In vitro and in vivo chemotaxis of guinea pig leukocytes toward leukotriene B4 and its w-oxidation products

Leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4 were studied for their relative activities towards guinea pig peritoneal eosinophils and neutrophils during in vitro chemotaxis in modified Boyden chambers. The leukotrienes were also injected into guinea pig skin, and the cellular infiltrate in 4 hour biopsies was evaluated histologically. Eosinophils migrated more actively than neutrophils towards LTB4 in vitro, while in vivo, more neutrophils were observed. 20-OH-LTB4 was markedly less active than LTB4 in vivo and in vitro, and 20-COOH-LTB was barely active at all. Crude ionophore-stimulated neutrophil supernatants (ECF) were more active towards eosinophils than towards neutrophils, both in vivo and in vitro, compared to the pure leukotrienes. The data confirm the potent chemotactic properties of LTB4 for eosinophils and neutrophils, with less activity of its w-metabolites.     

Catabolism of leukotriene B5 in humans

Human neutrophils, enriched by dietary supplementation with eicosapentaenoic acid, form leukotriene (LT)B5 in addition to LTB4 upon stimulation. LTB5 is one order of magnitude less biologically active than the potent chemokinetic and chemoattractant LTB4. Catabolites of LTB5 have not yet been characterized in vitro and ex vivo. It is unknown whether catabolism of LTB5 interferes with catabolism of LTB4. This report describes catabolism of LTB5 to 20-OH-LTB5, which in turn is catabolized to 20-COOH-LTB5. The structures of the two catabolites were established by UV-absorbance, behavior on reverse-phase high-performance liquid chromatography, enzymatic analysis of human neutrophils, and gas chromatography-mass spectrometry. In vitro, formation of LTB4 was delayed and formation of its catabolites was depressed by exogenous eicosapentaenoic acid. By supplementing the diet of six volunteers with 5 g eicosapentaenoic acid/day for 7 days, eicosapentaenoic acid quadrupled in neutrophil phospholipid fatty acids. Consequently, LTB5, 20-OH-LTB5, and 20-COOH-LTB5 were detected ex vivo. In contrast to the findings in vitro, however, levels of LTB4, 20-OH-LTB4, and 20-COOH-LTB4 were unaltered by the dietary intervention. Thus, in vitro, but not ex vivo, addition of eicosapentaenoic acid, and subsequent formation of LTB5, impeded catabolism of proinflammatory LTB4.     

Chemotaxis of human neutrophils and eosinophils towards leukotriene B4 and its 20-w-oxidation products in vitro

Peripheral blood neutrophils and eosinophils from 70 patients and controls were studied for their in vitro chemotactic and chemokinetic responses towards synthetic leukotriene B₄ (LTB₄), 20-OH-LTB₄ and 20-COOH-LTB₄. All three factors induced chemotaxis and chemokinesis of cells. 20-OH-LTB₄ was always less and 20-COOH-LTB₄ even less active than the parent compound. Cells from patients with atopic eczema and T cell lymphoma moved less than cells from normal controls or from patients with psoriasis. In the presence of LTB₄, 20-OH-LTB₄ and buffer alone, more eosinophils than neutrophils moved to the lower side of the filter, while this did not occur with platelet activating factor as chemoattractant. Studies of neutrophil and eosinophil chemotaxis in the presence of LTB₄ should therefore always take into account a high variability of the quantitative response which is donor and disease dependent.     

