leukotriene-b4 and Leukemia--Myeloid

Leukotrienes (LTs) are potent mediators of bronchial inflammation and are predominantly produced by myeloid cells. As myelocytic cells differentiate towards either eosinophils or neutrophils, the profile of leukotrienes they produce upon stimulation diverges. Eosinophils produce mainly cysteinyl leukotrienes whereas neutrophils predominantly synthesize 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (LTB delta). The mechanism by which this change in leukotriene composition occurs is unknown. In this study, we investigated the control of leukotriene biosynthetic enzymes during myeloid cell differentiation. Western-blot analyses of myelocytic leukemia cell lines, HL-60#7 and HL-60, differentiated towards eosinophilic or neutrophilic cell types, respectively, demonstrated that as myelocytic cells differentiate towards eosinophils or neutrophils, the protein levels of cytosolic phospholipase A2 (cPLA2) remain constant, whereas 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) levels are simultaneously elevated. As myelocytic cells become more eosinophil-like, 5(S)-hydroxy- 6(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (LTC delta) synthase activity and expression of both the protein and messenger RNA in the cells are dramatically increased (approximately 75-fold), while the LTC delta synthase level and activity in neutrophil-like cells remain constant at very low levels. In contrast, in neutrophilic cells, the amount of 5,6-oxido-7,9,11,14-eicosatetraenoic acid (LTA delta) hydrolase was elevated approximately 100-fold greater than the increase in LTA delta hydrolase from eosinophilic cells. These results indicate that as a myeloid cell differentiates towards a granulocyte, similar mechanisms of regulation may be applied to the leukotriene biosynthetic pathway up to the point at which the pathway diverges. At the stage in the leukotriene biosynthetic pathway where LTA delta may be converted to either LTC delta or to LTB delta, specific regulators of transcription may become activated as a myelocytic cell differentiates, thereby causing increased LTA delta hydrolase or LTC delta synthase expression.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Carrier Proteins; Cell Differentiation; Eosinophils; Epoxide Hydrolases; Glutathione Transferase; HL-60 Cells; Humans; Leukemia, Myeloid; Leukotriene B4; Leukotrienes; Membrane Proteins; Neutrophils; Phenotype

7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), a leukotriene B4 (LTB4) receptor antagonist with anti-inflammatory activity in animal models of colitis, was evaluated for effects on superoxide, LTB4 and prostaglandin E2 production. SC-41930 inhibited human neutrophil (PMN) superoxide generation maximally stimulated by f-Met-Leu-Phe (IC50 4 microM) and C5a (IC50 approximately 12 microM). Moreover, postreceptor stimulation of superoxide production by NaF (a G protein activator), but not by phorbol myristate acetate, was significantly inhibited by SC-41930, indicating that SC-41930 may act via attenuation of a G protein-mediated signal transduction. SC-41930 also inhibited A23187-stimulated LTB4 production (IC50 5.3 microM) in human PMN as well as LTB4 (IC50 2.1 microM) and prostaglandin E2 (IC50 2.9 microM) production in HL-60 cells. When coinjected intradermally (400 micrograms/site), SC-41930 inhibited A23187-stimulated increases in LTB4 levels in guinea pig skin. SC-41930 inhibited human synovial phospholipase A2 (IC50 72 microM), A23187-stimulated 5-hydroxy-eicosatetranoic acid production in human PMN (IC50 8.5 microM), and rat peritoneal leukotriene A4 hydrolase (IC50 20 microM), but not ram seminal vesical cyclooxygenase. The results suggest that the anti-inflammatory activity of SC-41930 could be attributed to postreceptor inhibition of inflammatory mediator production by PMN and other cells in addition to antagonism of PMN LTB4 receptors.

Topics: Animals; Benzopyrans; Dinoprostone; Eicosanoids; Epoxide Hydrolases; Guinea Pigs; Humans; Leukemia, Experimental; Leukemia, Myeloid; Leukotriene B4; Neutrophils; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Receptors, Immunologic; Receptors, Leukotriene B4; Skin; Superoxides; Tumor Cells, Cultured

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, Gi2, requires Mg2+ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.

Topics: Affinity Labels; Binding Sites; Complement C5a; Drug Synergism; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Leukemia, Myeloid; Leukotriene B4; Magnesium; Membrane Proteins; N-Formylmethionine Leucyl-Phenylalanine; Photic Stimulation; Receptors, Cell Surface; Ultraviolet Rays

A sesquiterpene thioacetate, 15-acetylthioxy-furodysinin (SK&F 105900) has been isolated from the sponge Dysidea SP. This compound can bind specifically to the human peripheral blood polymorphonuclear leukocyte (PMN) and to the differentiated human monocytic leukemic U-937 cell membrane leukotriene B4 (LTB4) receptors with high-affinity. This compound can also promote a concentration-dependent chemotaxis in PMNs and an intracellular calcium mobilization in U-937 cells that can be blocked by the LTB4 receptor antagonist, LY-223982. Furthermore, the calcium mobilization induced by SK&F 105900 can specifically cross-desensitize with the LTB4-induced calcium mobilization. These observations indicate that SK&F 105900 is a novel and specific high-affinity agonist that can bind to the LTB4 receptors and activate the receptor-mediated signal transduction processes in human PMN and U-937 cells.

