Effects of 60 Hz magnetic field exposure on polymorphonuclear leukocyte activation

We have investigated the effects of a sinusoidal 60 Hz magnetic field on free radical (superoxide anion) production, degranulation (beta-glucuronidase and lysozyme release) and viability in human neutrophils (PMNs). Experiments were performed blindly in very controlled conditions to examine the effects of a magnetic field in resting PMNs and in PMNs stimulated with a tumor promoter: phorbol 12-myristate 13-acetate (PMA). Exposure of unstimulated human PMNs to a 60 Hz magnetic field did not affect the functions examined. In contrast, exposure of PMNs to a 22 milliTesla (mT), 60 Hz magnetic field induced significant increases in superoxide anion (O2-) production (26.5%) and in beta-glucuronidase release (53%) when the cells were incubated with a suboptimal stimulating dose of PMA. Release of lysozyme and lactate dehydrogenase was unchanged by the magnetic field, whether the cells were stimulated or not. A 60 Hz magnetic field did not have any effect on O2- generation by a cell-free system xanthine/xanthine oxidase, suggesting that a magnetic field could upregulate common cellular events (signal transduction) leading to O2- generation and beta-glucuronidase release. In conclusion, exposure of PMNs to a 22 mT, 60 Hz magnetic field potentiates the effect of PMA on O2- generation and beta-glucuronidase release. This effect could be the result of an alteration in the intracellular signaling.     

Functional aspects of neutrophils in full-term infants

The measure of superoxide anion (.O2-) generation was carried out in the neutrophils (PMNs) of newborns. Comparison between .O2- generation in the resting state and after stimulation with zymosan was considered. PMNs were incubated in their own plasma and in plasma from healthy adult subjects. The results demonstrate a decrease of .O2- generation (in comparison with adult PMNs) in the PMNs from newborn when they were incubated in their own plasma while the .O2- generation reached the values equal to adult's PMNs when the cells of newborns were incubated in the adult's plasma. However PMNs from some newborns demonstrated a low .O2- generation even though they were incubated in adult's plasma. The data are suggestive of a predominant, but not always exclusive, responsibility of plasma factors in the decrease .O2- generation by PMNs of full term babies.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on transmembrane electrical potentials in granulocytes: relationship between enhancement of ligand-mediated depolarization and augmentation of superoxide anion (O2-) production

When human granulocytes that have been primed with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) are activated by ligands that stimulate the respiratory burst, the amount of superoxide anion (O2-) they generate is significantly increased. We have found that the accelerated rate of O2- release occurring under these conditions is accompanied by an antecedent increase in membrane depolarization. We examined the nature of the enhancement of membrane depolarization in GM-CSFrh-primed granulocytes and investigated its relationship to the increase in O2- generation by N-formyl methionylleucylphenylalanine (fMLP)-activated granulocytes. We found that augmented depolarization could not be accounted for by a change in the resting membrane potential induced by the growth factor and was still present after either blocking passive transmembrane Na+ movement with dimethylamiloride or by increasing the membrane's permeability to K+ with valinomycin. When their ability to depolarize was virtually eliminated by dissipating the transmembrane K+ gradient, GM-CSFrh-pretreated cells continued to generate more O2- after fMLP than did control cells. These results indicate that augmentation of the granulocyte's ability to generate O2- anions, which is induced by priming with GM-CSFrh, is independent both of the resting transmembrane potential and of alterations in the extent of membrane potential change induced by stimuli such as fMLP.     

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical (O(2)(-)) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O(2)(-) production. CD as well as PMA induced the translocation of p47(phox) to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCzeta antagonist with sequences based on the endogenous PKCzeta pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O(2)(-) production. On the other hand, the production of O(2)(-) induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.     

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.     

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.     

Inhibition of superoxide generation from fMLP-stimulated leukocytes by high concentrations of nitric oxide or peroxynitrite: characterization by electron spin resonance spectroscopy

This present study examined the effects of high concentrations of nitric oxide (NO*) and peroxynitrite (ONOO-) on superoxide (O2*-) production from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leukocytes (PMNs) by using electron spin resonance (ESR) and spin trapping with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). We demonstrated that ONOO- (100 microM) decreased the ESR signal of DEPMPO-OOH from fMLP-activated PMNs, indicating the inhibition of O2*- generation, while it enhanced the signal of DEPMPO-OH. Inhibition of the respiratory burst was also observed when PMNs were pre-exposed to high concentrations of NO* (100 microM), generated by the NO* donor NOR-1, 30 min prior to stimulation with fMLP. NOR-1 inhibited O2*- generation more effectively under conditions in which ONOO-was formed concurrently. The ability of high concentrations of either ONOO- or NO* to inhibit O2*-generation from fMLP-stimulated PMNs is relevant to pathophysiological conditions, such as severe inflammation, in which NO* or ONOO- production can be significantly elevated.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Superoxide production by phagocytes in myeloid Graffi tumor-bearing hamsters