Effect of structural modification at carbon atom 1 of leukotriene B4 on the chemotactic and metabolic response of human neutrophils

Human neutrophils biosynthesize the chemoattractant leukotriene B4 (LTB4) and metabolize LTB4 to omega oxidative products 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4). In this study, we prepared the C-1 methyl ester and N-methyl amide of LTB4 and then examined neutrophil chemotaxis and metabolism of these derivatives of LTB4. The results show that chemical modification of LTB4 at carbon atom 1 dramatically affects metabolism of the lipid molecule. The free acid form of LTB4 was taken up and metabolized by human neutrophils, while the methyl ester and N-methyl amide derivatives were poor substrates for omega oxidation. Although human neutrophils were poorly attracted to the methyl ester of LTB4, the amide derivative was a complete agonist of the neutrophil chemotactic response and displayed an ED50 for chemotaxis identical to that of LTB4. Therefore, we concluded that omega oxidation is not a requirement for the neutrophil chemotactic response induced by LTB4. These results also indicate that the N-methyl amide of LTB4 may be a useful ligand for the elucidation of molecular mechanisms operative in neutrophil chemotaxis to LTB4, since the C-1 derivative is not further metabolized. Two separate responses of human neutrophils are elicited by LTB4, resulting in both cellular activation and generation of omega oxidation products. It appears that putative receptors on the neutrophils can distinguish between LTB4 and certain derivatives that are structurally identical except for modification at the C-1 position (i.e., the methyl ester). LTB4 derivatives modified at the C-1 position do not undergo conversion to omega oxidation products by the neutrophil.     

Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils

Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD+ and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD+ and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for omega-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes omega-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1:2. The affinity of the dehydrogenase for 20-OH-LTB4 may be higher than that for 12-hydroxylaurate (Km = 70 microM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase.     

The identification and formation of 20-aldehyde leukotriene B4

Microsomes of human polymorphonuclear leukocytes (PMN) in the presence of 100 microM NADPH converted 0.6 microM leukotriene B4 (LTB4) to 20-OH-LTB4 (retention time = 18.0 min) and to two additional compounds designated I (retention time = 16.8 min) and II (retention time = 9.6 min) as analyzed by reverse-phase high performance liquid chromatography (HPLC). Compounds I and II were also formed from the reaction of 1.0 microM 20-OH-LTB4, PMN microsomes, and 100 microM NADPH; the identity of compound II was confirmed as 20-COOH-LTB4 by gas chromatography-mass spectrometry. Equine alcohol dehydrogenase in the presence of 100 microM NAD+ in 0.2 M glycine buffer (pH 10.0) converted 20-OH-LTB4 to 20-aldehyde (CHO) LTB4, which coeluted with compound I on reverse-phase HPLC. In the presence of 100 microM NADH in 50 mM potassium phosphate buffer (pH 6.5), equine alcohol dehydrogenase reduced both 20-CHO-LTB4 and compound I to 20-OH-LTB4, indicating the identity of compound I as 20-CHO-LTB4. Gas chromatography-mass spectrometry of trideuterated O-methyl-oxime trimethylsilyl ether methyl ester derivative of 3H-labeled compound I definitively established compound I as 20-CHO-LTB4. Addition of immune IgG to cytochrome P-450 reductase or 1.0 mM SKF-525A completely inhibited the formation of 20-CHO-LTB4 from 20-OH-LTB4, indicating that the reaction was catalyzed by a cytochrome P-450. LTB5 (3.0 microM), a known substrate for cytochrome P-450LTB and a competitive inhibitor of LTB4 omega-oxidation, completely inhibited the omega-oxidation of 1.5 microM 20-OH-LTB4 to 20-CHO-LTB4, indicating that the cytochrome P-450 was P-450LTB. Conversion of 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 by PMN microsomes was also dependent on NADPH and inhibited by antibody to cytochrome P-450 reductase, 1.0 mM SKF-525A, or 5.0 microM LTB5, indicating that this reaction was also catalyzed by cytochrome P-450LTB. These results identify the novel metabolite 20-CHO-LTB4 and indicate that cytochrome P-450LTB catalyzes three sequential omega-oxidations of LTB4 leading to the formation of 20-COOH-LTB4 via 20-OH-LTB4 and 20-CHO-LTB4 intermediates.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

Release of platelet-activating factor and the metabolism of leukotriene B4 by the human neutrophil when studied in a cell superfusion model

