of LTB₄ from leukocytes obtained from patients with APSGN. The current study provides further support for an anti-inflammatory role for 15-LO products in human nephritis through both antagonism and inhibition of leukotriene synthesis and its biological activity.     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB₄ formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipiddependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B₄ (LTB₄) in FMLP-stimulated neutrophils, the ID₅₀ was approximately 4.5 g/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB₄ nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB₄ production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

Characterization of arachidonic acid metabolism,superoxide production,and bacterial killing by bovine alveolar neutrophils elicited with leukotriene B4 and zymo-san-activated plasma

Leukotriene B₄ (LTB₄) and zymosan-activated plasma (ZAP) were each instilled into the lungs of steers to elicit alveolar neutrophils for subsequent functional analysis. Prior to instillation of either agent, bronchoalveolar lavage cell populations consisted of 95.8 ± 0.4% macrophages (mean ±SEM). Four hours after instillation of LTB₄ or ZAP, the lavage cell populations consisted of 75.0 ± 8.8% and 90.7 ± 0.7% neutrophils, respectively. Alveolar neutrophils elicited with LTB₄ and stimulated with the calcium ionophore A23187 released diminished amounts of LTB₄ and increased amounts of 5-hydroxyeicosatetraenoic acid (5-HETE) as compared to circulating neutrophils. Release of superoxide anion was decreased for LTB₄-elicited alveolar neutrophils as compared to circulating cells, while bacterial killing was unchanged. ZAP-elicited alveolar neutrophils released diminished amounts of LTB₄ when stimulated with A23187 as compared to circulating neutrophils. There were no differences observed in 5-HETE levels between the two cell populations. In addition, release of superoxide anion was diminished among ZAP-elicited alveolar cells, while bacterial killing was unchanged. Incubation of circulating neutrophils with LTB₄ did not influence the release of arachidonate metabolites, superoxide anion, or bacterial killing. However, incubation of circulating neutrophils with ZAP, followed by A23187 resulted in a reduction in the release of LTB₄, as compared to control cells. Prior exposure to ZAP did not influence the release of superoxide anion or bacterial killing by the circulating neutrophils.     

Serum from NSAID-treated patients attenuates the capacity of rat leukocytes to synthesize leukotriene B4

The synthesis of leukotriene B₄ by A23187-stimulated rat peritoneal leukocytes was studied in the presence of 0.1% normal human serum, serum from patients treated with NSAIDs for either an inflammatory (rheumatoid arthritis, RA) or a non-inflammatory condition (lumbar disc protrusion, LDP), and serum from RA patients drawn one week after withdrawal from NSAID treatment. The capacity for LTB₄ synthesis was significantly lower in the presence of serum from NSAID treated patients: thirty per cent less than observed in presence of normal serum in the RA group, and fifty per cent in the LDP group. When NSAIDs were withdrawn from RA patients, the LTB₄ production in presence of serum increased, but was not completely normalized after one week. These results indicate that NSAID treatment may down-regulate the capacity for leukotriene synthesis by an indirect effect.accepted by M. J. Parnham     

Interactions between synthesis of platelet-activating factor and leukotriene B4 in isolated human polymorphonuclear leukocytes

The aim of the present work was to study interactions between the synthesis of platelet-activating factor (PAF) and leukotriene B₄ (LTB₄) in human polymorphonuclear leukocytes (PMNs) in vitro. PAF, at nanomolar concentrations, stimulated calcium ionophore A23187-activated PMNs to release LTB₄ and 5-hydroxyeicosatetraenoic acid (5-HETE). This seems to be a receptor-mediated process as it was blocked by a PAF receptor antagonist WEB 2086 (IC₅₀ 6.6±3.9M). Moreover, LTB₄ stimulated the formation of PAF in activated PMNs. WEB 2086 did not, however, alter PMN migration towards either LTB₄ or the chemotactic peptide FMLP. This suggests that the enhancement of PAF synthesis in response to LTB₄ is a concomitant event rather than a mediating process in LTB₄-induced chemotactic movement of PMNs. These effects are implicated in the complex network of interactions between inflammatory mediators that results accumulation and activation of PMNs in the exacerbation of inflammatory processes.     

Leukotriene B4 metabolism in human leukocytes: Fact or artefact?