A progressive suppression of the phagocytic ability of peritoneal macrophages and polymorphonuclears (PMNs) in hamsters with transplanted myeloid tumors was previously established. The i.p. application of Cu/Zn SOD, isolated from the fungal strain Humicola lutea (HLSOD) (2 injections before and 5 injections after tumor transplantation) induced the mean survival time of the animals as well as a temporally stimulating action on the macrophage and PMNs phagocyting indices. In the present work, the superoxide production of peritoneal macrophages and PMNs during 30 days of tumor progression was followed. Effects of the application of HLSOD in an optimal protective dose on the superoxide production in peritoneal macrophages and blood PMNs were examined. The spontaneous and phorbol-myristate acetate (PMA)-inducible O2- production in both types of phagocytes was 4-5-fold increased in tumor-bearing hamsters (TBH), as compared to the controls, at day 14 after tumor transplantation (the day of tumor appearance in transplanted animals). Furthermore, O2- production was also similar to the control values for the following days of observation. HLSOD treatment of TBH induced a normalization of superoxide production in macrophages and PMNs. Therefore, the established decrease of superoxide anions in phagocyting cells of TBH indicates possible effects of HLSOD on the host antioxidant defense.     

Abnormal directed migration of blood polymorphonuclear leukocytes in rheumatoid arthritis. Potential role in increased susceptibility to bacterial infections.

Rheumatoid arthritis (RA) patients are at higher risks of bacterial infection than healthy subjects. Polymorphonuclear leukocytes (PMN) are the first line of nonspecific cellular defence against these infections. We tested the hypothesis that abnormal directed migration of PMN may be one reason for the increased infection rate of RA patients. PMN migration was investigated in 68 peripheral blood samples of 15 RA patients compared with 64 samples of healthy controls in a novel whole blood in vitro membrane filter assay. The migration of PMNs from RA patients and controls was stimulated using the bacterial chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Unstimulated PMN migration of RA patients was increased compared with healthy controls as measured by the following parameters: (a) absolute number of migrant PMNs (1954+/-87 vs. 1238 +/-58 PMN/mm2), (b) percentage of PMNs migrated into the filter (total migration index, TMI) (28.6+/-0.9 vs. 24.0+/-0.8%), (c) the distance half the migrating PMNs had covered (distribution characteristic, DC) (22.6+/-1.1 vs. 16.1+/-0.6 mm) and (d) the product of TMI and DC (neutrophil migratory activity, NMA) (669.0+/-45.0 vs. 389.0+/-18.9). fMLP stimulated PMNs of RA patients showed defective migration compared to unstimulated samples as shown by (a) a reduced number of migrant PMNs (1799+/-93 PMN/mm2), (b) lower TMI (26.1+/-0.9%), (c) unremarkable altered distribution characteristic (22.9+/-0.8 mm) and (d) significant reduced migratory activity (600.0+/-30.0). Our data suggest that the high incidence of infections in RA patients may partly be caused by defective migratory activity of PMNs to bacterial chemoattractants as demonstrated by fMLP.     

Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.