Stimulated human neutrophils are known to synthesize large quantities of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 5,12-dihydroxy-6,14-cis-8,10-trans-transeicosatetraenoic acid (LTB4). However, in an isolated cell suspension the majority of synthesized PAF appears to remain cell associated. In addition, LTB4 is rapidly metabolized to an omega-oxidation product (20-OH-LTB4). Experiments were designed to test the hypothesis that the degree of association of PAF with the neutrophils and the metabolism of LTB4 by the neutrophils is a result of the in vitro condition used during cell activation. Here we have compared in paired experiments ionophore A23187-induced production of PAF and LTB4 by human neutrophils in a concentrated cell suspension, a diluted cell suspension and in a system in which the cells are placed on a matrix and superfused with buffer at a constant flow rate (dynamic system). There was little difference in the amount of PAF synthesized in the concentrated cell suspension and the dynamic system. However, less PAF was produced by neutrophils in the dilution system. The percent of PAF released was consistently greater in the dynamic and dilution systems than in the concentrated cell suspension. For example, more than 40% of PAF measured by incorporation of [3H]acetate or gas chromatography/mass spectrometry was released in the dynamic system and dilution systems. In contrast, less than 15% of the PAF synthesized was released from the cells in the concentrated cell suspension. 1-0-Hexadecyl-2-acetyl-3-GPC was primarily released from the neutrophils. By contrast both 1-0-hexadecyl and 1-0-octadecyl linked species of PAF were found within the cells. Exogenous PAF added to neutrophils in the dynamic or dilution systems was taken up and metabolized at a significantly lower rate than that added to cells in the concentrated cell suspension. Most of the leukotrienes synthesized by the neutrophil during A23187 stimulation were released from the cells. However, studies of LTB4 metabolism revealed differences between the dynamic and concentrated cell suspension designs. By 20 min, most of the LTB4 was recovered as 20-OH-LTB4 in the concentrated cell suspension, whereas in the dynamic system little 20-OH-LTB4 was found in the superfusate over 20 min. These experiments suggest that a large proportion of PAF synthesized by neutrophils may be released. They also suggest that the omega-hydroxylation of LTB4 by neutrophils occurs after synthesized LTB4 is released and taken back up by the cell.     

Identification of an aldehyde dehydrogenase in the microsomes of human polymorphonuclear leukocytes that metabolizes 20-aldehyde leukotriene B4

We have previously reported that cytochrome P-450LTB in the microsomes of human polymorphonuclear leukocytes (PMN) catalyzes three omega-oxidations of leukotriene B4 (LTB4), leading to the sequential formation of 20-OH-LTB4, 20-CHO-LTB4, and 20-COOH-LTB4 (Soberman, R.J., Sutyak, J.P., Okita, R.T., Wendelborn, D.F., Roberts, L.J., II, and Austen, K. F. (1988) J. Biol. Chem. 263, 7996-8002). The identification of the novel final intermediate, 20-CHO-LTB4, allowed direct analysis of its metabolism by PMN microsomes in the presence of adenine nucleotide cofactors. Microsomes in the presence of 100 microM NAD+ or 100 microM NADP+ converted 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 with a Km of 2.4 +/- 0.8 microM (mean +/- S.E., n = 4) and a Vmax of 813.9 +/- 136.6 pmol.min-1.mg-1, for NAD+, as compared to 0.12 microM and 5.0 pmol.min-1.mg-1 (n = 2) for NADPH as a cofactor. The conversion of 1.0 microM of 20-CHO-LTB4 to 20-COOH-LTB4 in the presence of saturating concentrations (1.0 mM) of both NAD+ and NADP+ was not greater than the reaction in the presence of 1.0 mM of each cofactor separately, indicating that NAD+ and NADP+ were cofactors for the same enzyme. Antibody to cytochrome P-450 reductase did not inhibit the conversion of 20-CHO-LTB4 to 20-COOH-LTB4. When 1.0 microM 20-OH-LTB4 was added to microsomes in the presence of NADPH, approximately three-fourths of the product formed (63.7 +/- 5.1 pmol; mean +/- S.E., n = 3) was 20-CHO-LTB4 and approximately one-fourth (21.3 +/- 3.9 pmol; mean +/- S.E., n = 3) was 20-COOH-LTB4. In the presence of both NADPH and NAD+, only 20-COOH-LTB4 (85.5 +/- 9.9 pmol; mean +/- S.E., n = 3) was formed. PMN microsomes also contain an NADH-dependent aldehyde reductase which converts 20-CHO-LTB4 to 20-OH-LTB4, a member of the LTB4 family of molecules with biological activity. Based upon kinetic, cofactor and inhibition data, microsomal aldehyde dehydrogenase preferentially regulates the final and irreversible inactivation step in the LTB4 metabolic sequence.     

