prostaglandin-d2 and Leukemia--Basophilic--Acute

Mast cells produce chemical mediators, including histamine and arachidonate metabolites such as prostaglandin D(2) (PGD(2)) after antigen stimulation. Cyclopentenone prostaglandins of the J series, prostaglandin J(2) (PGJ(2)) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), are thought to be derivatives of PGD(2). In this study, the biphasic effects of the PGJ(2) and 15d-PGJ(2) on proliferation and apoptosis in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mast cells, were examined. At low concentrations, 1 or 3 microM PGJ(2) and 15d-PGJ(2) induced cell proliferation, respectively. At high concentrations (10-30 microM) both the inhibition of viability and decrease in histamine content in RBL-2H3 cells were dose dependent. These effects were independent of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), since troglitazone, an agonist of PPARgamma did not cause any effects in RBL-2H3 cells. Cell death induced by PGJ(2) and 15d-PGJ(2) was the result of apoptotic processes, since RBL-2H3 cells treated with 30 microM of the prostaglandins had condensed nuclei, DNA fragmentation and increase in activities of caspase-3 and -9. Moreover, PGJ(2) or 15d-PGJ(2)-induced apoptotic effects were prevented by the caspase inhibitor, z-VAD-fmk. In conclusion, the PGJ(2) or 15d-PGJ(2)-induced apoptosis in RBL-2H3 cells occurs mainly via mitochondrial pathways instead of by PPARgamma-dependent mechanisms.

Topics: Animals; Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Division; Cyclopentanes; Drug Interactions; Enzyme Inhibitors; Histamine; Leukemia, Basophilic, Acute; Prostaglandin D2; Prostaglandins; Rats; Tumor Cells, Cultured

Hematopoietic prostaglandin D synthase (H-PGDS) is a key enzyme in the production of prostaglandin D and its J series metabolites. We evaluated the antiinflammatory effect of retrovirally transfected H-PGDS in order to investigate the role of H-PGDS in monosodium urate monohydrate (MSU) crystal-induced acute inflammation.. Expression of endogenous PGDS in a murine air-pouch model of MSU crystal-induced acute inflammation was determined by real-time polymerase chain reaction. H-PGDS complementary DNA (cDNA) was retrovirally transfected into C57BL/6J fibroblasts, and the cells were designated as C57-PGDS cells. Production of prostaglandins by C57-PGDS cells was measured by enzyme immunoassay. The effect of C57-PGDS cells on crystal-induced inflammation was investigated.. Injection of the crystals caused a rapid decrease in H-PGDS expression by infiltrating cells and by the soft tissues around the air pouches. In contrast, expression of interleukin-1beta (IL-1beta) and macrophage inflammatory protein 2 (MIP-2) as well as cellular infiltration were significantly increased during the early stage of inflammation. C57-PGDS cells, but not control cells, produced an increased amount of PGD(2) in vitro, but suppressed production of PGE(2). Injection of C57-PGDS cells into air pouches inhibited cellular infiltration and MIP-2 and IL-1beta expression.. In this murine air-pouch model of MSU crystal-induced inflammation, retrovirally transfected H-PGDS cDNA could reduce cellular infiltration, at least partly by inhibiting MIP-2 and IL-1beta. These findings suggest that gene therapy with H-PGDS may be useful for treating inflammatory diseases.

Topics: Acute Disease; Animals; Arthritis, Gouty; Cell Line, Tumor; Chemokine CXCL2; Chemokines; Crystallization; Disease Models, Animal; Fibroblasts; Gene Expression Regulation, Enzymologic; Genetic Therapy; Interleukin-1; Intramolecular Oxidoreductases; Leukemia, Basophilic, Acute; Lipocalins; Macrophages; Male; Mice; Mice, Inbred C57BL; Prostaglandin D2; Rats; Retroviridae; Transfection; Uric Acid

