Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

Biosynthesis of 20,20,20-trifluoroleukotriene B4 from 20,20,20-trifluoroarachidonic acid: a metabolically stable analog of leukotriene B4 and its application to a study of stimulation of leukotriene B4 synthesis by immunoglobulin G1,2

Leukotriene B4 is rapidly metabolized through omega-oxidation, preventing its detection when it is produced under certain biological conditions. To investigate leukotriene B4 production in various physiological conditions, analogs of arachidonic acid which are converted to metabolically stable analogs of leukotriene B4 would be useful. We have synthesized 20,20,20-trifluoroarachidonic acid by the cis-selective Wittig reaction of the C12-C20 fragment with phosphonium salt. 20,20,20-trifluoroarachidonic acid was transformed into 20,20,20-trifluoroleukotriene B4 when incubated with human neutrophils in the presence of the calcium ionophore A23187. The product was identified by uv absorption spectrophotometry, gas chromatography-mass spectrometry, and coelution on high-performance liquid chromatography with 20,20,20-trifluoroleukotriene B4, which was enantioselectively synthesized by the reaction of the fluorine-containing C11-C20 fragment with the C1-C10 phosphonate. The fluorinated leukotriene B4 demonstrated as much chemotactic activity on human neutrophils as natural leukotriene B4 and was metabolically stable when incubated with human neutrophils, probably by blocking omega-oxidation. Also, enzymes catalyzing the transformation of arachidonic acid (AA) into leukotriene B4 did not discriminate the fluorinated precursors from the natural, nonfluorinated AA, thus 20-F3-AA is a valid analog of AA to be used in the study of AA metabolism. When 50 microM of the fluorinated acid was incubated with neutrophils stimulated with heat-aggregated human immunoglobulin G, a significant amount of fluorinated leukotriene B4 (4.3 ng/10(6) cells/40 min, at most) was formed in a dose-dependent manner while little leukotriene B4 was detected with incubation with 50 microM arachidonic acid, probably due to omega-oxidation of the product, leukotriene B4. 20,20,20-Trifluoroarachidonic acid appears to be a useful tool for studying the capacity of leukotriene B4 synthesis in various biological systems while long-lasting 20,20,20-trifluoroleukotriene B4 would serve as an excellent analog of leukotriene B4 in pharmacological studies to understand functions of leukotrienes B4.     

Human peritoneal macrophages synthesize leukotrienes B4 and C4

Macrophages were isolated from the dialysis fluid of patients undergoing continuous ambulatory peritoneal dialysis and separated by gradient centrifugation and purification on 50% Percoll. The cells were prelabeled with [14C]arachidonic acid for 1.5 h. The labeled cells were then incubated with calcium ionophore A23187 (1 microM), serum-treated zymosan (200 micrograms/ml), and a lipoxygenase inhibitor, nordihydroguairetic acid (1 X 10(-5) M). The arachidonate metabolites in the medium were separated on Sep-Pak columns, and finally purified by reverse-phase high-pressure liquid chromatography (HPLC). The labeled products co-chromatographed with authentic leukotriene B4 and leukotriene C4 standards. Serum-treated zymosan and A23187 significantly stimulated and nordihydroguairetic acid significantly inhibited leukotriene synthesis. Leukotriene D4 was not detected, which suggests that these cells contain low gamma-glutamyltranspeptidase or high dipeptidase activity. These results establish, for the first time, that human peritoneal macrophages synthesize the lipoxygenase products, leukotriene B4 and leukotriene C4.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Leukotriene A3. A poor substrate but a potent inhibitor of rat and human neutrophil leukotriene A4 hydrolase

Analysis of leukotriene B4 production by purified rat and human neutrophil leukotriene (LT) A4 hydrolases in the presence of 5(S)-trans-5,6-oxido-7,9-trans-11-cis-eicosatrienoic acid (leukotriene A3) demonstrated that this epoxide is a potent inhibitor of LTA4 hydrolase. Insignificant amounts of 5(S), 12(R)-dihydroxy-6-cis-8,10-trans-eicosatrienoic acid (leukotriene B3) were formed by incubation of rat neutrophils with leukotriene A3 or by the purified rat and human LTA4 hydrolases incubated with leukotriene A3. Leukotriene A3 was shown to be a potent inhibitor of leukotriene B4 production by rat neutrophils and also by purified rat and human LTA4 hydrolases. Covalent coupling of [3H]leukotriene A4 to both rat and human neutrophil LTA4 hydrolases was shown, and this coupling was inhibited by preincubation of the enzymes with leukotriene A4. Preincubation of rat neutrophils with leukotriene A3 also prevented labeling of LTA4 hydrolase by [3H]leukotriene A4. This result indicates that leukotriene A3 prevents covalent coupling of the substrate leukotriene A4 and inhibits the production of leukotriene B4 by blocking the binding of leukotriene A4 to the enzyme.     