Effects of leukotrienes and f-Met-Leu-Phe on oxidative metabolism of neutrophils and eosinophils

We assessed the effects of several leukotrienes and of f-Met-Leu-Phe on oxygen consumption in neutrophils and on the initial burst of chemiluminescence (CL) in both neutrophils and eosinophils. It was found that f-Met-Leu-Phe initiated 2.6 times higher oxygen consumption in neutrophils than did leukotriene B4 (LTB4). f-Met-Leu-Phe also stimulated five to 10 times more CL from both types of granulocytes than LTB4, which was at least five times more potent than its omega-hydroxylated metabolite, 20-OH-LTB4, whereas the corresponding 20-COOH derivative was effective only in eosinophils. The double dioxygenation product 5(S), 12(S)- DHETE caused no CL. Neutrophils from patients with chronic granulomatous disease did not respond with CL to any of the agents. The peak of CL occurred 50 to 60 sec after the addition of fMLP, whereas the LTB4-associated peak occurred after 5 to 6 sec and then rapidly subsided. The treatment of cells with sodium azide to inhibit the myeloperoxidase system did not change the kinetics or the rapid decline of the LTB4-induced CL. The CL response to LTB4 could be inhibited to 85% by 0.5 microgram/ml of superoxide dismutase, to 72% by 200 mg/ml of catalase, and to 50% by 80 microM of mannitol. The corresponding figures for f-Met-Leu-Phe-induced CL were 80, 58, and 16%, suggesting that, although a substantial part of the CL appears to be due to superoxide ion production, other oxygen radicals are involved in luminol-enhanced CL production. Thus, in contrast to some previous reports that leukotrienes do not stimulate an oxidative metabolic response in granulocytes despite their potent activity as chemotactic factors, our studies show that leukotrienes are definite inducers of granulocyte oxidative metabolism.     

Oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. Role of aldehyde dehydrogenase and leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega) in leukotriene B4 omega-oxidation

Leukotriene B4 (LTB4), a potent chemoattractant for leukocytes, is catabolized by human neutrophils via omega-oxidation. Neutrophil microsomes are known to oxidize 20-hydroxy-LTB4 (20-OH-LTB4) to its 20-oxo and 20-carboxy derivatives in the presence of NADPH. This activity has been ascribed to LTB4 omega-hydroxylase (cytochrome P-450LTB omega), a conclusion supported by our finding of the reversal of carbon monoxide inhibition by 450 nm light and by competitive inhibition studies. The oxidation of 20-oxo-LTB4 to 20-carboxy-LTB4 is also catalyzed by microsomes fortified with 1 mM NAD+, and this activity is not affected by cytochrome P-450LTB omega inhibitors. The evidence is compatible with involvement of a disulfiram-insensitive aldehyde dehydrogenase in this second oxidation pathway. Interaction of the two pathways is evidenced by facilitation of NADPH-dependent oxidation of 20-OH-LTB4 by the addition of NAD+. This synergism may be explained by removal of the aldehyde intermediate by the NAD(+)-dependent aldehyde dehydrogenase. Taken together with the finding that the NAD(+)-dependent activity is severalfold higher than the NADPH-dependent one, the dehydrogenase may be important in the oxidation of 20-OH-LTB4 to 20-carboxy-LTB4.     

