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.     

Action of leukotriene B4 in human granulosa cells in vitro

Leukotriene B4 directly enhanced progesterone release from superfusedhuman granulosa cells. This secretory effect was observed inconcentrations from 10^–12 to 10^–10 M. Lower andhigher concentrations failed to affect progesterone release.When we analysed the high performance liquid chromatographyprofile of supernatant from human granulosa cell cultures, wedetected a leukotriene B4 peak. In conclusion, these data supportthe hypothesis that leukotriene B4 may participate in the intracellularmechanism of progesterone release in human granulosa cells. arachidonate/human granulosa cells/leukotriene B4/5-lipoxygenase/progesterone     

Modulation of leukotriene formation by cellular composition and exogenous leukotriene A4.

We investigated the interactions of exogenous leukotriene A4 (LTA4) with isolated cells in the presence or absence of cellular stimuli. The majority of isolated cells are able to transform exogenous LTA4 into LTB4 as well as LTC4. In eosinophils, LTA4 induced 15-hydroxy-eicosatetranoic acid formation and was converted into LTB4. The Ca-ionophore-induced generation of LTB4 from polymorphonuclear leukocytes or from the cell fraction containing lymphocytes, monocytes and basophils was significantly suppressed with LTA4 while the formation of LTC4 was increased. Conversely, the Na-fluoride- and fMLP-induced generation of LTB4 was significantly increased. Our results suggest that the stimulus and the cellular composition determine the pattern of the generated inflammatory mediators.     

Production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human neutrophils is inhibited by Pseudomonas aeruginosa phenazine derivatives.

Pyocyanin, a phenazine pigment produced by Pseudomonas aeruginosa, and its metabolite 1-hydroxyphenazine inhibited leukotriene B4 and 5-hydroxyeicosatetraenoic acid production by up to 70% in human neutrophils stimulated with the calcium ionophore A23187 (5 microM). This potential anti-inflammatory effect was dose dependent and occurred at low concentrations (10 to 50 microM) that did not inhibit neutrophil viability.     

Tolfenamic acid inhibits leukotriene B4-induced chemotaxis of polymorphonuclear leukocytes in vitro

Inhibition of prostanoid synthesis is usually regarded as the mode of action of nonsteroidal antiinflammatory drugs (NSAIDs). In addition, some NSAIDs have been reported to have prostanoid-independent inhibitory effects on neutrophil functions. In the present study, we examined the effects of acetylsalicylic acid, diclofenac, indomethacin, ketoprofen, piroxicam and tolfenamic acid on leukotriene B₄ (LTB₄)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in vitro. Tolfenamic acid inhibited LTB₄-induced chemotaxis (IC₅₀ 59M), whereas the other compounds were ineffective. Tolfenamic acid inhibited also FMLP-induced chemotaxis at the same concentration range (IC₅₀ 46M). About 25% reduction in the chemotactic response was achieved with therapeutic concentrations of tolfenamic acid. We suggest that the inhibition of PMN chemotaxis is an additional mechanism in the antiinflammatory action of tolfenamic acid and that this action is not ligand specific.     

Leukotriene A4 modulates generation of leukotriene B4 and sulphidopeptide leukotrienes by human neutrophils.

We investigated the influence of exogenous leukotriene A4 (LTA4) on the reactivity of polymorphonuclear leucocytes (PMN). PMN were either prestimulated with LTA4 or incubated simultaneously with LTA4 and the Ca ionophore A23187 or sodium fluoride (NaF). The Ca ionophore A23187 and NaF induced generation of LTB4 from PMN was significantly diminished in the presence of LTA4 while the formation of LTC4 was enhanced. In contrast, preincubation of cells with LTA4 followed by subsequent stimulation with NaF synergistically increased the LTB4 generation from PMN. LTA4, either alone or in combination with the calcium ionophore A23187 or NaF, decreases GTPase activity in human PMN. This decrease was abolished when LTA4 pretreated cells were subsequently stimulated with NaF, but not with calcium ionophore A23187, suggesting a regulatory role of LTA4 on G-proteins. The results demonstrate dual functions of LTA4: it serves as a substrate for the generation of leukotrienes and also regulates the susceptibility of human PMN for subsequent response.     