The object of this study was to investigate the importance of omega oxidation in regulating leukotriene B₄ (LTB₄) levels in man. In human polymorphonuclear leukocytes metabolism of LTB₄ was rapid but was critically dependent on PMN number: greater than 1.5×10⁶ PMN/ml were required. Metabolism of LTB₄ was blocked in the presence of plasma. In whole blood and in PMN-rich rheumatoid synovial fluids no significant metabolism of LTB₄ was detected within 30 min at 37°C. We conclude that LTB₄ metabolism at inflamed sites will be regulated both by cellular content and the degree of plasma exudation. In most pathological conditions rapid exchange with the micro-vasculature will be more important than metabolism in limiting LTB₄ levels.     

Activation of human neutrophil LFA-1 (CD11a) by leukotriene B4

Homotypic aggregation (HA) of human neutrophils by the potent leukotactic factor, leukotriene B₄ (LTB₄), and phorbol myristate acetate (PMA) was evaluated by recording the net decrease in absorbency at 650 nm of suspensions of 10⁷ neutrophils/ml in a microtiter plate reader, which was found to correlate with microscopic evidence of aggregation. LTB₄-elicited HA was increased maximally by approximately one third above HA in buffer at 30 min, whereas PMA-induced HA reached a maximal level more than 21–fold higher than buffer control at 60 min. The involvement of LFA-1 in LTB₄-induced HA of neutrophils was suggested initially by the inhibitory effect of monoclonal anti-CD 18 and anti-CD11a antibodies. The binding to neutrophils of a monoclonal anti-LFA-1 antibody (NK1-L16) specific for an activation epitope of CD11a was increased a maximum of 28-fold and sixfold, respectively, after 1 and 5 min of preincubation with 10 nM LTB₄and fivefold after 5 min with PMA. Thus, both LTB₄ and PMA induce an activating conformational change in the CD11a adherence receptor of human neutrophils.     

Lipoxins A4 and B4 inhibit leukotriene B4 generation from human neutrophil leukocyte suspensions

Lipoxins A4 and B4 (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic and 5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acids, respectively) were examined in several biological systems and have proven to have many different activities from those of other eicosanoids. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit leukotriene B4 generation was assessed after incubation with the calcium ionophore A23187. The preincubation time of neutrophils with lipoxin A4 and B4 was 15 min. After that time the cells were incubated for 6 min with A23187 (5 microM) for the release of LTB4. We found that the pretreatment of neutrophils with lipoxins inhibited the release of LTB4 by A23187-stimulated PMNs. Nordihydroguaiaretic acid (NDGA) (10 microM), used as a control, strongly inhibited the generation of LTB4. Since LTB4 has been shown to be a modulator of cellular immunity, our data suggest that lipoxin A4 and B4 can contribute to the immunosuppression via inhibition of LTB4 generation. Moreover, the inhibition of LTB4 by lipoxins in neutrophils could have an important regulatory role in inflammation.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and opsonization synergistically enhance leukotriene B4 (LTB4) synthesis induced by phagocytosis in human neutrophils

Normal human blood neutrophils were studied for their capacity to synthesize leukotriene B₄ (LTB₄) and its -oxidized metabolites after phagocytosis of zymosan. Phagocytosis of serum-opsonized particles led to a higher release of LTs than did unopsonized zymosan. The most stricking effect of phagocytosis was observed when neutrophils were primed with granulocyte-macrophage colony-stimulating factor (GM-CSF): opsonization and GM-CSF synergistically increased LTB₄ synthesis by neutrophils.     

Effect of 5-lipoxygenase inhibitors onin situ LTB4 biosynthesis following calcium ionophore stimulation in the rat pleural cavity

The intrapleural injection of carrageenan in the rat induces exudate formation, cellular influx and leukotriene generation in the pleural cavity. We have demonstrated that the inflammatory response (exudate volume, and LTB₄ levels) is increased,in situ by the intrapleural administration of calcium ionophore A 23187 (100 nmol/rat) at 4, 16, 24, 48, and 72 h after the injection of carrageenan and that the A 23187-induced increase is dose-dependent. The oral administration of A-64077 and MK-886, two 5-lipoxygenase inhibitors (5-LOIs), at 10 mg/kg causes marked decreases in LTB₄ release at the above-mentioned time intervals. However, A 23187-induced augmented exudate formation is not affected by the treatment with 5-LOIs. The results suggest that the use of 5-LOIs to inhibit LTB₄ biosynthesis may be beneficial in various LTB₄-dependent pathological conditions.     

