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5-Oxo-eicosatetraenoic acid (5-oxoETE) stimulated human neutrophil (PMN) and eosinophil chemotaxis, PMN hexose uptake, and PMN membrane GTP/GDP exchange. Pertussis toxin (PT), a blocker of heterotrimeric G proteins (GP), completely inhibited these responses, but proved far less effective on the same responses when elicited by leukotriene B4, C5a, FMLP, platelet-activating factor, IL-8, or RANTES chemotactic factors. 5-OxoETE also specifically bound to the membrane preparations that conducted GTP/GDP exchange. This binding was down-regulated by GTPgammaS, but not ADPgammaS, and displaced by 5-oxoETE analogues, but not by leukotriene B4, lipoxin A4, or lipoxin B4. Finally, PMN expressed PT-sensitive GP alphaiota2 and PT-resistant GP alphaq/11- and alpha13-chains; eosinophils expressed only alphai2 and alphaq/11. We conclude that 5-oxoETE activates granulocytes through a unique receptor that couples preferentially to PT-sensitive GP. The strict dependency of this putative receptor on PT-sensitive GP may underlie the limited actions of 5-oxoETE, compared with other CF, and help clarify the complex relations between receptors, GP, cell signals, and cell responses.

STAT3 (signal transducer and activator of transcription 3) is a latent transcription factor that is activated by tyrosine phosphorylation (Tyr-705) in cells stimulated with cytokines or growth factors. Recent studies suggest that one or more cytoplasmic serine kinases also phosphorylate STAT3 and are necessary for maximal gene activation. Here we demonstrate, with a site-specific antibody, that STAT3 is phosphorylated on Ser-727 in human neutrophils stimulated with chemotactic factors (N-formyl-methionyl-leucyl-phenylalanine and complement C5a), cytokines [granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF)], or a protein kinase C activator (PMA). (2-Amino-3'-methoxyphenyl)oxanaphthalen-4-one (PD 98059), an inhibitor of extracellular signal-regulated protein kinase (ERK) activation, blocked the serine phosphorylation of STAT3 induced by chemotactic factors or PMA. The drug was less effective on cytokines: it virtually abolished the response to GM-CSF that occurred 5 min after stimulation but only partly decreased those at 15-30 min and did not appreciably alter responses to G-CSF regardless of incubation time. 1-(5-Isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of a putative STAT3 serine kinase, and 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) 1H-imidazole (SB 203580), an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not dampen any of these serine phosphorylation responses. We propose that neutrophils use both ERK-dependent and ERK-independent pathways to phosphorylate Ser-727 on STAT3. The former pathway is recruited by all ERK-activating stimuli, whereas the latter pathway uses an undefined serine kinase and is recruited selectively by cytokines.

Chemotactic factors, i.e., an N-formyl peptide, C5a, interleukin-8, and leukotriene B4, induced neutrophils to activate mitogen-activated protein (MAP) kinases, as defined by the tyrosine phosphorylation and decrease in electrophoretic mobility of immunodetected 44-, 42-, and 40-kDa proteins. PD 98059, an inhibitor of MAP kinase kinase activation, blocked these changes. The drug likewise blocked neutrophil chemotaxis but did not alter superoxide anion production and paradoxically enhanced degranulation responses to the stimuli. The MAP kinase pathway appears to have a highly selective role in mediating motility but not other cellular responses.