Studies on the uptake, binding and metabolism of leukotriene B4 by human neutrophils.

Human polymorphonuclear granulocytes (PMN) generate the inflammatory mediator leukotriene B4 (LTB4) as a response to cell activation. In addition, PMN inactivate LTB4 by omega-oxidation resulting in the formation of 20-OH- and 20-COOH-LTB4. The transport of exogenous LTB4 to the metabolizing enzymes is mediated via high- and low-affinity receptor subsets. Uptake of [3H]LTB4 by the cells was carried out in a time-dependent fashion, reaching maximal values after 5 min of incubation. No additional uptake of [3H]LTB4 then occurred. Prestimulation of PMN with phorbol myristate acetate or sodium fluoride resulted in the loss of high- and low-affinity receptors. Deactivating concentrations of LTB4 specifically reduced the high-affinity receptor subset. Prestimulation of PMN with cytochalasin B or with the membrane fluidizer butanol shifted the low-affinity receptors to the high-affinity state. The polyene antibiotic amphotericin B shifted high-affinity receptors to the low-affinity subset. The changes in the receptor expression pattern correlated with the respective conversion rate of exogenously added LTB4. Our results suggest that the distribution of high- and low-affinity receptors is regulated by GTP-binding proteins, the activation of protein kinase C and the organization of the membrane bilayer. In this way, human neutrophils control the respective level of the lipid mediator LTB4.     

Mepacrine inhibits fMLP-induced activation of human neutrophil granulocytes, leukotriene B4 formation, and fMLP binding.

Pulmonary sequestration and activation of polymorphonuclear leukocytes (PMNLs) are characteristic of many forms of acute lung injury. The present experiments were designed to study the effects of mepacrine on human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (fMLP). Mepacrine inhibited fMLP-induced superoxide production and degranulation in a dose-dependent manner with Kd values of 2.3 +/- 0.5 x 10(-7) M and 5.7 +/- 1.3 x 10(-6) M, respectively. Stimulation of PMNLs by 10(-6) M fMLP provoked the formation of barely detectable amounts of leukotriene B4 (LTB4) (< 5 pg/10(7) cells). Pretreatment of the cells with cytochalasin B augmented generation of LTB4 in response to fMLP (339 +/- 79 pg/10(7) cells). LTB4 formation was also inhibited by mepacrine (50% inhibitory concentration 1.0 +/- 0.5 x 10(-6) M). Furthermore, mepacrine inhibited the specific binding of [3H]fMLP to neutrophils with a Ki value of 1.4 +/- 0.4 x 10(-5) M. Mepacrine decreased the receptor binding affinity without altering the number of receptors. These findings demonstrate that the inhibitory effect of mepacrine is response dependent and suggest that this action of mepacrine could, in part, be attributed to a decrease in fMLP receptor affinity.     

In vitro studies of human monocyte migration across endothelium in response to leukotriene B4 and f-Met-Leu-Phe.

Relatively little is known about monocyte emigration from the vasculature or about the factors that regulate this process. In this study, a human in vitro model of a blood vessel wall was used for examination of monocyte transendothelial migration. Umbilical vein endothelial cells were grown to confluency on amnion connective tissue, and human monocytes were stimulated to cross the monolayer in response to the chemoattractants leukotriene B4 or f-Met-Leu-Phe. The pattern and time course of monocyte migration were similar for the two chemotactic factors. In both cases, approximately 40-50% of the adherent monocytes extended single or multiple pseudopods into the apical endothelial surface. This indenting behavior was also observed in the absence of chemotactic factors. It was not affected by the medium (M199 or Gey's) or method of monocyte isolation. Neutrophils also displayed this behavior, but only about half as many neutrophils as monocytes indented the endothelial surface. The integrity of the endothelium remained intact as the monocytes traversed the monolayer. When the monocytes reached the basal surface of the endothelium, they frequently wedged themselves between the basal surface of the endothelium and its basal lamina. The monocytes then invaded the basal lamina and accumulated in the connective tissue. In response to both f-Met-Leu-Phe and leukotriene B4, monocyte migration across the endothelium began as early as 10 minutes. The average rate of accumulation in the connective tissue peaked at 30 minutes; and by 60 minutes, 25-35% of the monocytes had traversed the monolayer. Approximately two to three times as many monocytes traversed the endothelium under conditions of chemotaxis as under conditions of chemokinesis or random migration. These studies provide the basis for understanding the process of monocyte migration out of the bloodstream and lay the foundation for the study of their differentiation into macrophages in the connective tissue.     

