Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.     

Recognition of human polymorphonuclear leukocyte receptors for leukotriene B4 by rabbit anti-idiotypic antibodies to a mouse monoclonal antileukotriene B4

Rabbit anti-idiotypic IgG antibodies to the combining site of a mouse monoclonal IgG2b antibody to leukotriene B4 (LTB4) cross-reacted with human polymorphonuclear (PMN) leukocyte receptors for LTB4. Anti-idiotypic IgG and Fab both inhibited the binding of [3H]LTB4, but not [3H]N-formylmethionyl-leucylphenylalanine (fMLP), to PMN leukocytes with similar concentration-effect relationships, whereas neither nonimmune rabbit IgG nor Fab had any inhibitory activity. At a concentration of anti-idiotypic IgG that inhibited by 50% the binding of [3H] LTB4 to PMN leukocytes, the antibodies preferentially recognized high affinity receptors. Anti-idiotypic IgG and Fab inhibited PMN leukocyte chemotactic responses to LTB4, but not fMLP, with concentration-effect relationships resembling those characteristic of the inhibition of binding of [3H] LTB4, without altering the LTB4-induced release of beta-glucuronidase. Chemotaxis and increases in the cytoplasmic concentration of calcium equal in magnitude to those elicited by optimal concentrations of LTB4 were attained at respective concentrations of anti-idiotypic IgG equal to and 1/25 the level required for inhibition of binding of [3H]LTB4 by approximately 50%. Thus, the anti-idiotypic antibodies bound to PMN leukocyte receptors for LTB4 with a specificity, preference for high affinity sites, and capacity to alter PMN leukocyte functions that were similar to LTB4.     

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.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

Selective modulation by guanine nucleotides of the high affinity subset of plasma membrane receptors for leukotriene B4 on human polymorphonuclear leukocytes

Isolated human polymorphonuclear (PMN) leukocyte plasma membranes express high affinity (mean Kd = 0.12 nM) and low affinity (mean Kd = 50 nM) receptors for the chemotactic factor leukotriene B4 (5(S),12(R)-dihydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid; LTB4) that are similar to those on intact PMN leukocytes. A portion of high affinity LTB4-R on PMN leukocyte membranes were converted to the low affinity state by GTP (mean +/- SE = 28.6 +/- 14.0%) and nonhydrolyzable GTP analogues, such as 5'-guanylylimidodiphosphate (GMP-PNP), in a concentration-dependent, nucleotide-specific, and reversible manner, without altering the intrinsic binding affinities of either class. [3H]GMP-PNP bound specifically to one class of receptors (mean Kd = 13 nM) on PMN leukocyte membranes. The interdependence of the LTB4-binding membrane protein and guanine nucleotide-binding protein was suggested by the capacity of LTB4 to enhance by a maximum of 150% the binding of [3H]GMP-PNP to PMN leukocyte membranes by increasing the number, but not altering the affinity, of receptors for GMP-PNP. Pertussis toxin, but not cholera toxin, reversed the enhancement of binding of [3H]GMP-PNP produced by LTB4. Guanine nucleotide-binding proteins and high affinity LTB4-R thus exhibit a mutual regulation that differs mechanistically from that of peptide chemotactic factor receptors on PMN leukocytes.     

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

Conversion of leukotriene B4 to dihydro and 19-hydroxy metabolites by rat polymorphonuclear leukocytes

Rat polymorphonuclear leukocytes metabolize leukotriene B4 (LTB4) by at least two major pathways. LTB4 is converted by a reductase in these cells to a dihydro metabolite in which one of the three conjugated double bonds has been reduced to give a conjugated diene with a UV absorption maximum at 230 nm. DihydroLTB4 appears to be a key intermediate in the metabolism of LTB4 by rat polymorphonuclear leukocytes, since a number of other metabolites, exhibiting UV absorbance at 235 nm, but not at 280 nm, have been detected by high pressure liquid chromatography. In addition, these cells contain a 19-hydroxylase, which converts LTB4 to 19-hydroxyLTB4, which has a typical leukotriene UV spectrum, exhibiting absorption maxima at 261, 270, and 282 nm.     

