Neutrophil cell responses and signal pathways elicted by the chemotactic arachidonic acid metabolites (6E, SZ, 1 lZ, 14Z)-5-oxo-icosatetraenoic acid and (6E, SZ, 1 IZ, 13E)-5-oxo-15-hydroxy-icosatetraenoic acid were studied and compared with those of other chemotaxins. Polyphosphoinositol lipid analysis revealed activation of phosphatidylinositol-bisphosphate 3-kinase by both agonists. Experiments with Fura-2 in the presence of EGTA indicated Ca2+ mobilization from intracellular stores by both 5-OXOicosanoids. A transient actin response and production of small amounts of superoxide anions upon stimulation with both agents was detected. The changes induced by 5-0x0-icosanoids were more moderate and transient than those obtained by other chemotaxins. Desensitization studies indicated cross-desensitization between both 5-oxo-icosanoids, but no interference with the response of other chemotaxins. All cell responses elicted by 5-0x0-icosanoids were inhibited by pertussis toxin suggesting involvement of Gproteins, a common activation mechanism for all known potent chemotaxins. In contrast to other chemotaxins, 5-0x0-icosanoids at concentrations 500-fold higher than the ED,, of other functions did not induce up-regulation of CD11 b and N-formyl-peptide receptors at the cell surface, and failed to potentiate Nformyl-peptide-induced superoxide anion production. These results indicate that 5-0x0-icosanoids trigger a unique pattern of neutrophil responses.Keywords: neutrophils ; 5-0x0-icosanoids ; Caz+ transients ; F actin; superoxide anions.Accumulation of neutrophils in tissue is characteristic of inflammation and observed in a large variety of pathological conditions. Leukocyte infiltration is presumably caused by different chemotaxins, which are generated by different mechanisms [I]. Extensively characterized chemotaxins are N-formyl peptides, the complement split product C5a (C5a), the chemokines interleukin-8 (IL-8) and GROa as well as the arachidonic acid metabolite leukotriene B, (LTB,) 11-61. In addition to chemotaxis, these substances stimulate with different capacity proinflammatory activities such as production of reactive oxygen metabolites, up-regulation of membrane proteins such as the complement receptor type 3 (CDllb) or the N-formyl-peptide receptor and priming of neutrophil responses [l -61. Activation of neutrophils by chemotaxins requires binding to ligand-specific cell-surface receptors [7]. The receptors for most chemotaxins interact with pertussis-toxin-sensitive or cholera-toxin-sensitive heterotrimeric guanine-nucleotide-binding proteins (G-proteins) 11, 7-91. Activation of G-proteins leads into different signal pathways initiated by phospholipase C-p2, phosphatidylinositol-bisphosphate (PtdInsP,) 3-kinase and actin polymerization [I, 2, 10, 1 I]. The second messengers generated by these differend signal pathways regulate the effector functions of leukocytes in concert [I, 2, 12-15].Recently, 5-oxo-icosanoids, i.e. (6E, SZ, 1 lZ, ~~Z ) -~-O X Oicosatetraenoic acid (5oETE) and (6E, 82, l l Z , 13E...
The arachidonic acid metabolites 5-oxo-[6E,8Z,11Z,14Z]-eicosatetraen oic acid (5oETE) and 5-oxo-15-hydroxy-[6E,8Z,11Z,13E]-eicosatetrae noi c acid (5oHETE) are potent eosinophil chemotaxins. Here, the activation profile of 5-oxo-eicosanoids in eosinophils was further characterized and compared to other eosinophil activators such as complement fragment C5a (C5a), platelet-activating factor (PAF), interleukin-5 (IL-5), and phorbol ester (PMA). Flow cytometric studies revealed a rapid and transient actin polymerization upon stimulation by both 5-oxo-eicosanoids. Desensitization studies using actin polymerization as the parameter indicated cross-desensitization between the two 5-oxo-eicosanoids but revealed no interference with the response to other chemotaxins. Fluorescence measurements with Fura-2-labeled eosinophils in the presence of EGTA indicated Ca2+-mobilization from intracellular stores by 5oETE and 5oHETE. Both 5-oxo-eicosanoids stimulated the production of reactive oxygen metabolites as demonstrated by lucigenin-dependent chemiluminescence, superoxide dismutase-inhibitable cytochrome C reduction, and flow cytometric dihydrorhodamine-123 analysis. At optimal concentrations the changes induced by 5-oxo-eicosanoids were comparable to those obtained by C5a and PAF, whereas IL-5 and PMA induced only a restricted pattern of cell responses. Cell responses elicited by 5-oxo-eicosanoids were inhibited by pertussis toxin, indicating coupling of the putative 5-oxo-eicosanoid-receptor to G-proteins. These results indicate that 5-oxo-eicosanoids are stong activators of eosinophils with comparable biologic activity to the eosinophil chemotaxins C5a and PAF. These findings point to a role of 5-oxo-eicosanoids in the pathogenesis of eosinophilic inflammation as chemotaxins as well as activators of pro-inflammatory activities.
Interleukin-8 (IL-8) and GRO alpha are leukocyte-attracting peptides of the chemokine family. To study the priming potential of these chemokines, we measured superoxide anion production and up-regulation of N-formyl peptide receptors in human neutrophils. IL-8 and GRO alpha themselves did not stimulate production of significant amounts of superoxide anions but potentiated N-formyl peptide-induced superoxide anion production in a concentration-dependent manner. Binding measurements by flow cytometry at 37 degrees C with fluorescein-labeled N-formyl peptide revealed enhanced total N-formyl peptide binding after pretreatment of neutrophils with IL-8 and GRO alpha. Binding measurements performed at 4 degrees C indicated that the chemokines stimulated the up-regulation of N-formyl peptide receptors at the cell surface but did not alter their affinity for the ligand. This study indicates that IL-8 and GRO alpha, in addition to their known chemotactic activity, prime neutrophils for superoxide anion production, presumably by up-regulating the number of receptors for strong superoxide-anion-triggering stimuli.
Tumor invasion and formation of metastases are major obstacles for a successful therapy of melanomas. Metastasis is thought to require multiple steps such as alpha v beta 3-integrin-mediated adhesion, proteolytic digestion of extracellular matrix by metalloproteinase-2, and reorganization of the actin cytoskeleton. To analyze the functional role of phosphatidylinositol 3-kinase in these processes, melanoma cells were treated with the fungal metabolite wortmannin. Wortmannin inhibited phosphatidylinositol 3-kinase activity in melanoma cells and migration in an equally concentration-dependent fashion. Flow cytometric analysis of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin-stained actin network indicated reduction of actin filaments by wortmannin. Fluorescence laser confocal microscopy experiments revealed breakdown of actin stress fibers. In addition, wortmannin inhibited alpha v beta 3-integrin-mediated adhesion of melanoma cells to vitronectin. Since flow cytometric measurements did not show altered expression of the alpha v beta 3-integrin at the cell surface, avidity changes of the alpha v beta 3-integrin by wortmannin are suggested. In contrast to the actin analysis and adhesion assays, wortmannin had no influence on mRNA expression or on protein secretion of metalloproteinase-2. These data provide evidence that phosphatidylinositol 3-kinase is an essential signal transduction protein required for migration of melanoma cells, regulating formation of the actin stress fiber as well as alpha v beta 3-integrin-mediated adhesion.
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