fMLP-stimulated Superoxide Research Articles

The effects of Bruton tyrosine kinase inhibition on chemotaxis and superoxide generation in human neutrophils.

The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP). Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously. Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25microM, while inhibited superoxide production by control neutrophils. Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.

Small GTPases Rap1 and RhoA regulate superoxide formation by Rac1 GTPases activation during the phagocytosis of IgG-opsonized zymosans in macrophages

Phagocytic NADPH oxidase plays a critical role in superoxide generation in macrophage cells. Small GTPases, including Rac1 and Rac2, have been implicated in the regulation of NADPH oxidase activity. Rap1, which has no effect in a cell-free system of oxidase activation, recently has been proven to colocalize with cytochrome b558. In addition, neutrophils from rap1A-/- mice reduce fMLP-stimulated superoxide production. Here, we tried to determine whether Rap1 also plays a role in the production of superoxide. IgG-opsonized zymosan (IOZ) particles treatment induced Rap1 activation and superoxide generation. Knock-down of Rap1 by si-Rap1 suppressed IOZ-induced superoxide formation. Sh-RhoA also reduced superoxide levels, but 8CPT-2Me-cAMP, an activator of Epac1 (a guanine nucleotide exchange factor (GEF) of Rap1), could recover the levels to the control value. When cells were stimulated by IOZ, Rap1 and Rac1 were translocated to the membrane, and then interacted with p22phox. 8CPT-2Me-cAMP rescued sh-RhoA-induced reduction of the interaction between Rac1 and p22phox, and enhanced lysophosphatidic acid (LPA)-induced increase of their interaction. Moreover, Rac1 activity was increased by both LPA and 8CPT-2Me-cAMP when treated with IOZ particles. Si-Vav2 impaired GTP-Rac1 levels in response to 8CPT-2Me-cAMP/IOZ. Phosphorylation of RhoA activates Rac1 in response to IOZ by the enhanced binding of phospho-RhoA to RhoGDI, leading to the release of Rac1 from the Rac1-RhoGDI complex. In conclusion, IOZ treatment induces Rap1 activation and phosphorylation of RhoA, which in turn cause Rac1 activation and promote Rac1 translocation to the membrane leading to binding with p22phox that activates NADPH oxidase and produces superoxide.

Ascorbic Acid Levels and Neutrophil Superoxide Production in Blood of Pre-, Early and Late Hypertensive Stroke-Prone Spontaneously Hypertensive Rats

We compared ascorbic acid (AA) levels in the blood and TPA- and fMLP-stimulated superoxide (O2•−) production in neutrophils of pre-, early, and late hypertensive stroke-prone spontaneously hypertensive rats (SHRSP) with those of age-matched Wistar Kyoto rats (WKY), or two other normotensive strains of rats. Plasma and lymphocyte AA levels were about two-fold higher in SHRSP as early as 4 weeks old compared to WKY, and also higher than those of Wistar and Sprague-Dawley (SD) rats. Levels of AA were high in the liver and adrenal glands of SHRSP, indicating congenitally high AA levels. The production of O2•− in neutrophils was about two-fold higher in SHRSP than in WKY even at 4 weeks of age, and increased with age in both strains. Among SHRSP, AA levels in lymphocytes decreased at the late hypertensive stages with a decrease in hepatic l-gulono-γ-lactone oxidase (GLO) activities. These data suggest that bi-phasic AA levels in the blood of SHRSP comprise congenitally high levels and a decrease after persistent hypertension due to enhanced O2•− production and a decrease in de novo AA synthesis through GLO.

Activation of the neutrophil respiratory burst by plasma from periodontitis patients is mediated by pro-inflammatory cytokines

To determine the effect of periodontitis patients' plasma on the neutrophil oxidative burst and the role of albumin, immunoglobulins (Igs) and cytokines. Plasma was collected from chronic periodontitis patients (n=11) and periodontally healthy controls (n=11) and used with/without depletion of albumin and Ig or antibody neutralization of IL-8, GM-CSF or IFN-α to prime/stimulate peripheral blood neutrophils, isolated from healthy volunteers. The respiratory burst was measured by lucigenin-dependent chemiluminescence. Plasma cytokine levels were determined by ELISA. Plasmas from patients were significantly more effective in both directly stimulating neutrophil superoxide production and priming for subsequent formyl-met-leu-phe (fMLP)-stimulated superoxide production than plasmas from healthy controls (p<0.05). This difference was maintained after depletion of albumin and Ig. Plasma from patients contained higher mean levels of IL-8, GM-CSF and IFN-α. Individual neutralizing antibodies against IL-8, GM-CSF or IFN-α inhibited the direct stimulatory effect of patients' plasma, whereas the ability to prime for fMLP-stimulated superoxide production was only inhibited by neutralization of IFN-α. The stimulating and priming effects of control plasma were unaffected by antibody neutralization. This study demonstrates that plasma cytokines may have a role in inducing the hyperactive (IL-8, GM-CSF, IFN-α) and hyper-reactive (IFN-α) neutrophil phenotype seen in periodontitis patients.