Molecular and cellular properties of human polymorphonuclear leukocyte receptors for leukotriene B4

The distinctive characteristics of human polymorphonuclear (PMN) leukocyte receptors for leukotriene B4 (LTB4) have been elucidated by studies of binding of [3H]LTB4, the structure of protein constituents of the receptors isolated from plasma membranes, and the effects of antireceptor antibodies. A high-affinity class of 4400 receptors with a KD of 0.4 nM mediates chemotaxis and increased adherence of PMN leukocytes, whereas a low-affinity class of 270,000 receptors with a KD of 61 nM mediates the release of lysosomal enzymes and increases in oxidative metabolism. The low-affinity receptors are composed of a 60,000-dalton protein-binding unit. The high-affinity receptors are composed of the same binding unit in association with a 40,000-dalton guanine nucleotide-binding protein. That antireceptor antibodies as well as LTB4 distinguish the two classes of receptors with different functional consequences suggests the possibility of unique approaches to the regulation of leukocyte function at the receptor level.     

Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase

LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.     

Some newly synthesized leukotriene B4 analogs inhibit LTB4-induced lysozyme release from rat polymorphonuclear leukocytes

Antagonistic activities of newly synthesized LTB4 analogs, which possess the same olefinic geometry and the same hydroxyl group stereochemistry as natural LTB4, were investigated by studying lysozyme release from rat polymorphonuclear leukocytes (PMNLs). 14,15-Dihydro LTB4(LTB3) induced lysozyme release from PMNLs as well as LTB4, while 14,15-dehydro LTB4 did not cause lysozyme release but instead clearly inhibit LTB4-induced lysozyme release. Compounds containing aminocarbonyl groups partially retained lysozyme releasing activity. A displacement of the hydrocarbon chain at C13-20 by cyclohexenyl and beta-cyclohexylethyl groups had little effect on lysozyme release, but did strongly inhibit release induced by LTB4. These results may be useful in developing LTB4 antagonists.     

Essential fatty acid deficiency inhibits the in vivo generation of leukotriene B4 and suppresses levels of resident and elicited leukocytes in acute inflammation

Essential fatty acid (EFA) deficiency exerts an anti-inflammatory effect in several models of inflammation. In an effort to understand underlying mechanisms, the effect of EFA deficiency on the generation of eicosanoids and the elicitation of leukocytes in a model of acute inflammation was examined. Acute inflammation was induced by the i.p. injection of zymosan in mice. The injection of zymosan in normal mice was followed by a short burst of eicosanoid synthesis lasting 2 hr. Leukotriene (LT)B4, LTC4, LTD4, and LTE4, thromboxane B2, and 6-keto-prostaglandin F1 alpha were detected using high pressure liquid chromatography and specific radioimmunoassays. This initial phase of eicosanoid production was followed by a more prolonged infiltration of leukocytes (predominantly polymorphonuclear neutrophils (PMN)) lasting 48 hr with little eicosanoid synthesis. When challenged with zymosan, EFA-deficient mice exhibited a marked decrease in the production of eicosanoids during the early phase. No LTB could be detected at all. The number of resident peritoneal macrophages in EFA-deficient mice was also substantially decreased, and the influx of PMN during the inflammatory response was markedly diminished. In order to establish that the generation of eicosanoids during the early phase of this model of acute inflammation played a causal role in the later infiltration of PMN, the effect of the mixed lipoxygenase/cyclooxygenase inhibitor, BW755C, on LTB formation and PMN influx in this model of inflammation was assessed in control animals. BW755C completely blocked LTB synthesis and inhibited the subsequent influx of PMN. In conclusion, EFA deficiency inhibits eicosanoid generation, depresses levels of resident macrophages, and markedly diminishes the influx of PMN in the acute inflammatory response. The decrease in PMN influx appears to result from the inhibition of the antecedent generation of LTB.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (Tc) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB4 can induce human Tc to exert suppressor cell activity, and incubation of lymphocytes with LTB4 for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40-90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O2 X-) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 +/- AA for icosanoid release; phorbol myristate acetate for O2 X-production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca2+-dependent K+permeability in PMNL was measured by the cell K+ release induced by A 23187. Tc and Tc subsets were determined using monoclonal antibodies (OKT3+, OKT4+ and OKT8+). A biphasic sequential release of the different substances (leukocytic icosanoids and O2 X-) was monitored: increase (approximately 36-48 h after Tl) and decrease (greater than or equal to 72 h after Tl). The increase in AA stimulation was more transient than that of O2 X -. The decline in the release of AA metabolites and O2 X-production was associated with the anergic phase (decrease OKT4+/OKT8+ ratio) and with the clinical outcome of the patients. The decrease in LTB4 and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca2+-dependent K+ permeability increased early up to 2 or 3 times normal. In order to go further with the mechanism of inhibition of LTB4 and O X-release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB4 secretion. A less important inhibition was observed with O2 X-release (-32%) and Ca2+-dependent K+ permeability (-30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): "suppressive factor(s) of membrane activation "SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromatography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O X 2-release.     