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B₄ (LTB₄) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB₄-induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB₄ pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.     

Role of leukotriene B4 in the interleukin-4-induced human mononuclear phagocyte activation.

Interleukin-4 (IL-4) induced a time- and dose-dependent production of leukotriene B4 (LTB4) by human resting monocytes indicating that IL-4 induced the activation of the 5-lipoxygenase pathway in resting human monocytes. Maximal effect was observed in the presence of 10 ng/ml IL-4, and in kinetics experiments LTB4 production plateaued 40 min after the onset of stimulation. When stimulated for 48 hr with IL-4, resting human monocytes expressed and released the low-affinity receptor for IgE (CD23) and were partially inhibited in the presence of a highly non-redox 5-lipoxygenase inhibitor (BW B70C), suggesting that the production of LTB4 partially contributed to the IL-4-induced CD23 expression and release. This hypothesis was strengthened by the fact that exogenous LTB4 (10 nM) was found to increase the effect of a suboptimal dose of IL-4 (1 ng/ml). In addition to these phenotypical changes, IL-4 primed the phorbol-12-myristate-13-acetate (PMA)-induced luminol-dependent chemiluminescence response (LDCL) by normal human monocytes, this priming effect being abrogated in the presence of BW B70C. Taken together, these data indicated that IL-4 induced the production of LTB4 by activation of the 5-lipoxygenase pathway in human monocytes, and that the activation of this pathway could upregulate the expression and release of CD23 and the respiratory burst of these cells.     

Effects of antirheumatic drugs on leukotriene B4 and prostanoid synthesis in human polymorphonuclear leukocytesin vitro

The effects ofd-penicillamine, sodium aurothiomalate, indomethacin, timegadine and tolfenamic acid on the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism were studied in human polymorphonuclear leukocytes (PMNs)in vitro. In short-term incubations,d-penicillamine and aurothiomalate did not affect leukotriene B₄ (LTB₄), prostaglandin E₂ (PGE₂) or thromboxane B₂ (TXB₂) production. Each of the three non-steroidal anti-inflammatory drugs (NSAIDs) used were potent inhibitors of prostanoid synthesis. In higher concentrations they also reduced LTB₄ production; timegadine and tolfenamic acid were effective in concentrations comparable to those measured in plasma during drug therapy, whereas indomethacin was needed in ten times higher concentrations. The different effects of NSAIDs on 5-lipoxygenase activity may be of importance in their therapeutic actions as well as in the appearance of some side-effects, e.g. gastric irritation and aspirin-induced asthma.     

The effects of 5-lipoxygenase inhibitors and leukotriene antagonists on the development of gastric lesions induced by nonsteroidal antiinflammatory drugs in mice

Conclusions The findings presented in this report show that: (1) 5-lipoxygenase inhibitors and some leukotriene antagonists protect the gastric mucosa against lesions induced by oral or parenteral administration of most NSAIDs, with the possibility of some variation in effects with aspirin.(2) These observations suggest that overproduction of leukotrienes and other products of 5-lipoxygenase activity may play a role in the development of acute gastric mucosal damage induced by NSAIDs.     

