Effect of carotenoid oxidation products on neutrophil viability and function

Human neutrophils are short-lived cells that play important roles in host defense and acute inflammation by releasing hydrolytic and cytotoxic proteins and reactive oxygen derivatives. Apoptosis, a physiological mechanism for cell death, regulates both production and survival of neutrophils, representing a basic biological mechanism for this type of cells. Carotenoids may react with toxic oxygen metabolites released by neutrophils to form a multitude of carotenoid cleavage products that exert, in turn, relevant prooxidative biological effects. Recent data suggest that carotenoid oxidation products may affect neutrophil viability and function by exerting proapoptotic activity and interfering with superoxide production by activated cells. The prooxidant and proapoptotic activities of carotenoid oxidation products could account, at least in some cases, for the procancerogenic properties of carotenoid rich diet.     

The role of cyclooxygenase inhibition in the effect of alpha-melanocyte-stimulating hormone on reactive oxygen species production by rat peritoneal neutrophils

The effect of alpha-MSH on reactive oxygen species (ROS) production by rat peritoneal neutrophils and the effect of cyclooxygenase (COX) inhibition were investigated using the chemiluminescence (CL) technique. Cells were obtained by peritoneal lavage 4h after administration of oyster glycogen to rats and were stimulated with lipopolysaccharide (LPS) from Salmonella enderitidis and phorbol 12-myristate 13-acetate (PMA). The increasing concentrations of alpha-MSH (10(-12)-10(-6) M) were added to stimulated cells alone or along with the COX inhibitors indomethacin, ketorolac or nimesulide (10(-8)-10(-5) M). Luminol and lucigenin CL levels were significantly increased in cells stimulated with LPS and PMA compared to unstimulated ones. alpha-MSH significantly reduced lucigenin CL values and this effect was completely reversed in the presence of indomethacin (10(-8) and 10(-7) M). In conclusion, alpha-MSH inhibits the production of superoxide radicals by activated rat peritoneal neutrophils and COX contributes to this effect.     

Exposure of human neutrophils to chemotactic factors potentiates activation of the respiratory burst enzyme

NADPH oxidase activity in particulate fractions from human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan was enhanced by prior exposure of the neutrophils to chemotactic factors. Enhanced activity was seen measuring both NADPH-dependent chemiluminescence and superoxide anion production. Enhancement was observed to be both time and dose dependent with several chemotactic stimuli, including casein, N-formyl-methionyl-leucyl-phenylalanine (f-MLP), and C5a. F-MLP and C5a showed similar patterns, with peak enhancement occurring within 2 to 15 min of preincubation and lasting up to 1 hr. In contrast, enhancement of PMA-stimulated oxidase activity by casein was more gradual and sustained, lasting up to 2 hr. Fractions from cells treated only with chemotactic factors and not stimulated with PMA showed no oxidase activity. Kinetic studies of this enhanced activity show that chemotactic factors induce increases in Vmax values but do not significantly alter Km values for the oxidase. Further experiments using agents that modulate degranulation suggest that enzyme release is not involved in this enhancement. These data suggest that pretreatment with chemotactic factors results in an increase in the amount of activated oxidase in membrane fractions obtained from PMA-stimulated neutrophils. This alteration of NADPH oxidase activity provides a subcellular basis for the enhanced bactericidal activity and increased oxidative metabolism seen in neutrophils treated with chemotactic factors.     

Adriamycin effects on transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbol myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

Adriamycin effects of transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbal myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production

Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite.     

Reduced neutrophil superoxide generation and plasma lipoperoxide levels in pigs fed with cod liver oil.

Circulating neutrophils, isolated from pigs fed for 8 weeks with a diet supplemented with CLO, had an accentuated n-3 fatty acid incorporation into the plasma membrane, as evidenced by an approximately four-fold greater n-3/n-6 ratio as compared with the control diet group. Moreover, the neutrophils of the CLO fed pigs produced less superoxide when stimulated with PMA or f-MLP, as well as showing a more prolonged latency period before O2(-)-generation. In the plasma of pigs fed with CLO there were higher levels of thiobarbituric reactive material and lipofuscin, while the content of GSSG was similar in both dietary groups. The results of this study indicate that dietary supplementation with CLO reduces the activation of circulating neutrophils and favours the presence in the plasma of lipoperoxides.     

Participation of protein kinase C in the activation of human neutrophils by phorbol ester

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.     

Neutrophil stimulation and priming by direct contact with activated human T lymphocytes.