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.     

Human B and T lymphocytes convert leukotriene A4 into leukotriene B4

Incubation of human tonsillar B lymphocytes and peripheral blood T lymphocytes with leukotriene A4 led to the formation of leukotriene B4. The purity of these cell suspensions was more than 99%, containing less than 0.5% monocytes. Incubation of purified B or T lymphocytes with the calcium ionophore A23187 did not lead to the formation of any detectable amounts of leukotrienes. Several established cell lines of B and T lymphocytic origin were also found to convert leukotriene A4 into leukotriene B4, showing that monoclonal lymphocytic cells possess leukotriene A4 hydrolase activity.     

Leukotriene B4 enhances activation, proliferation, and differentiation of human B lymphocytes

Highly purified human tonsillar B lymphocytes at different stages of activation were incubated with leukotriene B4 (LTB4). As a key marker for activation, we used the CD23 Ag. LTB4 enhanced the CD23 expression on resting B cells in synergy with B cell-stimulating factors from 4% to 50%. Maximal effect of LTB4 was observed at 10(-10) M to 10(-12) M. LTB4 also augmented the S and M phase entries as well as Ig secretion in synergy with IL-2 and IL-4. In contrast, 5S,12S-dihydroxyeicosatetraenoic acid, an isomer of LTB4, and leukotriene C4 lacked these effects. The results indicate that LTB4 amplifies lymphokine-driven activation, replication, and differentiation of human B lymphocytes.     

Leukotriene A4 hydrolase: analysis of some human tissues by radioimmunoassay

Leukotriene A4 hydrolase was quantitated by radioimmunoassay, in extracts from eight human tissues. The enzyme was detectable in all tissues, with the highest level (2.6 mg per g soluble protein) in leukocytes, followed by lung and liver. The polyclonal antiserum did not cross-react with cytosolic epoxide hydrolase purified from mouse or human liver. When incubated with leukotriene A4, formation of leukotriene B4 was evident in all tissues. Furthermore, enzymatic formation of (5S,6R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid from leukotriene A4, was found in extracts from liver, kidney and intestines.     

Catabolism of leukotriene A4 into B4, C4, and D4 by rat liver subcellular fractions

[3H]Leukotriene A4 was incubated with various subcellular fractions of rat liver homogenates. After solvent extraction and purification on C18 Sep-Pak cartridges, tritiated products migrating on reversed-phase HPLC with authentic unlabelled leukotriene C4, D4 and B4 were observed. The identity of leukotriene C4 was confirmed through enzymatic conversion into D4 by gamma-glutamyl transpeptidase as well as by bioassay on the rat stomach fundus after HPLC purification. The contractile response to the extracted material was blocked by the SRS antagonist, FPL 55712. Leukotriene B4 synthesis was located in the 100 000 X g supernatant, while C4 synthesis was present in the corresponding pellet. Leukotriene C4 formation was enhanced when reduced glutathione was supplemented in the incubation medium. These results demonstrate the presence in rat liver of various enzymatic steps in leukotriene A4 catabolism.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Metabolism of leukotriene A4 into C4 by human platelets

Tritium-labelled leukotriene A4 is converted by a suspension of human platelets into leukotriene C4. The conversion is stimulated by reduced glutathione and is dependent on the platelet concentration. Formation of leukotriene C4 is temperature and time dependent and is destroyed by heating the platelets at 100 degrees C for 5 min. Verification of leukotriene C4 formation was obtained by conversion into leukotriene D4 during reaction of the HPLC-purified platelet-derived leukotriene C4 with commercial gamma-glutamyl transpeptidase. In separate experiments we incubated authentic tritiated leukotriene C4 with human platelets and we showed the formation of tritiated leukotriene D4, demonstrating the presence of gamma-glutamyl transpeptidase activity in these cells. This activity could be blocked by the presence of reduced glutathione in the incubation mixture. In contrast, erythrocytes converted tritiated leukotriene A4 almost exclusively into leukotriene B4. Although platelets have been reported to lack 5-lipoxygenase activity, our study demonstrates that platelets possess the necessary machinery to transform leukotriene A4 into leukotrienes C4 and D4. Our results suggest that an intracellular interaction between platelets and leukotriene A4-forming cells, e.g., polymorphonuclear leukocytes, could lead to the formation of these potent peptidolipids in the circulation.     