Topics: Animals; Calcium; Chemotaxis, Leukocyte; Humans; Leukemia, Myeloid; Leukotriene B4; Neutrophils; Receptors, Immunologic; Receptors, Leukotriene B4; Sesquiterpenes; Tumor Cells, Cultured

Topics: Binding, Competitive; Cell Line; Humans; Kinetics; Leukemia, Myeloid; Leukotriene B4; Receptors, Immunologic; Receptors, Leukotriene; Receptors, Leukotriene B4; SRS-A

Basophils were isolated and propagated in large numbers from the blood of patients with chronic myelogenous leukemia. Propagation over 4-6 weeks of culture was dependent upon a growth factor(s) other than interleukin-2 obtained from a lectin-stimulated clone of the Jurkat cell line. Evidence that these basophils were dividing during culture included an increase in both the number of basophils and the histamine content of the cultures over time, as well as ultrastructural studies that demonstrated basophil cell division. The cells also had the capacity to be stimulated in an IgE-dependent manner characteristic of basophils. Cultured basophils passively sensitized with IgE underwent noncytotoxic degranulation after stimulation with specific antigen. Antigen-stimulated basophils released histamine, leukotrienes B4 and C4 and other 5-lipoxygenase products of arachidonic acid metabolism. Culture models such as this may permit the propagation and purification of sufficient numbers of basophils to allow biochemical and immunological analyses of basophil physiology.

Topics: Arachidonic Acid; Arachidonic Acids; Basophils; Epitopes; Humans; Immunoglobulin E; Lectins; Leukemia, Myeloid; Leukotriene B4; Lipoxygenase; SRS-A; Tumor Cells, Cultured

The metabolism of arachidonic acid through the lipoxygenase pathway was studied in suspensions of fresh human bone marrow cells from eight patients with chronic myelocytic leukemia (CML) and 10 normal controls. After the cells were incubated with the calcium ionophore A23187 and arachidonic acid, a technique including reverse- and straight-phase high-pressure liquid chromatography (HPLC) was employed to isolate and detect different lipoxygenase-mediated compounds. The detected compounds included leukotriene B4 (LTB4), with its two major nonenzymatic isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4 5S,12S-DHETE, and the monohydroxy eicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. The pattern of lipoxygenase-mediated products from the bone marrows was similar to that previously described from human peripheral blood. Of eight bone marrow samples from CML patients, five expressed values above 600 ng LTB4/10(8) nucleated cells, as compared to only one out of 10 controls. In contrast, the CML patients produced significantly lower amounts of both the double-dioxygenation product 5S,12S-DHETE (56.8 +/- 16.0 ng [mean +/- SE] versus 146.1 +/- 31.3 ng; p less than 0.05) and the monohydroxy acid 12-HETE (965 +/- 351 ng versus 4390 +/- 1801 ng; p less than 0.05), indicating a 12-lipoxygenase deficiency. The present results show that leukotrienes are formed by human bone marrow cells and further suggest the existence of altered lipoxygenase activity in CML.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Bone Marrow; Humans; Hydroxyeicosatetraenoic Acids; Leukemia, Myeloid; Leukotriene B4; Lipoxygenase

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.

Topics: Binding Sites; Binding, Competitive; Cell Differentiation; Cell Line; Chemotaxis, Leukocyte; Dexamethasone; Dimethyl Sulfoxide; Female; Granulocytes; Humans; Kinetics; Leukemia, Myeloid; Leukotriene B4; Phospholipids; Receptors, Immunologic; Receptors, Leukotriene B4; Temperature; Triglycerides

The arachidonate metabolism by leukocytes and platelets was studied in 14 patients with myeloproliferative disorders including 7 patients with chronic myeloid leukemia (CML), 5 with polycythemia vera (PV) and 2 with essential thrombocythemia (ET). When the leukocytes were incubated with arachidonate and A23187, leukotriene B4 and hydroxyeicosatetraenoic acids (HETEs) were constantly detected using reversed-phase high-performance liquid chromatography in normal subjects, while selective deficiency of 5-lipoxygenase products (leukotriene B4 and 5-HETE) was found in 4 patients with CML. this novel abnormality of the leukocytes seemed to be derived from the possible deficiency of 5-lipoxygenase in these patients' polymorphonuclear neutrophils (PMNNs). The formation of 15-HETE appeared to be almost normal in all the patients. Platelet 12-lipoxygenase deficiency was detected in 2 patients with PV and 2 with CML in whom one was associated with the deficiency of 5-lipoxygenase products. These bicellular abnormalities of the arachidonate metabolism might contribute to understand dysfunctions of PMNNs and platelets in some patients with myeloproliferative disorders.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Humans; In Vitro Techniques; Leukemia, Myeloid; Leukocytes; Leukotriene B4; Lipoxygenase; Myeloproliferative Disorders; Polycythemia Vera; Prostaglandin-Endoperoxide Synthases; Thrombocytosis