The concept that T lymphocytes regulate neutrophil function has an important implication in the understanding of the role of these cells in immunity against infection and in inflammatory diseases, but evidence for this concept is primarily derived from the effects of lymphokines on neutrophils. We now present evidence to show that living or paraformaldehyde-fixed mitogen-activated T lymphocytes, as well as an activated T cell line (HUT-78), induce by cell-cell contact, an oxygen-dependent respiratory burst measured by both the lucigenin-dependent chemiluminescence assay and superoxide production. Neutrophils reacted with purified human T lymphocytes which had been activated by culture in the presence of PHA and PMA for 72 h showed a marked and significant respiratory burst compared with neutrophils treated with T lymphocytes cultured in the absence of these mitogens. Similar results were observed with the paraformaldehyde-fixed T cell line (HUT-78). The ability to stimulate neutrophils required intact paraformaldehyde-fixed T cells, and neutrophil stimulation failed to occur if the T cells and neutrophils were separated by membrane filters. mAb to TNF-alpha, and TNF-beta blocked the ability of rTNF-alpha and TNF-beta to stimulate neutrophils but did not block the neutrophil response induced by activated T cells. Pretreatment of neutrophils with the activated T lymphocytes enhanced the response to the tripeptide, FMLP. It is therefore conceivable that activated T lymphocytes attracted at sites of inflammation influence neutrophil activity by direct plasma membrane interaction which clearly represents an efficient microbial defence mechanism, minimizing tissue damage during inflammation.     

Contribution of CD54 to human eosinophil and neutrophil superoxide production.

We have reported that CD54 on eosinophils is involved in eosinophil degranulation. However, the role of CD54 in eosinophil and neutrophil superoxide production is still uncertain. We assessed the effect of CD54 on eosinophils and neutrophils in recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF)- or phorbol myristate acetate (PMA)-induced superoxide production through CD18. Anti-CD54 monoclonal antibody attenuated leukocyte aggregation and superoxide production of rGM-CSF- or PMA-stimulated neutrophils and PMA-stimulated eosinophils. Anti-CD18 monoclonal antibody or theophylline attenuated superoxide production of eosinophils and neutrophils stimulated by either stimuli. Flow cytometric analysis demonstrated CD54 expression on freshly isolated neutrophils but not on freshly isolated eosinophils. CD54 newly expressed on eosinophils reached its peak expression 30 min after PMA stimulation. The increase in CD18 and CD54 expression on neutrophils caused by rGM-CSF stimulation was partially inhibited by theophylline. These data demonstrated that CD54 and CD18 interaction of eosinophils or neutrophils is involved in superoxide production and that the inhibition of superoxide production by theophylline may be at least partly due to the inhibition of CD54 and CD18.     

Generation of biologically active C-reactive protein peptides by a neutral protease on the membrane of phorbol myristate acetate-stimulated neutrophils

The association of human C-reactive protein (CRP) with nonstimulated and PMA-stimulated human neutrophils and the concomitant degradation of CRP (monitored by TCA-soluble peptides and SDS-PAGE analysis) has been studied. Maximum association of 125I-labeled CRP with neutrophils and 125I-labeled CRP degradation during association with these cells was achieved by stimulating the neutrophils with PMA at 10 ng/ml; a concentration in which azurophil granule release was not significant. For PMA-stimulated neutrophils, the association of 125I-labeled CRP was 1.8 times higher and PMA-stimulated neutrophil-mediated degradation of the ligand was three times faster than that for nonstimulated cells. The neutrophil-associated 125I-labeled CRP in the absence and presence of PMA proved on SDS-PAGE analysis to be approximately 50% degraded. There was a positive correlation between the extent of CRP degradation and the association of 125I-labeled CRP with neutrophils. In addition to generation of neutrophil associated CRP intermediates, small soluble CRP peptides were generated during association of CRP with neutrophils. These peptides inhibited superoxide production from opsonized zymosan-activated neutrophils by approximately 40% at 10 micrograms/ml. 125I-labeled CRP degradation mediated by nonstimulated neutrophils, and neutrophil-conditioned medium (from both non-stimulated and PMA-stimulated cells) was inhibitable by alpha 1-antitrypsin and approximately seven times less at 1 h than that occurring during 125I-labeled CRP-association with PMA-stimulated neutrophils. The degradation of 125I-labeled CRP mediated by PMA-stimulated neutrophils was not fully inhibitable by alpha 1-antitrypsin. The data point to the involvement of a membrane-associated serine protease, which is maximally activated by PMA, in the degradation of 125I-labeled CRP during association with neutrophils. Our results indicate that at an inflammatory site CRP-derived peptides can be produced that inhibit the action of activated neutrophils.     

Effect of isorhapontigenin on respiratory burst of rat neutrophils

The effect of Isorhapontigenin (Iso) isolated from Belamcanda chinensis on respiratory burst of rat neutrophils was investigated. Iso (1, 10, 100 mmol/l) showed an inhibitory effect on superoxide anion and hydrogen peroxide production in phorbol myristate acetate (PMA) activated rat neutrophils in a concentration-dependent manner. Scanning electron microscopy detected that Iso (100 mmol/l) protected against surface changes in rat neutrophils stimulated with PMA. Also, 100 mmol/l Iso inhibited the release of beta-glucuronidase from the activated neutrophils. Electron-spin resonance (ESR) detected that Iso scavenged oxygen free radicals generated in the PMA activated Neutrophils. These results suggest that Iso inhibits respiratory burst of PMA-activated rat neutrophils by scavenging oxygen free radicals.     