Anti-proteinase 3 antibodies both stimulate and prime human neutrophils

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are postulated to injure vascular endothelium by inducing cytokine-primed neutrophils to release proteolytic enzymes and generate reactive oxygen species. Anti-PR3 induce exocytosis, and since priming is associated with upregulation of plasma membrane proteins by exocytosis of intracellular granules, we tested the hypothesis that anti-PR3 prime neutrophils in the absence of cytokines. Isolated human neutrophils were incubated with or without anti-PR3. Superoxide release was determined by measuring the reduction of ferricytochrome C. Exocytosis of secretory vesicles and specific granules was determined by measuring the expression of CD35 and CD66b, respectively, using flow cytometry. Anti-PR3 (15 mug/mL) directly stimulated superoxide production and enhanced FMLP-stimulated superoxide production. Anti-PR3 (0.5 mug/mL) did not stimulate superoxide production but did enhance FMLP-stimulated superoxide production. Incubation of neutrophils with anti-PR3 resulted in time-dependent exocytosis of secretory vesicles and specific granules. Anti-PR3-induced exocytosis, but not superoxide production, was dependent on p38 mitogen-activated protein kinase. Conclusions. These data demonstrate that anti-PR3 can directly stimulate production of reactive oxygen species by neutrophils without cytokine priming, and that anti-PR3 prime neutrophils for increased FMLP-stimulated reactive oxygen species production. Anti-PR3 also induce exocytosis via a mechanism separate from their effect on reactive oxygen species production. These findings suggest that anti-PR3 ANCA may activate neutrophils and cause endothelial cell injury by multiple pathways, including some that are independent of priming by a second agent.

Rap1a Null Mice Have Altered Myeloid Cell Functions Suggesting Distinct Roles for the Closely Related Rap1a and 1b Proteins

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a-/- embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a-/- mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a-/- mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.

Neutrophil caveolin-1 expression contributes to mechanism of lung inflammation and injury

Caveolin-1 present in immune cells may be involved in regulation of the inflammatory response. Here, using caveolin-1-null (Cav-1(-/-)) mice, we addressed the role of caveolin-1 in polymorphonuclear neutrophils (PMNs) in regulating PMN activation-mediated lung injury. In lungs of wild-type (Cav-1(+/+)) mice perfused at constant flow with Krebs-Henseleit solution, addition of Cav-1(+/+) PMNs (4 x 10(6) cells) into the perfusate followed by their activation with formyl-Met-Leu-Phe (fMLP, 1.0 muM) plus platelet-activating factor (1.0 nM) increased pulmonary microvessel filtration coefficient by 150% and wet-to-dry lung weight ratio by 50% as well as PMN accumulation in lungs. These responses were markedly reduced in lungs perfused with Cav-1(-/-) PMNs followed by addition of the same activating agents. fMLP-stimulated adhesion of Cav-1(-/-) PMNs to pulmonary microvascular endothelial cells and migration of Cav-1(-/-) PMNs across endothelial monolayers were also impaired compared with Cav-1(+/+) PMNs. Cav-1(-/-) PMNs showed 50-80% reduction in PMA- or fMLP-stimulated superoxide production compared with Cav-1(+/+) PMNs. In addition, Cav-1(-/-) PMNs had decreased migratory activity (50%) and adhesion to fibrinogen (40%) in response to fMLP. Rac1 and Rac2 were activated in Cav-1(+/+) PMNs after stimulation of fMLP but not in Cav-1(-/-) PMNs. Exogenous expression of caveolin-1 in COS-phox cells augmented the fMLP-induced Rac1 activation and superoxide production, indicating a direct role of caveolin-1 in the mechanism of superoxide production. Thus caveolin-1 expression in PMNs plays a key role in mediating PMN activation, adhesion, and transendothelial migration and in PMN activation-induced lung inflammation and vascular injury.

