Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotreine B₄ has been developed. The assay is sensitive; 5 pg LTB₄ caused significant inibition of binding of [³H]-LTB₄ and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B₂ and arachidonic acid do not exhibit detectable cross-reactions (< 0.03%). Flowever, some non-cyclic dihydroxy- and monohydroxy-eicosatetraeonic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-$\text{trans}$-14-$\text{cis}$-elcosatetraenoic and 12-hydroxy-5,8,10,14-elcosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB₄ from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB₄ by reverse-phase high liquid chromatography follwing solvent extraction and silinic acid chromatography.     

Characterization of leukotriene A4 and B4 bioysnthesis

We have studied LTA₄ and LTB₄ synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB₄ from arachidonic acid are localized in the soluble fraction (100, 000 x g supernatant) of these cells. The formation of LTA₄ and LTB₄ is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 μM, the synthesis of LTB₄ leveled off at 30 μM and of LTA₄ at 100 μM while 5-HETE had not reached a plateau at 300 μM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA₄ and only a relatively small amount of LTB₄. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA₄ to LTB₄. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA₄ synthesis did not produce any additional LTA₄ or LTB₄. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD₄. This material however had a different retention time from LTD₄ when subjected to HPLC and co-chromatographed with synthetic LTE₄. In addition to LTE₄ a substance which had properties indistinguisable from those of LTB₄ when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB₄. The adventitia and intima were the richest source of LTE₄, the adventitia releasing slightly more than the intima. The output of LTB₄ and LTE₄ was inhibited by 6,9-deepoxy-6,9-(phenylimino)-Δ^6,8 prostaglandin I₁ (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE₄.     

Studies on the conjugation of leukotriene B4 with proteins for development of a radioimmunoassay for leukotriene B4

Leukotriene B₄ (LTB₄) (I) has been converted to its N-(3-aminopropyl)amide derivative (III) and to its hydrazide derivative (VII) via LTB₄ δ-lactone. The amide (III) was coupled with Bovine Serum Albumin using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. The hydrazide (VII), was coupled with Hemocyanin (Keyhole Limpet) (KLH) using 6-N-maleimidohexanoic acid chloride as coupling agent.     

Isomers of leukotriene B4 possess different biological potencies

Rat elicited polymorphonuclear leucocytes (PMNs), when exposed to the ionophore A23187, release three isomers of leukotriene B₄. The three isomers have been purified and tested for their ability to induce the chemokinesis of human PMNs in vitro, the aggregation of rat PMNs in vitro and changes in vascular permeability in rabbit skin in vivo in the presence of PGE₂. The results demonstrate that all three isomers are biologically active and that the enzymatically produced isomer, in which the conjugated triene contains one and two double bonds, is more potent than the two diastereoisomers of LTB₄ which contain all double bonds in the conjugated triene and which are produced by non-enzymatic hydrolysis.     

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B₄ (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [³H]-leukotriene B₄ resulted in the rapid conversion of leukotriene B₄ to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B₄) and 5(S),12(R),19-trihydroxy-6,8,10,14-eicosatetraenoic acid (19-hydroxy-leukotriene B₄). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B₄) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoic acid (Δ16,17–18-carboxy-19,20-dinor-leukotriene B₄) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B₄ by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B₄ from inflammatory sites.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB₄ (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB₄ and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo- LTB₄. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB₄ was at 10⁻⁷M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB₄ was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at C-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB₄. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta⁶ double bond are all important structural features for chemotactic activity with delta⁶ stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies. The data suggested that they acted at the same receptor since even the less active isomers were able to desensitive the neutrophils to LTB₄.     

Benzenepropanoic acids containing chromanone or naphthalenone moieties are potent and orally active leukotriene B4 antagonists

Systematic structural modification of peptidoleukotriene antagonists of the o-hydroxyacetophenone class has led to the discovery of certain [[(3,4-dihydro-4-oxo-8-propyl-2H-1-benzopyran-7-yl)oxy]alkyl]benzenepropanoic acids and related compounds (7), which appear to be potent and selective antagonists of the proinflammatory mediator leukotriene B₄.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

Effects of two leukotriene B4 (LTB4) receptor antagonists (LY255283 and SC-41930) on LTB4-induced human neutrophil adhesion and superoxide production

Leukotriene B4 (LTB4) induces a number of functional changes in human neutrophils, including both superoxide release and CD11b/CD18 (Mo1)-mediated adherence to various substrates, such as keyhole limpet hemocyanin (KLH). These effects are both time- and concentration-dependent. Neutrophil adhesion was at least 10-fold more sensitive to the stimulatory action of LTB4 than superoxide production. Two LTB4 receptor antagonists, LY255283 (1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)heptyloxy )- phenyl)ethanone) and the sodium salt of SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H- 1-benzopyran-2-carboxylic acid) were evaluated for effects on human neutrophil superoxide production and adhesion. Despite being more sensitive to LTB4-induced stimulation, neutrophil adhesion was at least 100-fold less sensitive to inhibition by LY255283 and SC-41930 than superoxide production. Both LTB4 receptor antagonists behaved similarly in these models. These compounds did not inhibit neutrophil responses induced by granulocyte/macrophage colony-stimulating factor (GM-CSF).     