The newly described products of 5-hydroxyeicosanoid dehydrogenase, 5-oxo-6,8,11,14-eicosatetraenoic acid (ETE) and 5-oxo-15(OH)ETE, induced directional migration and actin polymerization of human monocytes in vitro. At peak concentrations, the two eicosanoids had a chemotactic activity of about 40% of that observed in the presence of an optimal concentration of FMLP and twice the activity elicited by the related eicosanoid 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). 15-Oxo-ETE showed a very low but detectable chemotactic activity. All of these chemotactic responses were blocked by Bordetella pertussis toxin, but were resistant to LY255283, a leukotriene B4 (LTB4) receptor antagonist. 5-Oxo-ETEs and 5-HETE induced homologous desensitization of chemotactic response, but did not cross-desensitize to other chemotactic agonists (e.g., monocyte chemotactic protein (MCP)-1 and LTB4). 5-Oxo-ETEs increased in a synergistic fashion the monocyte migration to MCP-1 and MCP-3. In the same range of concentrations, 5-oxo-ETE increased MCP-1-induced release of arachidonic acid from labeled monocytes. No synergistic interaction was observed when FMLP was used as chemoattractant. Thus, this study identifies monocytes as cells responsive to 5-oxo-ETEs and shows that monocyte activation by 5-oxo-ETEs occurs through an LTB4 receptor-independent mechanism that associates with pertussis toxin-sensitive G proteins. The synergistic interaction between 5-oxo-ETEs and C-C chemokines, two families of mediators both synthesized by phagocytic cells, may be relevant in vivo for the regulation of monocyte accumulation at sites of allergic and inflammatory reactions.

The effects of chemotactic factors on rabbit neutrophils were evaluated measuring cell migration in modified Boyden chambers and under agarose, in lysosomal enzyme release, leukocyte aggregation, and in vivo neutropenia. Chemotactins employed included the complement-derived C3 and C5 fragments, the bacterial chemotactic factor from culture supernatant fluids of Escherichia coli, and the synthetic chemotactic factors Met-Leu-Phe and formyl-Met-Leu-Phe. A consistent parallelism was found in all the leukocyte responses to a given chemotactic factor. In no instance, with any of the five chemotactic factor preparations, did cells responding in one assay system fail to respond in the four other assay systems, suggesting a common event in all of the cell responses. Boyden chamber chemotaxis was consistently the most sensitive assay; the agarose assay was, in general, less sensitive by a factor of 100 fold. Enzyme release approached, in cell sensitivity to chemotactic factors, that of the Boyden chamber assay. In general, in vitro leukocyte aggregation and in vivo neutropenia were considerably less sensitive assays. Chemotactic factor inactivator (CFI) purified from human serum destroyed in parallel all biological activities of C3 and C5 chemotactic factors but had no effect on the bacterial chemotactic factor and the activities of synthetic chemotactic peptides.

Chemotactic substances stimulate neutrophils in suspension to aggregate. Arachidonic acid (but not several structurally related fatty acids) induces a similar neutrophil response. We now report that two blockers of arachidonic acid metabolism, indomethacin and 5,8,11,14-eicosatetraynoic acid, inhibit this arachidonic acid-mediated response. Moreover, both blockers also inhibit aggregation stimulated by a synthetic chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, and their potency in doing so parallels their potency in inhibiting the response to arachidonic acid. These results suggest that metabolites of arachidonic acid may stimulate certain neutrophil functions and be involved in cellular responses to at least some chemotactic substances.

The ability of several stimuli, all of which have been reported to stimulate eosinophil motility, to augment uptake or extracellular deoxyglucose (DOG), a glucose analogue, was studied. DOG uptake was stimulated in a concentration-dependent manner by the more potent chemotaxins--zymosan-activated serum, partially purified C5a. N-formyl-methionyl-leucyl-phenylalanine, and 5- and 11-hydroxyeicosatetraenoic acids. Less potent stimuli--the eosinophil chemotactic factor of anaphylaxis, histamine, and prostaglandin E2--did not stimulate DOG uptake. However, the correlation between the abilities of the agents to stimulate motility and DOG uptake was not completely uniform. Prostaglandin F2 alpha was a potent stimulant of DOG uptake yet is known to enhance only weakly chemokinesis and to be not chemotactic for eosinophils. The combined data from this and other studies indicate that these stimuli elicit specific and different sets of functional responses from eosinophils.