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.     

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.     

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.     

Eicosapentaenoic acid modulates neutrophil leukotriene B4 receptor expression in cystic fibrosis.

In patients with cystic fibrosis (CF), high intrapulmonary concentrations of the neutrophil chemotaxin leukotriene B4 (LTB4) are associated with specific reduction of LTB4-induced chemotaxis of circulating neutrophils. The chemotactic abnormality is partially corrected by dietary supplementation with eicosapentaenoic acid (EPA). LTB4-induced neutrophil chemotaxis is mediated by specific, high-affinity, cell surface LTB4 receptors. The hypotheses that neutrophil LTB4 receptors are down-regulated in CF, and that EPA normalizes receptor expression, were tested by measuring the number (Rmax) and affinity (Kd) of LTB4 receptors on neutrophils from eight CF patients before and after EPA (6 weeks of 2.7 g/day), and from nine normal individuals. High-affinity receptor Rmax was depressed in CF patients (0.6 +/- 0.2 x 10(4)/cell (mean +/- s.d.) versus 1.8 +/- 0.7 x 10(4)/cell in normals), but corrected to normal (2.0 +/- 1.9 x 10(4)/cell) after EPA. High-affinity receptor Kd was depressed in CF patients (0.4 +/- 0.3 nM versus 1.4 +/- 0.5 nM in normals), and also corrected to normal with EPA (1.4 +/- 1.2 nM). Low-affinity receptors were depressed, but did not change significantly with EPA. These results indicate that neutrophil responses in chronic inflammatory lung disease can be influenced directly by LTB4 receptor modulation, and that this effect of EPA predominates over alterations in neutrophil signal transduction in situations of chronic exposure to LTB4.     

Peripheral blood neutrophil leukotriene B4 release and migration in rheumatoid arthritis

The present study was designed to compare peripheral blood neutrophil migration and leukotriene (LT) release between patients with rheumatoid arthritis (RA) and healthy controls and to correlate the neutrophil functions with clinical disease activity. Nineteen patients with moderately active RA and 19 age and sex matched healthy volunteers participated in this study. Isolated peripheral blood neutrophils from RA patients released equal amounts of LTB₄ but their random migration was enhanced as compared with neutrophils from healthy controls. LTB₄ release in whole blood was significantly lower in samples from RA patients than in those from the healthy volunteers (13.5±1.4 and 19.1±1.4 ng/10⁶ neutrophils respectively; P<0.001). LTB₄ release from isolated RA neutrophils correlated with the levels of C-reactive protein, duration of morning stiffness and Ritchie articular swelling index. Concentrations of hyaluronate, cyclic AMP and 13,14-dihydro-15-keto-prostaglandin were not different between patients with RA and healthy volunteers. Neither was there any difference in TXB₂ production by platelets during blood clotting. In conclusion, peripheral blood neutrophils of RA patients seem to be primed and/or activated as their random migration is enhanced as compared with those of healthy volunteers. In RA, LTB₄ release from peripheral blood neutrophils seems to reflect the clinical activity of the disease. However, RA neutrophils released smaller (in whole blood) or equal (isolated cells) amounts of LTB₄ as compared with the respective controls. These contradictory findings suggest that LTB₄ release from peripheral blood neutrophils has no major role in the regulation of disease activity in rheumatoid arthritis.     

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.     

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.     

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.     

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.     

Biological activities of leukotriene B4

Leukotriene B₄ isomer III is released from polymorphonuclear leucocytes, monocytes, eosinophils and macrophagesin vitro and has been detected and measured in human synovial fluidin vivo. Its most prominent biological activities are to induce the aggregation of and to stimulate the movement (chemokinesis and chemotaxis) of leucocytesin vitro and it acts as a cytotaxinin vivo. In addition, it increases vascular permeabilityin vivo when administered together with the vasodilator, PGE₂.     

