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 microM 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 suggests 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.     

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.     

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.     

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.     

Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega-oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.     

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.     

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(-7)M. 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies against rabbit omega-hydroxylases of prostaglandins and fatty acids cross-react with leukotriene B4 omega-hydroxylase in human neutrophils (cytochrome P-450LTB omega)

Leukotriene B4 (LTB4) omega-hydroxylase activity in human neutrophil microsomes was significantly inhibited by antisera against three rabbit omega-hydroxylase P-450s, lung prostaglandin omega-hydroxylase (P-450p-2), small intestine prostaglandin A omega-hydroxylase (P-450ia), and kidney fatty acid omega-hydroxylase (P-450kd). In contrast, the activity is not affected by antibodies raised against the phenobarbital-inducible forms of P-450s from both rabbits and rats. These findings suggest that the LTB4 omega-hydroxylase (P-450LTB omega) is structurally related to a group of rabbit omega-hydroxylase P-450s. The antiserum raised against P-450p-2 also inhibited the NADPH-dependent oxidation of 20-hydroxy LTB4 to 20-oxo LTB4 and 20-carboxy LTB4 by the microsomes, supporting that P-450LTB omega is able to catalyze the subsequent oxidation of 20-hydroxy LTB4 as well as the omega-hydroxylation of LTB4.     

Intracellular retention of the 5-lipoxygenase pathway product, leukotriene B4, by human neutrophils activated with unopsonized zymosan

The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Divergent effects of alpha1-antitrypsin on neutrophil activation, in vitro

alpha1-Antitrypsin (AAT) is a major circulating serine proteinase inhibitor in humans. The anti-proteinase activity of AAT is inhibited by chemical modification. These include inter- or intramolecular polymerisation, oxidation, complex formation with target proteinases (e.g., neutrophil elastase), and/or cleavage by multi-specific proteinases. In vivo, several modified forms of AAT have been identified which stimulate biological activity in vitro unrelated to inhibition of serine proteinases. In this study we have examined the effects of native and polymerised AAT and C-36 peptide, a proteolytic cleavage product of AAT, on human neutrophil activation, in vitro. We show that the C-36 peptide displays striking concentration-dependent pro-inflammatory effects on human neutrophils, including induction of neutrophil chemotaxis, adhesion, degranulation, and superoxide generation. In contrast to C-36 peptide, native and polymerised AAT at similar and higher concentrations showed no effects on neutrophil activation. These results suggest that cleavage of AAT may not only abolish its proteinase inhibitor activity, but can also generate a powerful pro-inflammatory activator for human neutrophils.     

Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis

Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.     

Schistosoma japonicum: identification and characterization of neutrophil chemotactic factors from egg antigen

Two neutrophil chemotactic factors were identified in soluble egg antigen preparations of Schistosoma japonicum. The higher-molecular-weight neutrophil chemotactic factor was not separable from eosinophil chemotactic factor by means of gel filtration, anion-exchange chromatography, isoelectric focusing, or affinity chromatography; this neutrophil chemotactic factor is apparently identical to the higher-molecular-weight eosinophil chemotactic factor which we purified previously from the soluble egg antigen. The chemotactic activity of the eosinophil chemotactic factor for neutrophils was stable to periodate oxidation but was notably affected by heating or Pronase digestion, suggesting that the determinant for neutrophil chemotaxis exists on the peptide moiety of the eosinophil chemotactic factor. The lower-molecular-weight neutrophil chemotactic factor was separable from the higher-molecular-weight eosinophil chemotactic factor by gel filtration or anion-exchange chromatography. This neutrophil chemotactic factor was rather hydrophobic and heat-stable, but was sensitive to Pronase or carboxypeptidase A digestion. These results suggest that the receptors on the surfaces of neutrophils and eosinophils for those chemoattractants would be different from each other. We suppose that neutrophil chemotactic factors and eosinophil chemotactic factors from the eggs are responsible for neutrophil and eosinophil accumulation around the eggs in schistosomiasis japonica.     

Neutrophil chemotaxis and enzyme release competitive inhibition by a diazoacetamide pepsin inhibitor

Treatment of human neutrophils with a reagent (diazoacetylnorleucine methyl ester plus copper ion) which covalently labels the active site of the acid proteases, pepsin and cathepsin D, inhibits neutrophil chemotaxis and enzyme release stimulated by the chemoattractants pepstatin and formylmethiony peptides. In contrast, chemotaxis and enzyme release in response to zymosan activated serum are not affected. Furthermore, diazoacetylnorleucine methy ester plus copper competes with [3H]formylmethionyl leucylphenylalanine for binding to neutrophils. Since pepstatin shares binding sites with formylmethionyl leucylphenylalanine, the present data suggest that diazoacetylnorleucine methyl ester plus copper reacts with the neutrophil receptor for pepstatin and formylmethionyl peptides, and thus may be useful in further characterization of this structure.     

Inhibition of neutrophil leukotriene B4 production by a novel synthetic N-3 polyunsaturated fatty acid analogue, beta-oxa 21:3n-3

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the beta-position, beta-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the beta-oxa-PUFA substantially depresses the production of leukotriene B(4) (LTB(4)) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, beta-oxa 21:3 n-6, is also a strong inhibitor of LTB(4) production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB(4). The parent beta-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB(4) formation. beta-Oxa-21:3n-3 inhibits the conversion of [(3)H]20:4n-6 to [(3)H]5-hydroxyeicosatetraenoic acid and [(3)H]LTB(4) by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. Beta-oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of beta-oxa-21:3n-3 to a 16-monohydroxy-beta-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, beta-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, beta-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.     

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.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

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.     

Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product

Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.