Arachidonate activation of the NADPH-oxidase in intact neutrophils and in a cell-free O2- generation system was compared to synergistic activation in response to arachidonate and agents that effect protein phosphorylation. In intact neutrophils, suboptimal doses of retinal which increase protein phosphorylation, or 4B-phorbol 12-myristate 13-acetate (PMA) an activator of protein kinase C, induced minimal O2- release, but primed neutrophils to release enhanced amounts of O2- in response to 2.5 microM arachidonate. In contrast to retinal or PMA, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, did not induce any release of O2-, but significantly increased the maximal rate and duration of O2- release in response to arachidonate. In the cell-free system, only arachidonate induced O2- generation. Consistent with previous findings, activation of the cell-free system was dependent of the presence of light membranes, cytosol, NADPH, Mg2+, and 82 microM arachidonate. Pretreatment of neutrophils with suboptimal doses of PMA or retinal had little effect on the arachidonate-stimulated release of O2- in cell-free preparations of these cells. However, cytosol (but not light membranes) from PMA or retinal-primed neutrophils was more effective in completing resting membrane NADPH-oxidase activity when compared to cytosol from resting cells. The addition of protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine decreased the effectiveness of PMA-primed cytosol to complete the cell-free system, but had little effect on cytosol obtained from cells primed with retinal. The addition of protein phosphatase inhibitors, p-nitrophenyl phosphate or okadaic acid to neutrophil cavitates increased 3-fold the release of O2- in cell-free preparations of these cells. Okadaic acid and p-nitrophenyl phosphate also increased the effectiveness of both cytosol and light membranes to complete the cell-free system when combined with cytosol or light membranes from resting neutrophils, respectively, indicating that both fractions are affected by the inhibition of protein phosphatase activity. These data indicate that increases in protein phosphorylation alone do not lead to the activation of the NADPH-oxidase, but in addition to the requirement of an anionic amphiphile, the release of O2- from intact neutrophils or in the cell-free system is increased by stimulus activation of protein kinase C or more impressively by inhibition of protein phosphatase activity.     

Spin Trapping of Superoxide from Glass Adherent Polymorphonuclear Leukocytes Induced by N-Formylmethionyl-Leucyl-Phenylalanine

Dahinden et al. reported that N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide release from polymorphonuclear leukocytes (PMNs) lasted more than 60 min when the cells were allowed to attach to a petri dish before induction. In contrast, it lasted only for 2.5 min when cells were in suspension (J. Clin. Invest. 72: 113-121, 1983). In spite of this report, the effect of cell adhesion has been ignored in most spin trapping studies of superoxide release from PMNs. This study shows that most PMNs in a quartz flat electron paramagnetic resonance (EPR) cuvette which was placed horizontally adhered to the wall within 3 min. In contrast, if the cuvette was placed vertically, only 20-30% of the cells became adherent in 30 min. We performed spin trapping studies using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, and monitored the effect of cell adhesion on superoxide generation. When spin trapping was conducted on PMNs in suspension, the EPR signal of superoxide adduct (DMPO-OOH) was undetectable after stimulation with fMLP. However, PMNs which were allowed to adhere to the cuvette after stimulation generated superoxide for hours. Moreover, when PMNs were allowed to adhere prior to the stimulation, the magnitude of superoxide release was augmented three-to fourfold. Unlike fMLP, phorbol myristate acetate (PMA), which has been most commonly used in spin trapping studies, induced superoxide release which was not influenced by cell adhesion. We emphasize the importance of specifying the cell-adhesion-state in spin trapping studies.     

Effect of the 5-lipoxygenase inhibitor piriprost on superoxide production by human neutrophils

The effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1 (Piriprost) on the oxidative response was studied in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate, 13-acetate (PMA) or opsonized zymosan. Piriprost inhibited the stimulatory effect of fMLP on superoxide anion (O2-) generation, at concentrations higher than those which depress leukotriene B4 (LTB4) formation. This inhibition was overcome by increasing the concentration of fMLP. Neither exogenous LTB4 nor indomethacin were able to reverse the inhibitory effect of piriprost on fMLP action. In contrast, piriprost did not inhibit the stimulation of O2- production induced by PMA or zymosan. Piriprost behaves thus as a specific and apparently competitive antagonist of fMLP: this action does not seem to involve lipoxygenase inhibition and might be exerted at the level of the fMLP receptor or its associated mechanisms of transduction.     

Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system

We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 160,000. Ca(2+) stimulates the basal H(2)O(2) generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H(2)O(2) production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This "immature" protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22(Phox). Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H(2)O(2)-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91(Phox) in the leukocyte; and (4) the thyroid H(2)O(2)-generating system is analogous to the leukocyte system with regard to ThOX's and gp91(Phox) but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid.     

Characterization of nitric oxide consumption pathways by normal,chronic granulomatous disease and myeloperoxidase-deficient human neutrophils

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H(2)O(2) substrate since all superoxide (O(2)(-.) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O(2)(-.) generation, significantly faster than expected from 1:1 termination of NO with O(2)(-.). Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.     