Murine gp49, a 49-kDa type I transmembrane glycoprotein, is a member of the Ig-like receptors expressed on the surface of cells involved in natural immunity such as mast cells, NK cells, and macrophages. The two major subtypes, gp49A and gp49B, are encoded by two different genes adjacent to each other. gp49B contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic region and is known to function as an inhibitory molecule. In contrast, gp49A does not harbor any specific motif for signal transduction, nor has its physiological role been determined. Here we report on the stimulatory nature of gp49A by analyzing biochemical characteristics of chimeric molecules consisting of an ectodomain of Fc receptor and a C-terminal half of gp49A, namely the pretransmembrane, transmembrane, and cytoplasmic portions, expressed on the rat basophilic leukemia mast cell line. Cross-linking of the chimeric receptors evoked cytoplasmic calcium mobilization, PGD(2) release, and transcription of IL-3 and IL-4 genes, but did not elicit degranulation of the cells. The chimeric molecule could be expressed as a singlet and a homodimeric form on the cell surface. A pretransmembrane cysteine residue of gp49A was necessary for dimer formation. Dimerization was be necessary for their incorporation into glycolipid-enriched membrane fraction (GEM) upon cross-linking stimuli. The calcium mobilization response was inhibited by treatment of cells with methyl-beta-cyclodextrin, an inhibitor of GEM formation. Together with these results, it was strongly suggested that gp49A could be expressed as a homodimer and elicit activation signals that lead to calcium mobilization, eicosanoid production, and cytokine gene transcription through its incorporation into GEM.

Topics: Animals; Antigens, Surface; Calcium Signaling; Cell Degranulation; Cysteine; Cytokines; Dimerization; Leukemia, Basophilic, Acute; Mast Cells; Mast-Cell Sarcoma; Membrane Glycoproteins; Membrane Microdomains; Mice; Prostaglandin D2; Rats; Receptor Aggregation; Receptors, IgG; Receptors, Immunologic; Recombinant Fusion Proteins; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

The implications of phospholipase D (PLD) in cytosolic phospholipase A2 (cPLA2) activation were studied in a mast cell line, RBL-2H3, upon stimulation with antigen. Antigen-stimulated prostaglandin D2 generation was apparently suppressed by ethanol with a concomitant decrease in phosphatidic acid (PA) formation. The prostaglandin D2 generation was also inhibited almost completely by methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA2, but not by diacylglycerol lipase inhibitor. Furthermore, stimulation with antigen resulted in an increase in lysophosphatidic acid formation, which was suppressed by MAFP in parallel with an increase in PA formation. These results suggest that PA formed by the catalytic action of PLD is used as a substrate for cPLA2, thus PLD regulates cPLA2 activation in antigen-stimulated RBL-2H3 cells.

Topics: Animals; Antigens; Arachidonic Acids; Cytosol; Dinitrophenols; Enzyme Activation; Enzyme Inhibitors; Leukemia, Basophilic, Acute; Organophosphonates; Phosphatidic Acids; Phospholipase D; Phospholipases A; Phospholipases A2; Prostaglandin D2; Rats; Serum Albumin, Bovine; Tumor Cells, Cultured

Exposure to ozone has been reported to cause increased immediate bronchial reactivity to inhaled allergen in asthmatics. The purpose of these studies was to determine whether ozone induces either spontaneous physiological degranulation or enhanced immunoglobulin E (IgE)-mediated degranulation of mast cells, thus accounting for the in vivo effects noted in asthmatics. A rat mast cell line (RBL-2H3) was exposed to different levels of ozone (0.1, 0.3, 0.5, and 1.0 ppm), covered by different amounts of buffer, and both cytotoxic and nontoxic exposure conditions were determined. In addition to cytotoxicity, spontaneous release of granule products and prostaglandin D2 (PGD2) associated with ozone exposure were assessed. RBL-2H3 cells were also exposed to ozone under noncytotoxic conditions followed by stimulation with alpha-IgE to cross-link membrane-bound IgE and A23187 so that the effect of ozone on stimulated degranulation could be examined. Only exposure conditions associated with cytotoxicity were associated with spontaneous release of mast cell serotonin, indicating no physiologic degranulation due to ozone exposure. Data presented herein also demonstrate that ozone substantially inhibited both IgE- and A23187-induced degranulation. Neither catalase nor superoxide dismutase protected cells from the inhibitory effect of ozone, indicating that ozone does not act through generation of H2O2 or superoxide. Additionally, ozone caused a modest increase in spontaneous PGD2 generation only under cytotoxic conditions. Thus ozone appears to inhibit mast cell degranulation after IgE- or A23187-mediated stimulation and causes direct release of mast cell granule products and PGD2 only under conditions associated with membrane cytotoxicity.