Evaluation of synthetic sphingosine, lysosphingolipids and glycosphingolipids as inhibitors of functional responses of human neutrophils.

Human neutrophils, when exposed to soluble stimuli, aggregate, release oxygenated products of arachidonic acid and generate active oxygen species. Sphingolipid-derived products such as sphingosine and lysosphingolipids have been shown to exert selective actions on a variety of cell types, including neutrophils. Therefore, to determine the structural basis for selective inhibition of neutrophil responses by naturally occurring sphingolipids, seven compounds were prepared by total organic synthesis, and their impact on neutrophils in suspension has been studied. The compounds synthesized included sphingosine, psychosine, lactosyl lysosphingolipid, globotriaosyl (Gb3) lysosphingolipid, galactosyl cerebroside, lactosyl ceramide and Gb3 ceramide. The neutrophil responses studied were aggregation, leukotriene generation and superoxide anion production. When exposed to non-cytotoxic levels of the synthetic compounds, as monitored by exclusion of Trypan Blue, none of the synthetic sphingolipids inhibited A23187-induced aggregation of neutrophils. Only lactosyl lysosphingolipid, at a concentration of 1 microM, significantly inhibited aggregation induced by fMetLeuPhe; the other compounds in this series including sphingosine were without effect at equal molar concentrations (1 microM). Aggregation induced by phorbol 12-myristate 13-acetate (PMA) (0.1 microM) was significantly blocked by only two of the synthetic sphingolipids (1 microM). At concentrations below 1 microM, these inhibitory actions were not evident, nor was it possible to assign a structure-activity relationship for this series of compounds. None of the synthetic sphingolipids effectively inhibited the generation of superoxide anions induced by PMA. In addition, neither synthetic sphingosine nor psychosine affected either the formation or metabolism of leukotriene B4. Taken together, the results provide further evidence that sphingolipids, when added to intact cells, are not potent selective inhibitors of functional responses of human neutrophils.     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst

Zinc is an essential trace element for the immune system. It is known to be essential for highly proliferating cells, especially for cells of the immune system. However, zinc and other divalent cations are known to inhibit the human neutrophilic NADPH oxidase. Differentiated HL-60 cells were found to accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH oxidase by phorbol esters (PMA). This increase in vitamin C transport is due to the generation of superoxide and subsequent oxidation of ascorbate to dehydroascorbate (DHA) which is preferentially taken up by the cells. We found that zinc reversibly inhibits both PMA-stimulated ascorbate uptake and superoxide generation with a half-maximal effect at 20 microM of free zinc ions. Higher residual extracellular ascorbate concentrations were measured with increasing zinc concentrations, indicating that less ascorbate was oxidized and taken up by the cells. When the fluorescent dye diSC3(5) was used to monitor shifts in membrane potential, we found that depolarization with PMA was prolonged after preincubation of the cells with zinc. Suppression of the respiratory burst as well as inhibition of the uptake of the antioxidant vitamin C may disturb the balance between oxidative damage of invading particles and antioxidant protection in activated neutrophils.     

Phospholipase A2 modulates respiratory burst developed by neutrophils in patients with rheumatoid arthritis

Activated by bacterial peptides, phorbol esters, calcium ionophores and other agonists, neutrophils (PMNs) release the proinflammatory mediator, arachidonic acid (AA) via the intervention of phospholipase A(2) (PLA(2)). AA may play an essential role in activation of NADPH-oxidase, which is involved in the generation of superoxide anion by neutrophils. The present study is focused on the involvement of PLA(2) in the respiratory burst developed by PMNs isolated from patients with rheumatoid arthritis (RA). PLA(2) exists in very high levels in diseases such as rheumatoid arthritis and may cause acute inflammatory and proliferative changes in synovial structures. The respiratory burst was evaluated as superoxide anion release, using an amplified chemiluminescence method. The assays were performed using PMNs untreated or treated with different doses of stimulatory reagents (phorbol 12-myristate-13-acetate (PMA), calcium ionophore (A23187)). Our data suggested that PMA stimulated the production of superoxide anion in a dose-response manner, as compared with A23187, which did not induce a significant release of superoxide anion in PMNs-RA. The exogenous addition of AA significantly amplified the superoxide anion release by PMNs-RA stimulated with PMA and to a lesser extent, by PMNs stimulated with A23187. AA has also reversed the inhibitory effect of arachidonyl-trifluorometylketone and E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BEL) on the superoxide anion release by PMNs-RA. In conclusion, the differential responses to these two agents suggested that different isoforms of PLA(2) were activated by A23187 or PMA, and support the idea that activation of these different PLA(2) served distinct functions of PMNs. Therefore, the inhibition of PLA(2) enzymes might be of great importance in the immunotherapy of rheumatoid arthritis.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils.

NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.     

Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O₂-) and its dismutation product, hydrogen peroxide (H₂O₂). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 μg/ml) decreased H₂O₂ yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H₂O₂ release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O_2;^- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H₂O₂ and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O₂^- and H₂O₂ generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O_2;^-and *NO production.