Involvement of p38 MAP Kinase in Not Only Activation of the Phagocyte NADPH Oxidase Induced by Formyl-methionyl-leucyl-phenylalanine but Also Determination of the Extent of the Activity

Activated NADPH oxidase in neutrophils produces superoxide. We investigated the role of p38 MAP kinase in activating NADPH oxidase stimulated by the bacteria-derived peptide fMLP. fMLP-stimulated superoxide production was completely abolished by SB203580, a p38 MAP kinase inhibitor, whereas anisomycin, a p38 MAP kinase activator, did not induce superoxide production, indicating that p38 MAP kinase was essential, but not sufficient, for NADPH oxidase activation. Anisomycin pretreatment strongly activated p38 MAP kinase in fMLP-stimulated cells, accompanied by greatly increased superoxide production, suggesting that p38 MAP kinase determines the extent of the fMLP-stimulated NADPH oxidase activity. Furthermore, superoxide production was remarkably reactivated by cytochalasin B addition after fMLP-stimulated production had disappeared, and this was correlated with highly activated p38 MAP kinase. These results suggest that p38 MAP kinase is involved not only in activating NADPH oxidase stimulated by fMLP but also in determining the extent of its activity.

Aminopeptidase N (CD13) Regulates Tumor Necrosis Factor-α-induced Apoptosis in Human Neutrophils

Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-alpha (TNFalpha) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFalpha in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFalpha-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFalpha-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFalpha-induced apoptosis, cell polarization, and TNFalpha-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFalpha-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFalpha-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.

Immune‐Modulating Effects of Melatonin, N‐Acetylserotonin, and N‐Acetyldopamine

Melatonin and N-acetylserotonin (NAS) have antioxidant properties. In the present study, we examined whether melatonin, NAS, and N-acetyldopamine (NAD) have a modulatory effect on tumor necrosis factor-alpha (TNF-alpha) synthesis and superoxide production. Differentiated THP-1-derived human monocytes were coincubated with Escherichia coli lipopolysaccharide (LPS) and rising concentrations of melatonin, NAS, or NAD. After 24 h, TNF-alpha was measured in cell supernatants. In addition, the production of superoxide by HL-60-derived human neutrophils upon stimulation with 4-beta-phorbol 12-beta-myristate 13-alpha-acetate (PMA) or N-formyl methionyl-leucyl-phenylalanine (fMLP) and increasing concentrations of melatonin, NAS, or NAD was determined. Incubation of THP-1-derived monocytes with increasing concentrations of melatonin, NAS, or NAD resulted in a marked decrease in LPS-stimulated TNF-alpha production, which was dose-dependent and on the order of 96-98%. Incubation of HL-60-derived neutrophils with increasing concentrations of melatonin, NAS, or NAD resulted in a modest decrease in PMA-stimulated superoxide production, which was dose-dependent. At the 100 microM dose, melatonin, NAS, or NAD resulted in a 14 +/- 4%, 30 +/- 1%, and 29 +/- 1% decrease in PMA-stimulated superoxide production, respectively. Coincubation of HL-60 cells with melatonin, NAS, or NAD also resulted in a modest dose-dependent decrease in fMLP-stimulated superoxide production. At the 100 microM dose, melatonin, NAS, or NAD resulted in a 13 +/- 1%, 14 +/- 1%, and 14 +/- 1% decrease in superoxide production, respectively. Our results indicate that the inhibitory effect of melatonin, NAS, or NAD on LPS-induced TNF-alpha production is robust and dose-dependent. These compounds are equally effective in attenuating the generation of oxidant radicals, although to a lesser degree.