Eosinophil and neutrophil production of leukotriene C4 and B4: comparison of cells from asthmatic subjects and healthy donors

Production of LTC4 and LTB4 by eosinophils and neutrophils was compared between nine asymptomatic asthmatic subjects and eight healthy donors. We observed a statistically significant difference in LTC4 generated by eosinophils, but not LTB4 produced by neutrophils. These findings suggest a quantitative difference in eosinophils between asthmatic subjects and healthy donors.     

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.     

Enzymes involved in the biosynthesis of leukotriene B4 and prostaglandin E2 are active in sebaceous glands

The expression of enzymes involved in leukotriene and prostaglandin signalling pathways, of interleukins 6 and 8 and of peroxisome proliferator-activated receptors in sebaceous glands of acne-involved facial skin was compared with those of non-involved skin of acne patients and of healthy individuals. Moreover, 5-lipoxygenase and leukotriene A₄ hydrolase were expressed at mRNA and protein levels in vivo and in SZ95 sebocytes in vitro (leukotriene A₄ hydrolase > 5-lipoxygenase), while 15-lipoxygenase-1 was only detected in cultured sebocytes. Cyclooxygenase-1 and cyclooxygenase-2 were also present. Peroxisome proliferator-activated receptors were constitutively expressed. Enhanced 5-lipoxygenase, cyclooxygenase 2 and interleukin 6 expression was detected in acne-involved facial skin. Arachidonic acid stimulated leukotriene B₄ and interleukin 6 release as well as prostaglandin E₂ biosynthesis in SZ95 sebocytes, induced abundant increase in neutral lipids and down-regulated peroxisome proliferator-activated receptor-, but not receptor-1 mRNA levels, which were the predominant peroxisome proliferator-activated receptor isotypes in SZ95 sebocytes. In conclusion, human sebocytes possess the enzyme machinery for functional leukotriene and prostaglandin pathways. A comprehensive link between inflammation and sebaceous lipid synthesis is provided.T. Alestas and R. Ganceviciene contributed equally to the study     

Effects of auranofin on leukotriene production and leukotriene stimulated neutrophil function

Arachidonic acid is metabolized in neutrophils by lipoxygenase to leukotrienes, which are suggested to play a central role in inflammation. The antirheumatic drug auranofin (4 g/ml) was found not to inhibit neutrophil production of the lipoxygenase products 5-HETE, 15-HETE and LTB₄,in vitro when stimulated with the calcium ionophore A23187. Auranofin, however, modulated neutrophil aggregation, enzyme release and chemotaxis induced by LTB₄. The results suggest that auranofin may exert some of its antirheumatic effects through affecting neutrophil responses to leukotrienes.Supported by grants from: Swedish National association against Rheumatism, King Gustav V 80 years Fund, Tore Nilssons Fund, P and A Hedlunds Fund, Swedish Society for Medical Research, Förenade Liv Mutual Group Life Insurance Company, Stockholm, Sweden.     

Identification of leukotriene B4 as the neutrophil chemotactic factor released by antigen challenge from passively sensitized guinea pig lungs

Neutrophils are prominent in some IgE-mediated allergic reactions and may contribute to the pathophysiology of immediate hypersensitivity. Antigen challenge of fragments of guinea pig lung tissue that were passively sensitized with IgE or IgG antibody evoked the release of neutrophil chemotactic activity (NCA) in parallel with histamine. The NCA released from lung tissue by both IgG- and IgE-dependent stimulation coeluted from a column of Sephacryl S-300 with synthetic leukotriene B4 (LTB4). The NCA in eluates from the Sephacryl S-300 column contained LTB4, as determined by high-performance liquid chromatography and specific radioimmunoassay, in quantities that accounted for the observed chemoattractant activity in the eluates. Furthermore, the NCA of supernatants from antigen-challenged lung fragments was reduced by a mean of 80% after absorption with a monoclonal antibody to LTB4. LTB4 thus constitutes the major functional constituent of NCA released after anaphylactic challenge of IgE- and IgG-sensitized guinea pig lung tissue.     

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.