Metabolism of leukotriene A4 by human erythrocytes. A novel cellular source of leukotriene B4

Human erythrocytes transformed leukotriene A4 into leukotriene B4. Metabolism was proportional to the erythrocyte concentration, even at subphysiological levels (0.08-4 X 10(9) erythrocytes/ml). Comparative metabolic studies excluded the possibility that leukotriene B4 originated from trace amounts of polymorphonuclear leukocytes or platelets present in the purified erythrocyte suspensions. For example, suspensions of isolated platelets (100-500 X 10(6) cells/ml) failed to convert leukotriene A4 into leukotriene B4; and conversion by suspensions of isolated polymorphonuclear neutrophils was insufficient to account for the amounts of leukotriene B4 formed by erythrocytes. Leukotriene B4 formation was maximal within 2 min and substrate concentration dependent. Enzymatic activity originated from a 56 degrees C labile nondialyzable (Mr greater than 30,000) soluble component in the 100,000 X g supernatant obtained from lysed erythrocytes. In contrast to the contemporary view, our results indicate that human erythrocytes are not metabolically inert in terms of eicosanoid biosynthesis. The role of human erythrocytes during inflammatory or pulmonary disorders deserves re-examination in this context.     

Characterization of biological properties of synthetic and biological leukotriene B3

Leukotriene B3 was chemically synthesized and its ability to aggregate rat polymorphonuclear leukocytes (PMN) and to enhance chemokinesis of human leukocytes demonstrated. In both these assays the potency of synthetic leukotriene B3 was marginally less than that of leukotriene B4. Rat PMN incubated with leukotriene A3 were very inefficient in the enzymatic conversion of this epoxide to leukotriene B3. However, the leukotriene B3 produced was able to aggregate rat PMN. These results suggest that unlike leukotriene B5, the proinflammatory properties of leukotriene B3 are similar to those of leukotriene B4. However, since the enzymatic conversion of leukotriene A3 to leukotriene B3 is extremely poor it seems unlikely that leukotriene B3 itself has any major role in vivo.     

Metabolism of leukotriene E4 by rat tissues: formation of N-acetyl leukotriene E4

Leukotriene E4 was incubated with subcellular fractions from rat liver homogenates. A product identified as 5-hydroxy-6-S-(2-acetamido-3-thiopropionyl)-7,9-trans-11,14- cis-eicosatetraenoic acid (N-acetyl leukotriene E4) was formed. Enzymes catalyzing the reaction were associated with particulate fractions sedimenting between 600 and 8500 g and 20,000 and 105,000 g. Acetyl coenzyme A served as the donor of the acetyl group. N-Acetyl leukotriene E4 was also formed by the 105,000g sediment fractions from kidney, spleen, skin, and lung. The myotropic activity of N-acetyl leukotriene E4 on isolated guinea pig ileum was reduced over 100-fold compared to that of leukotriene D4.     

Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.     

Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.     

Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.     

Appearance of specific leukotriene B4 binding sites in myeloid differentiated HL-60 cells

Exposure of HL-60 cells for 6 days to a combination of 1.25% (v/v) dimethyl sulfoxide and 10 microM dexamethasone induces myeloid differentiation which results in a cell with many of the characteristics of a mature granulocyte. At 4 degrees C myeloid differentiated, but not undifferentiated, monocytic differentiated or eosinophilic differentiated HL-60 cells display marked specific leukotriene B4 binding. Leukotriene B4 binding at 4 degrees C reaches a maximum within 10 min, is readily reversed by unlabeled leukotriene B4, and is stereospecific. Only molecules with structural and biological similarity to leukotriene B4 can competitively inhibit leukotriene B4 binding. Scatchard analysis at 4 degrees C in differentiated cells shows two classes of binding sites. The high affinity sites have a Kd of 0.27 nM and a Bmax of 14.8 fmol/10(7) cells; the low affinity sites have a Kd of 0.58 microM and a Bmax of 2453 fmol/10(7) cells. The appearance of specific leukotriene B4 binding sites in the myeloid differentiated cells correlates with their ability to chemotax in response to leukotriene B4. Undifferentiated cells do not chemotax to leukotriene B4. At 37 degrees C leukotriene B4 is incorporated into phospholipid and triglyceride species in both undifferentiated and myeloid differentiated HL-60 cells making binding studies at 37 degrees C in intact cells impossible. No evidence of omega-hydroxylase activity was found in HL-60 cells. These data suggest that the HL-60 cell may be an excellent model system for the study of leukotriene B4 receptor binding, processing, and gene expression.