Role of PI3-kinase–dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival

Phosphoinositide 3-kinase (PI3-kinase)-dependent phosphorylation of the proapoptotic Bcl-2 family member Bad has been proposed as an important regulator of apoptotic cell death. To understand the importance of this pathway in nontransformed hematopoietic cells, we have examined the effect of survival cytokines on PI3-kinase activity and Bad expression and phosphorylation status in human neutrophils. Granulocyte macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) both reduced the rate of apoptosis in neutrophils cultured in vitro for 20 hours. Coincubation with the PI3-kinase inhibitor LY294002, which in parallel experiments abolished GM-CSF-primed, fMLP-stimulated superoxide anion production and GM-CSF-stimulated PtdIns(3,4,5)P(3) accumulation, inhibited the GM-CSF and TNF-alpha survival effect. In contrast, the MAP kinase kinase (MEK1/2) inhibitor PD98059 and the protein kinase A inhibitor H-89 had only a marginal effect on GM-CSF-mediated neutrophil survival. GM-CSF substantially increased Bad phosphorylation at Ser112 and Ser136 and increased the cytosolic accumulation of Bad. GM-CSF also regulated Bad at a transcription level with a marked decrease in mRNA levels at 4 hours. TNF-alpha caused a biphasic effect on the rate of morphologic apoptosis, which corresponded to an early increase, and a late inhibition, of Bad mRNA levels. LY294002 inhibited GM-CSF- and TNF-alpha-mediated changes in Bad phosphorylation and mRNA levels. These data suggest that the survival effect of GM-CSF and TNF-alpha in neutrophils is caused by a PI3-kinase-dependent phosphorylation and cytosolic translocation of Bad, together with an inhibition of Bad mRNA levels. This has important implications for the regulation of neutrophil apoptosis in vivo.

Priming by grepafloxacin on respiratory burst of human neutrophils: its possible mechanism.

Grepafloxacin is a broad-spectrum fluoroquinolone derivative that has good tissue penetration. We demonstrated that grepafloxacin showed a priming effect on neutrophil respiratory burst, triggered by either a chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4), but not by the phorbol ester phorbol 12-myristate 13-acetate (PMA). The priming effect of grepafloxacin on fMLP-stimulated superoxide generation by human neutrophils correlated with the penetration of grepafloxacin into cells. Removal of extracellular grepafloxacin did not inhibit the priming effect on fMLP-stimulated superoxide generation. Furthermore, grepafloxacin induced the translocation of p47-phox and p67-phox to the membrane fraction of neutrophils, whereas tyrosine phosphorylation was hardly observed in neutrophils exposed to grepafloxacin. The priming effect of grepafloxacin on superoxide generation from neutrophils was not inhibited by treatment with pertussis toxin, a protein-tyrosine kinase inhibitor (ST-638) or a protein kinase C inhibitor (calphostin C), or chelation of extracellular calcium. Grepafloxacin did not change the fMLP receptor-binding properties. Taken together, these findings suggest that grepafloxacin evokes a priming effect on neutrophil superoxide generation intracellularly through the translocation of p47-phox and even p67-phox protein to the membrane fractions. GTP binding protein, protein-tyrosine phosphorylation and protein kinase C activation are not involved in the priming effect.

Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42ERK2and p38SAPK and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A2

AbstractArachidonic acid (AA) generated by phospholipase A2(PLA2) is thought to be an essential cofactor for phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Both enzymes are simultaneously primed by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor–α (TNF-α). The possibility that either unprimed or cytokine-primed responses of PLA2 or NADPH oxidase to the chemotactic agents formyl-methionyl-leucyl-phenylalanine (FMLP) and complement factor 5a (C5a) could be differentially inhibited by inhibitors of the mitogen-activated protein (MAP) kinase family members p42ERK2 (PD98059) and p38SAPK(SB203580) was investigated. PD98059 inhibited the activation of p42ERK2 by GM-CSF, TNF-α, and FMLP, but it did not inhibit FMLP-stimulated superoxide production in either unprimed or primed neutrophils. There was no significant arachidonate release from unprimed neutrophils stimulated by FMLP, and arachidonate release stimulated by calcium ionophore A23187 was not inhibited by PD98059. In contrast, PD98059 inhibited both TNF-α– and GM-CSF–primed PLA2 responses stimulated by FMLP. On the other hand, SB203580 inhibited FMLP-superoxide responses in unprimed as well as TNF-α– and GM-CSF–primed neutrophils, but failed to inhibit TNF-α– and GM-CSF–primed PLA2 responses stimulated by FMLP, and additionally enhanced A23187-stimulated arachidonate release, showing that priming and activation of PLA2 and NADPH oxidase are differentially dependent on both the p38SAPK and p42ERK2 pathways. Studies using C5a as an agonist gave similar results and confirmed the findings with FMLP. In addition, methyl arachidonyl fluorophosphonate (MAFP), the dual inhibitor of c and iPLA2 enzymes, failed to inhibit superoxide production in primed cells at concentrations that inhibited arachidonate release. These data demonstrate that NADPH oxidase activity can be dissociated from AA generation and indicate a more complex role for arachidonate in neutrophil superoxide production.