Novel priming compounds of cystathionine metabolites on superoxide generation in human neutrophils

Human peripheral blood polymorphonuclear leukocytes were preincubated with cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria. Among the cystathionine metabolites, cystathionine ketimine and N-acetyl-S-(3-oxo-3-carboxy-n-propyl) cysteine (NAc-OCPC) significantly enhanced the N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide generation, but cystathionine, NAc-cystathionine, and cyclothionine did not enhance the superoxide generation. Cystathionine ketimine and NAc-OCPC also enhanced superoxide generation induced by opsonized zymosan (OZ) but not that induced by arachidonic acid (AA) and phorbol 12-myristate 13-acetate (PMA). Superoxide generation induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein, an inhibitor of tyrosine kinase, and was enhanced by 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C. Cystathionine ketimine and NAc-OCPC markedly also increased phosphorylation of 45-kDa protein in human neutrophils and the phosphorylation depended on the concentrations of cystathionine ketimine and NAc-OCPC. The phosphorylation of 45-kDa protein induced by cystathionine ketimine and NAc-OCPC was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by H-7 and staurosporine, inhibitors of protein kinase C. Cystathionine metabolites and l-cystathionine sulfoxides were separated into two diastereoisomers, CS-I and CS-II. CS-I enhanced the superoxide generation induced by AA and PMA but not that induced by fMLP and OZ. In contrast, CS-II enhanced the superoxide generation induced by fMLP and OZ, but not that induced by AA and PMA.     

MEK-1/2 inhibition prevents 5-lipoxygenase translocation in N-formylpeptide-challenged human neutrophils

In order to elucidate the role of mitogen-activated protein kinase kinase (MEK-1/2) in 5-lipoxygenase (5-LO) activation we studied the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced 5-LO translocation in human blood neutrophils (PMNs). In non-primed, Ca(2+)-repleted PMNs, fMLP consistently stimulated MEK-1/2 phosphorylation, but induced 5-LO translocation and product formation (430+/-128 pmol; SEM, n=13) only in 13 of 18 PMN preparations from different healthy donors. In fMLP-responsive cells, the MEK-1/2 inhibitor PD098059 (50 microM) attenuated MEK phosphorylation and abolished 5-LO activation at the translocation step. The fMLP-mediated 5-LO product formation was also sensitive to MEK inhibition by U0126 and to p38 inhibition by SB203580. But in contrast to PD098059, U0126 at 10 microM and SB203580 at 20-50 microM impaired 5-LO activity in the cell-free assay setting, suggesting direct actions of higher concentrations of U0126 and SB203580 on 5-LO apart from MEK and p38 inhibition, respectively. These data show that fMLP initiates 5-LO product formation in non-primed, Ca(2+)-repleted human blood PMNs from healthy donors, and that MEK signaling is pivotal, but not sufficient for 5-LO activation in response to the receptor agonist fMLP.     

Studies of signal transduction in the respiratory burst-associated stimulation of fMet-Leu-Phe-induced tubulin tyrosinolation and phorbol 12-myristate 13-acetate-induced posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils and HL-60 cells

A specific stimulation of tubulin tyrosinolation in human neutrophils (PMNs) is induced by the synthetic peptide chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and this stimulation is closely associated with activation of the NADPH oxidase-mediated respiratory burst (Nath, J., and Gallin, J. I. (1983) J. Clin. Invest. 71, 1273-1281). In contrast, along with tubulin tyrosinolation, a distinctly different respiratory burst-associated random posttranslational incorporation of tyrosine into multiple PMN proteins is observed in PMNs stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol (DAG). In studies exploring the mechanism(s) of signal transduction for these distinct neutrophil responses, we found that the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation in PMNs and in differentiated HL-60 cells is completely blocked by pertussis toxin, while the PMA-induced random incorporation of tyrosine is not inhibited. We also found that expression of the fMet-Leu-Phe-mediated stimulation of tubulin tyrosinolation in HL-60 cells is correlated with increases in the specific activity of protein kinase C and with the acquisition of respiratory burst activity which occur during induced myeloid maturation of these cells. Furthermore, both the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation and the PMA or DAG-induced random posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils, were found to be reversibly inhibited (greater than 70%) by the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)piperazine (C-I) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), in parallel with inhibition of superoxide (O2-) generation. In related studies, we also found that fMet-Leu-Phe-stimulated O2- production is comparably inhibited by C-I and H-7, but in a highly temperature-dependent manner. Inhibition was observed only when C-I or H-7 is added to PMNs at physiologic temperature, i.e. 37 degrees C. Interestingly, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. Considered together, these findings argue against the suggestion that there is a protein kinase C-independent pathway for activation of the respiratory burst in neutrophils stimulated with N-formyl peptides.     

Effect of N-acetyl- -cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.     

S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.