Topics: Animals; Asthma; Calcimycin; Catalase; Cell Line; Cell Survival; Cytoplasmic Granules; Dose-Response Relationship, Drug; Humans; Immunoglobulin E; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Ozone; Prostaglandin D2; Rats; Superoxide Dismutase; Tumor Cells, Cultured

We recently reported that delta 12-prostaglandin (PG) J2 caused various cells to synthesize heme oxygenase, HO-1 (Koizumi, T., Negishi, M., and Ichikawa, A. (1992) Prostaglandins 43, 121-131). Here we examined the molecular mechanism underlying the delta 12-PGJ2-induced HO-1 synthesis. delta 12-PGJ2 markedly stimulated the promoter activity of the 5'-flanking region of the rat HO-1 gene from -810 to +101 in rat basophilic leukemia cells. From functional analysis of various deletion mutant genes we found that the delta 12-PGJ2-responsive element was localized in a region from -690 to -660, containing an E-box motif, which was essential for the delta 12-PGJ2-stimulated promoter activity. When the region containing the delta 12-PGJ2-responsive element was combined with a heterologous promoter, SV40 promoter, in the sense and antisense direction, the element showed an enhancer activity in response to delta 12-PGJ2. Gel mobility shift assays demonstrated that delta 12-PGJ2 specifically stimulated the binding of two nuclear proteins to the E-box motif of this region. These results indicate that delta 12-PGJ2 induces the expression of the rat HO-1 gene through nuclear protein binding to a specific element having an E-box motif.

Topics: Animals; Antineoplastic Agents; Base Sequence; Blotting, Northern; Cell Line; Chloramphenicol O-Acetyltransferase; Gene Expression; Heme Oxygenase (Decyclizing); Leukemia, Basophilic, Acute; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Plasmids; Point Mutation; Promoter Regions, Genetic; Prostaglandin D2; Rats; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Simian virus 40; Transfection; Tumor Cells, Cultured

We determined the ability of hydrocortisone to inhibit rat basophilic leukemia cell mediator release induced by anti-IgE and by neutrophil-derived histamine-releasing activity (HRA-N). Serotonin release induced by HRA-N and anti-IgE was inhibited by 78 +/- 5 and 70 +/- 4%, respectively (IC50 7.5 x 10(-7)M) by hydrocortisone (10(-5)M). HRA-N does not cause arachidonic acid metabolism, however, anti-IgE induced the generation of PGD2 and leukotriene (LT)C4, and the generation of both mediators was inhibited by 10(-5)M hydrocortisone (IC50 = 4.8 x 10(-7)M, and 3.6 x 10(-9)M, respectively). Inhibition required at least 5 to 6 h of hydrocortisone exposure and was maximal after 22 h. The observed effects of hydrocortisone could be reproduced by human recombinant lipocortin-I (5 x 10(-7)M). Hydrocortisone, 10(-5)M, was a less potent inhibitor of calcium ionophore A23187-mediated serotonin release and PGD2 and LTC4 generation (inhibition of 20 +/- 2, 17 +/- 10, and 37 +/- 10%, respectively). Inasmuch as A23187-induced stimulation is not dependent on receptor coupling, the enhanced ability of hydrocortisone to inhibit IgE- and HRA-N-mediated events as compared with A23187 suggests that one possible site of action of hydrocortisone may be interruption of receptor-effector signals. In the presence of arachidonic acid, hydrocortisone-treated cells released as much LTB4 and PGD2 as control cells, however, serotonin release and LTC4 generation were inhibited 50 and 55%, respectively. Thus, these data suggest that hydrocortisone has three possible sites of action: 1) inhibition of phospholipase A2 activity, 2) inhibition of glutathione-s-transferase, and 3) inhibition of serotonin release by a third mechanism, possibly by interrupting the coupling of receptor and effector systems.

Topics: Animals; Annexins; Arachidonic Acid; Arachidonic Acids; Calcium-Binding Proteins; Cytokines; Dose-Response Relationship, Drug; Histamine Release; Humans; Hydrocortisone; Immunoglobulin E; In Vitro Techniques; Leukemia, Basophilic, Acute; Mast Cells; Neutrophils; Prostaglandin D2; Rats; Serotonin; SRS-A; Tumor Cells, Cultured

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Drug Synergism; Leukemia, Basophilic, Acute; Phospholipases; Phospholipids; Prostaglandin D2; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Topics: Animals; Arachidonate 5-Lipoxygenase; Butyrates; Butyric Acid; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rats; Tumor Cells, Cultured