CGS 22745: A selective orally active inhibitor of 5-lipoxygenase

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) synthesis in guinea pig leukocytes (IC₅₀=0.6M). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB₄ in blood (IC₅₀'s of 4.3, 0.56 and 3.2 M for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB₄ formationex vivo after 5 min. Its effective biological half-life was >160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibitedex vivo A23187-stimulated LTB₄ formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.     

Biosynthesis of leukotriene B4

Leukotriene B₄ (LTB₄) is a lipid mediator derived from arachidonic acid (AA) by the sequential action of 5-lipoxygenase (5-LOX), 5-lipoxygenase-activating protein (FLAP) and LTA₄ hydrolase (LTA₄H). It was initially recognized for its involvement in the recruitment of neutrophils and is one of the most potent chemotactic agents known to date. A large body of data has indicated that LTB₄ plays a significant role in many chronic inflammatory diseases, such as arthritis, chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer and more recently, metabolic disorder. In this review, we focus on the biosynthesis of LTB₄ and its biological effects. In particular, we will describe a basic biochemical understanding integrated with recent developments in the field of structural biology of the three key enzymes (5-LOX, FLAP and LTA₄H) in LTB₄ biosynthesis, and also summarize the most outstanding work on in vivo biological and pathogenic roles of these enzymes and the development of enzyme inhibitors.     

Effect of a leukotriene B4 receptor antagonist on leukotriene B4-induced neutrophil chemotaxis in cavine dermis

Leukotriene B₄ (LTB₄) is a proinflammatory product of arachidonic acid metabolism that has been implicated as a mediator in a number of inflammatory diseases. When injected intradermally into the cavine, LTB₄ elicits a dose-dependent immigration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-l-benzopyran-2-carboxylic acid, a potent LTB₄ receptor antagonist inhibited the chemotactic actions of LTB₄ when coadministered into the dermal site and when given intravenously or orally with ED₅₀ values of 200 ng, 0.5 mg/kg, and 0.6 mg/ kg respectively. This compound may well have application in disease states, such as inflammatory bowel disease and psoriasis, where LTB₄ is implicated as a proinflammatory mediator.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophilsin vitro. Evidence of formation of antiinflammatory compounds

Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B₄ (LTB₄), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB₄ by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 M) and different concentrations (0–100 M) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB₄ was dose dependently inhibited by both LA (IC₅₀=45 M) and DGLA (IC₅₀=40M). This inhibition of LTB₄ formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB₄ formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB₄ formation (IC₅₀=7.5 M and IC₅₀=0.2 M). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB₄ formation (IC₅₀=0.75 M). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB₄ formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB₄ formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.     

Stimulus-dependent effects of CI-986 on arachidonic acid metabolism by human neutrophils

CI-986 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1,3,4-thiadiazole-2(3H)-thione, choline salt) was evaluated for its effect on arachidonic acid metabolism by human neutrophils in response to different stimuli. Leukotriene B₄ (LTB₄) release in response to calcium ionophore A23187 was 15 to 35 fold greater than the responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP) or serum-opsonized zymosan (SOZ), respectively, while the thromboxane B₂ (TXB₂) release response was similar for the three stimuli tested. CI-986 inhibited the release of LTB₄ and TXB₂ in response to A23187 with IC₅₀s of 63.4 and 1.6 µM, respectively. In comparison, the compound inhibited SOZ-stimulated LTB₄ release with an IC₅₀ of 11.2µM, while having no effect on TXB₂ at concentrations up to 100 µM. Conversely, CI-986 inhibited FMLP-stimulated LTB₄ release by 42% at 100 µM, while inhibiting TXB₂ release with an IC₅₀ of 0.13 µM. These results demonstrate a stimulus-dependent inhibitory effect of CI-986 on human neutrophil eicosanoid metabolism.     