Specific binding of leukotriene B4 to guinea pig lung membranes

We have demonstrated binding sites for LTB4 in guinea pig lung membranes. Binding of [3H]-LTB4 was of high affinity (Kd = 0.76 nM), saturable and linear with protein concentration (0.2-1.2 mg/ml). Scatchard and Hill's plot analysis indicated a single class of binding site with a Hill's coefficient of 0.99 +/- 0.08 (n = 4). [3H]-LTB4 was unmetabolized during incubation with membrane preparations, as indicated by high performance liquid chromatography. Divalent cations such as Mg2+ and Ca2+ enhanced binding capacity without changing the Kd. Na+ ions decreased binding in a concentration-dependent manner. Guanine nucleotides, GTP, GTP gamma S and Gpp(NH)p also decreased the number of binding sites. Finally, competition experiments demonstrated the following order of potency for displacement of [3H]-LTB4 from its receptor site: LTB4 greater than 20-OH-LTB4 much greater than 20-COOH-LTB4 = 6-trans-12-epi-LTB4 greater than LTC4 = LTD4 = 5-HETE. These data indicate that a specific LTB4 receptor, in addition to the previously documented LTC4 and LTD4 receptors, exists in guinea pig lung.     

Transition of affinity states for leukotriene B4 receptors in sheep lung membranes.

Leukotriene B4 (LTB4) is a pro-inflammatory arachidonate metabolite. We have characterized the LTB4 receptors in sheep lung membranes and have assessed the contribution of the guanine-nucleotide-binding (G) protein in the regulation of receptor affinity states. Saturation isotherms have demonstrated a single class of LTB4 receptor with a Kd of 0.18 +/- 0.03 nM and a density (Bmax.) of 410 +/- 84 fmol/mg of protein in sheep lung membranes. The effect of the G-protein on receptor affinity was assessed in the presence of non-hydrolysable GTP analogues (e.g. GTP[S]) and in membranes following alkali treatment (pH 12.1) to remove the G-protein. Saturation isotherms produced either in the presence of GTP[S] (Kd.GTP[S] = 0.51 +/- 0.02 nM) or with alkali-treated membranes (Kd.alk. = 0.52 +/- 0.02 nM) demonstrated a 3-fold shift in receptor affinity for [3H]LTB4 binding. In competition experiments, the rank order of affinity of LTB4 analogues was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than 20-COOH-LTB4, using either untreated or alkali-treated membranes, both in the presence and absence of GTP[S]. These findings demonstrate that, in sheep lung membranes, there is only one class of LTB4 receptor. Removal of the G-protein or uncoupling of the receptor from the G-protein shifted the agonist-binding affinity of the receptor by 3-4-fold, without affecting the specificity of the LTB4 receptor in either the high- or the low-affinity state.     

Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated with calcium ionophore A23187