Inhibition of leukotriene B4 biosynthesis by a novel phosphodiesterase inhibitor

TVX 2706, a 3-ethyl-1-(3-nitrophenyl)-2,4-[1H, 3H] quanzazolidione was found to exert strong antiinflammatory properties in vivo. This antiinflammatory potency obviously depends on a pronounced inhibition of phosphodiesterase (PDE) activity, shown in cell culture systems as well as in homogenates of rat PMNL, that causes a marked elevation of intracellular cAMP. In the present study, we have examined the effect of TVX 2706 on the inhibition of leukotriene B₄ (LTB₄) biosynthesis in rat PMNL by the aid of HPLC. TVX 2706 causes an inhibition of LTB₄ generation in an biphasic manner, obviously characteristic for this substance. Within the range of ^1x10^?4M (100%) to 5×10^?6M (40%) the inhibition is concentration-dependent, but all lower concentration down to 5×10^?5 M reduced LTB₄ synthesis to 30–40% of control values on a dose independent manner. No other substance tested until now, produces this characteristics, reproducible pattern of marked leukotriene inhibition. Our results suggest, that inhibition of LTB₄ biosynthesis may be induced by elevated intracellular CAMP levels. In accordance with the biphasic cellular response there is a proved different inhibitory activity of TVX 2706 on high and low affinity PDE that could be responsible for this substance specific effect. Although the exact mode of action of TVX 2706 remains unexplained, all in vivo and in vitro results prove TVX 2706 to be a very potent antiinflammatory substance with an interesting pharmacological profile.     

Failure of leukotriene B4 to translocate calcium across phosphatidylcholine membranes

It has been reported that leukotriene B4 can translocate calcium across model membranes (Serhan et. al., (1982) J. Biol. Chem., 257: 4746). Such ionophoretic behavior could account for its biological effects. We have examined the effect of chromatographically pure leukotriene B4 on Ca2+ permeability when added exogenously at 3 microM to phosphatidylcholine liposomes and when incorporated at 5 mole % in the lipid mixture used to prepare liposomes. No effect was observed with either procedure. An oxidized preparation of leukotriene B4 stimulated calcium permeability, however, suggesting that oxidation may account for the previously reported ionophoretic behavior of leukotriene B4.     

Biosynthesis and biological activity of leukotriene B5

Several studies indicate that increased intake of eicosapentaenoic acid (EPA) in the diet may lead to decreased incidence of thrombotic events. Most investigators agree that this is achieved by competitively inhibiting the conversion of arachidonic acid (AA) to thromboxane A₂ in the platelets. The effect of high EPA-intake on the formation of prostacyclin is less clear. However, EPA is a good substrate for lipoxygenase enzymes which results in formation of hydroperoxy- and hydroxy-acids, and, in some cases, leukotrienes. The biological activities of the leukotrienes derived from arachidonic acid suggest that they mediate or modulate some symptoms associated wth inflammatory and hypersensitivity reactions. In order to clarify the possible effect of dietary manipulation of inflammatory processes, leukotriene B₅ (LTB₅) was prepared and its biological activities assessed. LTB₅ was biosynthesised by incubating EPA with glycogen-elicited polymorphonuclear neutrophils (PMN) from rabbits in the presence of the divalent cation ionophore, A23187. The LTB₅ was extracted from the incubate using minireverse phase extraction columns (Sep-pak) and purified by reverse-phase high pressure liquid chromatography (RP-HPLC). The purity of the product assessed by repeat RP-HPLC and straight phase (SP) HPLC was greater than 95%. Ultra-violet spectrophotometry of the product confirmed its purity and also provided assessment of the yield. The biological activity of LTB₅ was assessed and compared with that of LTB₄ in the following tests: aggregation of rat neutrophils, chemokinesis of human PMN, lysosomal enzyme release from human PMN and potentiation of bradykinin-induced plasma exudation. In all these tests. LTB₅ was considerably less active (at least 30 times) than LTB₄.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