Enhanced levels of leukotriene B(4) in synovial fluid in Lyme disease

The purpose of this study was to evaluate the potential role of LTB(4) and cysteinyl leukotrienes in Lyme disease (LD). Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB(4) as well as LTC(4) secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB(4) levels in synovial fluid were significantly increased in patients with Lyme arthritis (median 142 ng/ml, range 88-296) when compared to the control subjects with NIA (median 46 ng/ml, range 28-72) (p < 0.05). No statistical difference of urinary LTE(4) levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB(4) production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB(4) in synovial fluid of patients with Lyme arthritis underline the involvement of LTB(4) in the pathogenesis of this disease.     

Inflammation and pain sensitivity: effects of leukotrienes D4, B4 and prostaglandin E1 in the rat paw

Leukotrienes (LT's) and prostaglandins (PG's) have been proposed as mediators of vascular permeability changes in inflammatory reactions. Also, prostaglandins, especially of the E-type, have been shown to enhance pain responses. In the present studies in rats, the effects of LTB4 and LTD4 on edema and pain thresholds were examined in combination with PGE1 and/or brewer's yeast. Subplantar injections of LTD4 or LTB4 induced small increases in paw thickness which were potentiated by the co-administration of PGE1. LTD4 alone had no significant effect on the development of the yeast paw edema. LTB4 was found to reduce significantly the yeast edema and this reduction could be reversed by administration PGE1. A small but significant decrease in pain threshold was caused by PGE1 and this was significantly enhanced in the presence of LTD4. LTB4, like PGE1, was found to cause slight hyperalgesia but no synergy between the two agents was observed. LTD4 was found to have no effect on the initial hypoalgesia or subsequent development of hyperalgesia caused by brewer's yeast. Both LTB4 and PGE1, however, prevented the initial hypoalgesia and significantly reduced the latency for development of yeast induced hyperalgesia. These effects of LTB4 are discussed in terms of possible release of cyclooxygenase products.     

Biological properties of dihydro-leukotriene B4, an alternative leukotriene B4 metabolite

Dihydro-leukotriene B4 (a 5,12-dihydroxy-eicosatrienoic acid) has been shown to be the primary metabolite of leukotriene B4 (LTB4) in a variety of cells other than human polymorphonuclear leukocytes (PMNLs). In this report we show that dihydro-LTB4 is significantly less active than LTB4 in different biological assay systems, i.e. leukocyte chemotaxis, chemokinesis, aggregation, adhesion to endothelium and superoxide anion production. This suggests that primary reduction constitutes a second so far unknown deactivation pathway for LTB4.     

Leukotriene B4 production by stimulated whole blood: comparative studies with isolated polymorphonuclear cells

A new method was developed to study leukotriene B4 (LTB4) production by stimulated whole blood. The calcium ionophore A23187 and serum-treated zymosan induced LTB4 production, measured by radioimmunoassay, in a dose- and time-dependent manner. The pattern of LTB4 production by whole blood differed markedly from that observed with isolated, purified polymorphonuclear leukocytes. Higher levels of LTB4 were reached and maintained in whole blood. The system allowed to detect drug effects on LTB4 synthesis in vitro. This new method to study the synthesis of LTB4 takes into account the complex interactions between different cell types which can modulate LTB4 metabolism.     

Role of leukotriene B4 in the pathogenesis of hepatic ischemia-reperfusion injury in the rat.