Human blood eosinophils and neutrophils that had been incubated with the supernatants of cultures of lipopolysaccharide (LPS)-stimulated blood mononuclear cells demonstrated respective enhanced abilities to produce immunoreactive leukotriene C4 (LTC4) and immunoreactive leukotriene B4 (LTB4) after activation by the calcium ionophore A23187. Under optimal conditions, the enhancing effect was observed with the eosinophils (n = 21) and the neutrophils (n = 14) from all but one donor of each type of granulocyte. Enhancement was maximum when granulocytes were preincubated with a 1/3 dilution of LPS-stimulated mononuclear cell culture supernatants for 1 to 2.5 min and were then stimulated with 2.5 microM ionophore for 1 to 2 min (neutrophils) or 15 min (eosinophils). Maximal enhancement ranged from 20 to 4500% for LTC4 generation by eosinophils (geometric mean, 87%) and from 30 to 1600% for LTB4 generation by neutrophils (geometric mean, 105%). There was no enhancement of leukotriene biosynthesis when the LPS-stimulated mononuclear cell culture supernatants and ionophore were added simultaneously to the granulocytes. The enhancing activity for LTC4 generation by eosinophils was removed by washing the cells after the addition of the LPS-stimulated mononuclear cell culture supernatants and before the introduction of ionophore. This enhancing activity was produced by Ig-, Leu-1- adherent blood mononuclear cells, which are presumed to be monocytes; supernatants of adherent cells augmented A23187-induced LTC4 generation by eosinophils from 21 to 2300% (geometric mean, 402%) in 11 experiments and LTB4 generation by neutrophils from 7 to 200% (geometric mean, 60%) in 10 experiments. There was an inverse correlation between the percent enhancement and the LTC4 levels produced by stimulated eosinophils in the absence of the monokine(s) (r = -0.79, p less than 0.01), but not between percent enhancement and the LTB4 levels generated by ionophore-activated neutrophils in the control buffer. The activity of the monocyte-derived enhancing material on each type of granulocyte was relatively heat stable. Enhancement of eosinophil production of LTC4 was associated with an acidic group of monocyte-derived molecules having isoelectric points of 4.2 to 4.3, 4.5 to 4.6, and 4.9, and exhibiting marked heterogeneity in size.(ABSTRACT TRUNCATED AT 400 WORDS)     

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.     

Functional properties of guinea pig eosinophil leukotriene B4 receptor.

It is currently thought that pulmonary eosinophils play a proinflammatory role in bronchial asthma. Leukotriene B4 (LTB4) is being considered as an important mediator in regulating eosinophil function because of its potent activities in inducing leukocyte chemotaxis, chemokinesis, degranulation, and aggregation. Because the LTB4 receptor has not been characterized in eosinophils, we report in this study the presence of a functional high affinity receptor for LTB4 on guinea pig (GP) eosinophils. Scatchard analysis of saturation binding studies yielded a Kd of 1.4 +/- 0.2 nM (mean +/- SEM, n = 3) and a Bmax of 1.6 +/- 0.4 pmol/mg of protein for LTB4 in GP eosinophil membranes. A linear Scatchard plot was obtained, suggesting that GP eosinophil membranes expressed only a single high affinity LTB4 receptor population. Saturation binding studies in whole cells also yielded a linear Scatchard plot, with a Kd of 2.8 +/- 0.96 nM (mean +/- SEM, n = 4) and a Bmax of 4 x 10(4) +/- 6 x 10(3) receptors/cell. Competitive binding studies using several compounds with structures similar to that of LTB4 showed that these agents bound to the receptor in the following descending order of affinity (Ki, nM): LTB4 (0.96) less than TB3 (1.0) greater than 20-hydroxy-LTB4 (3.5) greater than 12(R)-hydroxy-5,8,14-cis,10-trans-eicosatetraenoic acid (20) greater than 12(S)-hydroxy-5,8,14-cis,10-trans-eicosatetraenoic acid (231) greater than 20-carboxy-LTB4 (350) greater than 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid (541). This rank order of potency in binding affinity correlates closely with the ability of these compounds to induce both chemotaxis and superoxide anion generation. Analysis of the structure-activity relationship suggests that the 12R-hydroxyl group and a cis double bond at the C-6 position are important for optimal agonist binding to the LTB4 receptor present in GP eosinophil membranes. The results suggest that LTB4 may be an important chemoattractant for eosinophils in GP and may induce the release of reactive oxygen species from this cell.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism

As a model to perhaps better indicate potential in vivo tissue inflammatory events, the generation of leukotriene (LT)B4, 20-OH-LTB4, sulfidopeptide LT, and platelet-activating factor (PAF) from human whole blood stimulated with zymosan was compared with that produced by isolated human neutrophils suspended either in buffer or plasma. Several reports have shown that substantial LTB4 biosynthesis could be induced after addition of zymosan to whole blood, but little was known concerning the generation of other important lipid mediators, or the cellular source of these. We have shown that, in spite of some subject variation, the zymosan-induced production of 20-OH-LTB4, LTB4, and LTE4 reached maxima within 30 to 60 min with 1.1, 2.8, and 0.60 ng/10(6) neutrophils, respectively. These concentrations would be sufficient to induce significant biologic effects. Studies with isolated cell mixtures suggested that the neutrophil was the primary source of the lipid mediators or their precursors in this system, although a number of other cell types contributed as accessory cells to the final amounts and mix of mediators produced. The ratio of neutrophils to accessory cells in mixed cell experiments dramatically modified the metabolic pattern of leukotriene generation. The concentration of LTB4 was increased in the presence of RBC and that of LTE4 when platelets were present. These results suggested that cellular cooperation and transcellular biosynthesis played a key role in the overall production of eicosanoids such as LTB4 and LTC4. The concomitant synthesis of PAF in isolated cells and in whole blood was also determined as another member of the complex lipid mediator network. Maximal production of cell-associated PAF was observed within 30 min after the initiation of phagocytosis and reached levels of 3 to 5 ng PAF/10(6) neutrophils. When other cells were present in a coincubation system, the time course for production of PAF was not altered, but maximal concentration of PAF was lower, perhaps as a result of enhanced PAF metabolism. Study of eicosanoids and other lipid mediator production in mixed cell populations provides insight into those events occurring within tissues, where cross-cell signaling and transcellular biosynthesis may occur.     

Measurement of icosanoids. Report of the Group for Standardization of Methods in Icosanoid Research

This statement from laboratories highly qualified in icosanoid analysis identifies the urgent need for the availability of the following compounds in labeled (deuterium and tritium) and unlabeled form: PGE2 PGF2 alpha PGD2 6-keto-PGF1 alpha Thromboxane B2 9 alpha,20-dihydroxy-11,15-dioxo-2,3- dinorprost -5-enoic acid 9 alpha-hydroxy-11,15-dioxo-2,3,18,19- tetranorprost -5-ene-1,20-dioic acid 15-keto-13,14-dihydro-PGE2 15-keto-13,14-dihydro-PGF2 alpha 5 alpha-7 alpha-dihydroxy-11- ketotetranorprosta -1,16-dioic acid 7 alpha-hydroxy-5,11-diketo- tetranorprosta -1,16-dioic acid 2,3 dinor-thromboxane B2 2,3 dinor-6-keto-PGF1 alpha 2,3 dinor-6,15-diketo 13,14 dihydro-20-carboxyl-PGF1 alpha 2,3 dinor-13,14-dihydro-6,15-diketo-PGF1 alpha LTB4 LTC4 LTD4 LTE4 LTF4 20-OH-LTB4 20-COOH-LTB4 5-HETE 12-HETE 15-HETE omega-OH-12-HETE 5S, 12S-di HETE 5S, 15S-di HETE HHT other hydroxylated polyunsaturated fatty acids and their epoxides.     

Leukotriene B4 binding to human neutrophils

[3H] Leukotriene B4 (LTB4) binds concentration dependently to intact human polymorphonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4 degrees C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [3H] LTB4 indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 X 10(-9)M and Bmax of 1.96 X 10(4) sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 X 10(-9)M and a Bmax of 45.16 X 10(4) sites per neutrophil. Competitive binding experiments with structural analogues of LTB4 demonstrate that the interaction between LTB4 and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25 degrees C [3H] LTB4 is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB4. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [3H] LTB4 binding, suggesting that LTB4 is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB4 in neutrophils are tightly coupled processes.