The metabolism leukotriene B4 in synovial fluid and whole human blood

The metabolism of synthetic leukotriene B₄ (LTB₄) in synovial fluid from rheumatoid arthritis and osteoarthritis patients and in whole blood from these same patient groups and from normal volunteers has been studied. A linear relationship existed between a plot of the time of incubation of samples with LTB₄ and the percentage of the initial concentration of LTB₄ at each time point. The slope of this line, the rate constant for metabolism, has been used to compare different samples. LTB₄ was metabolised more rapidly in the synovial fluid of rheumatoid arthritis patients than osteoarthritis patients. Furthermore, LTB₄ was metabolised more rapidly in the blood of rheumatoid arthritis patients that either osteoarthritis patients or normal volunteers. These differences in metabolism correlate with the polymorphonuclear leukocyte (PMN) and albumin content of samples. It is suggested that binding of LTB₄ to albumin will in part determine the available concentration of LTB₄ in inflammatory lesions.     

The metabolism of leukotriene B4 by peripheral blood polymorphonuclear leukocytes in psoriasis

The formation of leukotriene B₄ and its ω-oxidised metabolites has been compared in calcium ionophore-stimulated polymorphonuclear leukocytes, in the absence of exogenous substrate, from fourteen psoriatic subjects and thirteen healthy controls. Although there was no significant difference in the levels of leukotriene B₄, the psoriatic cells synthesised significantly greater amounts of ω-oxidation products than control cells. This difference was confirmed in an experiment comparing the time course of formation of the ω-oxidation products of leukotriene B₄, under similar conditions, in polymorphonuclear leukocytes from four psoriatic subjects and three healthy controls. The kinetic constants for the metabolism of exogenous leukotriene B₄ by 20-hydroxylase were determined by a radiochromatographic enzyme assay in polymorphonuclear leukocytes from three patients with psoriasis and three healthy controls. No significant differences were found in the apparent K_m and V_max values. It is concluded that the increased formation of ω-oxidation products in psoriatic cells may be secondary to increased synthesis of leukotriene B₄ by these cells, with consequent increased metabolism, rather than to an inherent abnormality of the 20-hydroxylase system. Further work is needed to determine the kinetics of the enzymes involved in leukotriene B₄ synthesis in the psoriatic polymorphonuclear leukocyte, and also to assess the contribution of the leukotriene B₄ and ω-oxidation products from polymorphonuclear leukocytes infiltrating the skin to the pathogenesis of the psoriatic lesion.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotrien (LT) B₄ in unstimulated and in A23187-stimulated human whole blood. LTB₄ was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37°C for 60 min produced only trace amounts of LTB₄ (0.16±0.05 ng/ml, mean±SD, n=3). LTB₄-like immunoreactivity (ir-LTB₄) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrealed to cyclooxygenasep or lipoxygenase activity. Incubation of human whole blood with A23187 (2–10 μM) resulted in a concentration-dependent stimulation of LTB₄ production. At 10 μM A23187, ir-LTB₄ was 18±2.4 ng/ml (mean±SEM, n=28). In A23187-stimulated serum samples, LTB₄ concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added caused a dose-dependent inhibition of A23187-stimulated ir-LTB₄ production with an IC₅₀ of 17 μM.     

The leukotriene B4 receptor, BLT1, is required for the induction of experimental autoimmune encephalomyelitis

Leukotriene B₄ (LTB₄) is a potent chemoattractant and activator of neutrophils, macrophages and T cells. These cells are a key component of inflammation and all express BLT1, a high affinity G-protein-coupled receptor for LTB₄. However, little is known about the neuroimmune functions of BLT1. In this study, we describe a distinct role for BLT1 in the pathology of experimental autoimmune encephalomyelitis (EAE) and T_H1/T_H17 immune responses. BLT1 mRNA was highly upregulated in the spinal cord of EAE mice, especially during the induction phase. BLT1^−/− mice had delayed onset and less severe symptoms of EAE than BLT1^+/+ mice. Additionally, inflammatory cells were recruited to the spinal cord of asymptomatic BLT1^+/+, but not BLT1^−/− mice before the onset of disease. Ex vivo studies showed that both the proliferation and the production of IFN-γ, TNF-α, IL-17 and IL-6 were impaired in BLT1^−/− cells, as compared with BLT1^+/+ cells. Thus, we suggest that BLT1 exacerbates EAE by regulating the migration of inflammatory cells and T_H1/T_H17 immune responses. Our findings provide a novel therapeutic option for the treatment of multiple sclerosis and other T_H17-mediated diseases.