A common feature to most models of ischemia-reperfusion injury is the accumulation of polymorphonuclear leukocytes (PMNs) into the post-ischemic tissue during the reperfusion period. Interventions that lead to decreased PMN infiltration protect against tissue injury and therefore a knowledge of the chemotactic mediators leading to PMN accumulation is essential to understanding the pathogenesis of the injury and to the development of successful therapeutic strategies. Leukotriene B4 (LTB4), a metabolite formed via the 5-lipoxygenase pathway from arachidonic acid, is one of the most potent chemotactic mediators known. We have investigated the formation of LTB4 in a well characterized model of hepatic ischemia-reperfusion injury in the rat and made use of a specific leukotriene biosynthesis inhibitor, L663,536, to determine the importance of LTB4 in the pathogenesis of the injury. LTB4 concentrations were measured with a specific and sensitive gas chromatographic-mass spectrometric method previously developed in our laboratory. In liver tissue LTB4 levels were below the detection limit of 20 pg/g before 45 min ischemia and did not increase during the first 6 h of reperfusion. However, at 15 h and 24 h reperfusion LTB4 concentrations had increased to levels 50-fold those in control liver (867 +/- 267 pg/g). The increase of plasma alanine aminotransferase (ALT) activities indicated two phases of injury, an initial phase during the first few hours of reperfusion, and a second more severe injury phase between 6 h and 24 h reperfusion. PMNs accumulated in tissue throughout the reflow period reaching 700 +/- 49 per 50 high power fields (HPF) at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of stereoisomers of leukotriene B4 to dihydro and tetrahydro metabolites by porcine leukocytes

We have previously shown that porcine leukocytes convert leukotriene B4 (LTB4) to two major products, 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4. Although we did not detect these products after incubation of LTB4 with human polymorphonuclear leukocytes, these cells converted 12-epi-6-trans-LTB4 to the corresponding 6,11-dihydro metabolite (i.e., there appeared to be a shift in the positions of the remaining double bonds). The objective of the present investigation was to determine whether 6-trans isomers of LTB4 are metabolized by porcine leukocytes by a pathway similar to LTB4, or whether they are metabolized by a pathway analogous to that in human leukocytes. We found that 6-trans-LTB4 and 12-epi-6-trans-LTB4 are metabolized more much extensively than LTB4 by porcine leukocytes. 6-trans-LTB4 appears to be converted by two different reductase pathways to two dihydro products differing in the positions of the two remaining double bonds between carbons 5 and 12. Dihydro-12-oxo and dihydro-5-oxo metabolites are also formed from this substrate. Porcine leukocytes also convert 6-trans-LTB4, presumably by a combination of the above two pathways, to tetrahydro, tetrahydro-12-oxo and tetrahydro-5-oxo metabolites, none of which possesses any conjugated double bonds. 12-epi-6-trans-LTB4 is also converted to tetrahydro metabolites by these cells. Experiments with deuterium-labeled 6-trans-LTB4 indicated that the deuterium in the 5-position was almost completely lost during the formation of tetrahydro-6-trans-LTB4, whereas about 80-85% of the deuterium in the 12-position was lost, suggesting a requirement for a 5-oxo intermediate. As with LTB4, 12-epi-8-cis-6-trans-LTB4, the product of the combined actions of 5-lipoxygenase and 12-lipoxygenase, was converted principally to dihydro and dihydro-12-oxo metabolites. Only a relatively small amount of the tetrahydro metabolite was detected.     

Inhibitory effect of LY 255283 on the synthesis of leukotriene B(4) and thromboxane A(2) in human peripheral blood polymorphonuclear leukocytes and monocytes

LY 255283 [(1-(5-ethyl-2-hydroxy-4-(6-methyl-6-)1H-tetrazol-5-yl)-heptyloxy) phenyl)ethanone], a specific leukotriene B(4) (LTB(4)) receptor antagonist, inhibited the production of LTB(4) in human peripheral blood polymorphonuclear leukocytes (PMNL) and in monocytes activated by calcium ionophore A23187. In human monocytes activated by ionophore it inhibited also the production of thromboxane B(2) (TXB(2)). The effect of LY 255283 on 5-lipoxygenase (5-LO) and LTA(4) hydrolase activities which catalyse the production of LTB(4) and LTA(4) has not been studied yet. It is thought that LY 255283 may inhibit the production of LTB(4) and TXA(2) by antagonising the effect of ionophore-induced LTB(4) on 5-lipoxygenase and cyclooxygenase in human peripheral blood PMNL and monocytes.