Carnosine and related compounds inhibit chemiluminescence of human plasma lipoproteinsin vitro

The antioxidant activity of carnosine and related compounds (5 and 10 mM) is assessed from the chemiluminescence curve using a model of Fe-initiated lipid peroxidation of human serum apoB-lipoproteins. These agents lower to different degrees the amplitudes of “fast” and “slow” bursts, the reaction rate, and the chemiluminescence light sum; the latency is prolonged, which may occur upon reduction of lipid hydroperoxides and/or inactivation of free radicals. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 2, pp. 152–154, February, 1995 Presented by I. P. Ashmarin. Member of the Russian Academy of Medical Sciences     

Inhibition of leukotriene B4 in neutrophils by lipoxins A4 and B4

Lipoxins are derived from the oxygenation products of arachidonic acid in human leukocytes. They have exhibited selective biological effects different from those of other eicosanoids. We have examined the effect of lipoxin A₄ and B₄ (LXA₄, LXB₄) on the production of leukotriene B₄ (LTB₄) in human neutrophils. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit LTB₄ generation was assessed after incubation with calcium ionophore A23187. We found that the pretreatment of neutrophils with lipoxins inhibit the release of LTB₄ by A23187 stimulated PMNs. Our data suggests that LXA₄ and B₄ can contribute to immunosuppression in an inflammatory state via the inhibition of LTB₄ synthesis.     

Epstein-Barr virus primes human polymorphonuclear leucocytes for the biosynthesis of leukotriene B4.

In the present study, we have investigated the effect of the short-term incubation of polymorphonuclear leucocytes (PMN) with infectious Epstein-Barr virus (EBV) on leukotriene B(4) (LTB(4)) biosynthesis. Pre-exposure of PMN to EBV led to an increased production of LTB(4) upon stimulation with either the ionophore A23187, the chemotactic peptide fMLP, or phagocytic particles (zymosan). Experiments performed with viral particles pretreated with a neutralizing antibody raised against the gp350 of the viral envelope revealed that a specific interaction between the PMN surface and the viral glycoprotein gp350 is required for the priming effect of EBV. Preincubation of PMN with EBV resulted in an increased release of arachidonic acid upon stimulation with a second agonist. Moreover, LTB(4) biosynthesis in EBV/A23187-treated PMN was greatly diminished in the presence of an inhibitor of the cytosolic phospholipase A2 (cPLA(2)), suggesting that cPLA(2) plays a critical role in the priming effect of EBV. Accordingly, EBV by itself promoted Ser-505 phosphorylation of cPLA(2) and strongly enhanced fMLP-induced phosphorylation of p38 MAP kinase, an enzyme known to phosphorylate cPLA(2) in human PMN. Furthermore, fMLP-induced translocation of cPLA(2) was strongly enhanced when PMN were previously exposed to EBV. These data indicate that binding of EBV to human PMN results in the activation of intracellular events involved in the release of pro-inflammatory lipid mediators.     

Zymosan-induced leukotriene B4 generation by human neutrophils is augmented by rhTNF-alpha but not chemotactic peptide.

Tumour necrosis factor (TNF) is a mediator of inflammation that has been shown to enhance neutrophil responses to soluble and particulate stimuli. The release of leukotriene B4 (LTB4) by human neutrophils stimulated by unopsonized zymosan was measured in the presence or absence of recombinant human TNF-alpha (rhTNF-alpha) preincubation. There was a threefold increase in the LTB4 response at an optimal TNF concentration of 10(-9) M and an optimal preincubation time of 10-20 min. A similar time and dose dependency was observed for CR3 receptor expression and for the release of the secondary lysosomal granule marker, vitamin B12-binding protein. In contrast, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), although stimulating an increase in both CR3 receptor number and in particle phagocytosis, failed to induce an increase in LTB4 release in response to zymosan. In addition, the present study demonstrated that, unlike FMLP, the exocytotic mechanism for secondary granule release by rhTNF-alpha functioned in the absence of a rise in cytosolic free calcium. Furthermore, it was independent of changes in cyclic nucleotide concentrations and did not require an intact cytoskeleton. Thus the capacity of rhTNF-alpha to amplify the neutrophil response to zymosan through the CR3 receptor appears to be related to the amplification of post-membrane events as well as to an increase in the number of functionally active receptors.