Development and application of a membrane receptor assay for leukotriene B4

5(S),12(R)-dihydroxy-6-cis-8,10 tran-14-cis-eicosatetraenoic acid (LTB₄) is a potent inflammatory mediator generated by human cells. A receptor assay using membranes from cultured HL-60 cells has been developed to quantitate LTB₄ with a range of sensitivity from 10 pg to 5 ng. The initial metabolite of LTB₄, 20-OH LTB₄, has a cross reactivity of 28% while other, lipoxygenase products do not significantly compete. This assay has been used to study ionomyocin-induced formation of LTB₄ by human neutrophils. The use of membranes from HL-60 cells for the measurement of LTB₄ provides a sensitive and highly selective alternative to radioimmunoassay for the determination of the levels of this important eicosanoid in biological fluids and should be useful in the development of antagonists of the LTB₄ receptor.     

Priming effects of leukotriene B4 on endothelial cell injury induced by tpa-activated leukocytes

Among arachidonic acid metabolites, leukotriene B₄ (LTB₄) plays an important role in inflammation, such as in the activation, adhesion, chemotaxis, and invasion of leukocytes. In this paper, we examined the effect of LTB₄ on endothelial cell injury induced by polymorphonuclear leukocytes (PMNLs).⁵¹Crrelease, a marker of cellular injury, was elicited from prelabeled endothelial cells when the cells were cocultured with PMNLs activated by phorbol ester (TPA, 12-0-tetradecanoyl-phorbol-13-acetate). Under this condition, pretreatment of PMNLs with LTB₄ enhanced their injury in a dose-dependent manner (0.2–2 M). However, LTB₄ alone at any dose could not induce any cellular injury. We also determined the amount of active oxygen species produced by PMNLs in response to TPA. The intensity of luminoldependent chemiluminescence, a marker of active oxygen production, in PMNLs was also increased by pretreatment with 1 M LTB₄. These data suggest that LTB₄ enhances endothelial cell injury by the priming effect on active oxygen production in activated PMNLs.     

Leukotriene B4 and inflammatory disease

Polymorphonuclear leukocytes (PMNL) are prominent at sites of acute inflammation. Their infiltration is stimulated under pathological conditions by a variety of agents which include bacteria, immune complexes and complement derived chemotactic peptides. Recently attention was focussed on the 5-lipoxygenase product leukotriene B₄ (LTB₄) which has been demonstrated to induce the key features associated with an acute inflammatory reaction. However, evidence supporting a pro-inflammatory role for LTB₄, and therefore the anti-inflammatory efficacy of 5-lipoxygenase inhibitors, is largely circumstantial. Moreover, there are concerns that other chemotactic factors, notably C₅a, may compensate for the absence of LTB₄. Here we challenge this view and, on the basis of recent experimental and clinical data suggest that LTB₄ does not simply duplicate the activity of C₅a. Instead we propose that their predominant site(s) of action differ in such a way that they may synergise in mediating PMNL recruitment.     

Leukotriene B4 production by blood neutrophils in allergic rhinitis—effects of cetirizine

Background: Mucosal inflammatory processes in late phase of allergic diseases involve cytokine production, cell adhesion molecule overexpression and release of inflammatory mediators with chemotactic activity, such as leukolriene B₄ (LTB₄), We had previously observed increased production of LTB₄ by neutrophils in patients with allergic rhinitis and discussed the role of granulocyte macrophage-colony stimulating factor (GM-CSF) priming. Some antihislaminic compounds were shown to diminish the production of leukotrienes by neutrophils. Objective: In a first step, we evaluated in ex vivo and in vitro studies, the effects of cetirizine on LTB₄ production by blood neutrophils from allergic and healthy subjects. In a second step, we studied the in vitro effect of cetirizine on LTB₄ production by neutrophils from healthy subjects during GM-CSF priming of these cells. Methods: Neutrophils from both populations were purified from venous blood and LTB₄ production was measured using high performance liquid cromatography (HPLC) method. Results: In ex vivo studies, cetirizine treatment induced a decreased LTB₄ production by neutrophils in allergic rhinitis. This effect of decreased LTB₄ production was reproduced in vitro with 10^?8–10^-6 cetirizine. Nevertheless, this anti-Hl compound had no effect on neutrophil priming with GM-CSF. Conclusion: As LTB₄ is an important chemotactic factor, Cetirizine could act on inflammatory cell recruitment by inhibiting LTB₄ production by neutrophils.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.