Clustering of Urokinase Receptors (uPAR; CD87) Induces Proinflammatory Signaling in Human Polymorphonuclear Neutrophils

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 +/- 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of beta-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.

Tumor necrosis factor-α-mediated signal transduction in human neutrophils: Involvement of sphingomyelin metabolites in the priming effect of tnf-α on the fMLP-stimulated superoxide production

We investigated the mechanism underlying the priming effect of TNF-α on fMLP-stimulated superoxide production in human neutrophils. Tnf-α enhanced fMLP-stimulated superoxide production in a concentrationdependent manner. Tnf-α also induced sphingomyelin (SM) hydrolysis and increased the formation of its metabolite, sphingosine-1-phosphate (SP-1-P). The treatment of neutrophils with sphingomyelinase also resulted in a similar priming effect. C 2 ceramide produced a concentration-dependent inhibition of fMLP-stimulated superoxide production within the concentration range of 1–30 μM. Sphingosine had a dual effect on fMLP-stimulated superoxide generation, exhibiting a priming effect at lower concentrations (0.2–1 μM), but an inhibitory effect at higher concentrations (1–30 μM). SP-1-P (1–30 μM), showed a concentration-dependent enhancement of fMLP stimulated superoxide production. Furthermore, after treating neutrophils with DL-threo-dihydro-sphingosine, a competitive inhibitor of sphingosine kinase, TNF-α produced a similar dual effect as observed with sphingosine. These results strongly suggest that SM hydrolysis plays a key role in the intracellular signal transduction mediating the TNF-α-mediated priming effect.

Depressant Effects of Ambroxol on Lipopolysaccharide- or fMLP-stimulated Free Radical Production and Granule Enzyme Release by Alveolar Macrophages

In order to explore the depressant action of ambroxol, a bronchial expectorant, on the activated alveolar macrophage responses, its effect on lipopolysaccharide (LPS)- or N-formyl-methionyl-leucyl-phenylalanine (fMLP)- stimulated free radical production and granule enzyme release by rat lung alveolar macrophages was investigated. Ambroxol attenuated the 100 ng/ml LPS- or 1 μM fMLP-stimulated superoxide, H2O2and nitric oxide production and releases of acid phosphatase and lysozyme by alveolar macrophages. Ambroxol attenuated phorbol myristate acetate-stimulated superoxide and nitric oxide production that was inhibited by 100 nM staurosporine. N,N-dimethylsphingosine (DMS, 4.5 and 9 μM) alone stimulated superoxide production by macrophages, while 45 μM of the compound did not show a stimulatory effect. However, DMS decreased nitric oxide production in a dose-dependent manner. Ambroxol did not alter the DMS effect on free radical production that was affected by 10 μM genistein. A preincubation of macrophages with ambroxol (10 and 100 μM), staurosporine and genistein attenuated the elevation of [Ca2+]icaused by LPS. The results suggest that ambroxol exerts a depressant effect on LPS- or fMLP-stimulated free radical production and granule enzyme release by rat alveolar macrophages, which may be attributed to its inhibitory action on the activation process, protein kinase C, but its action on protein tyrosine kinase is not suggested.

Role of the extracellular signal-regulated protein kinase cascade in human neutrophil killing of Staphylococcus aureus and Candida albicans and in migration.

Killing of Staphylococcus aureus and Candida albicans by neutrophils involves adherence of the microorganisms, phagocytosis, and a collaborative action of oxygen reactive species and components of the granules. While a number of intracellular signalling pathways have been proposed to regulate neutrophil responses, the extent to which each pathway contributes to the killing of S. aureus and C. albicans has not been clearly defined. We have therefore examined the effect of blocking one such pathway, the extracellular signal-regulated protein kinase (ERK) cascade, using the specific inhibitor of the mitogen-activated protein kinase/ERK kinase, PD98059, on the ability of human neutrophils to kill S. aureus and C. albicans. Our data demonstrate the presence of ERK2 and a 43-kDa form of ERK but not ERK1 in human neutrophils. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the activities of both ERK2 and the 43-kDa form were stimulated. Despite abrogating the activity of both ERK forms, PD98059 only slightly reduced the ability of neutrophils to kill S. aureus or C. albicans. This is consistent with our finding that PD98059 had no effect on neutrophil adherence or degranulation, although pretreatment of neutrophils with PD98059 inhibited fMLP-stimulated superoxide production by 50%, suggesting that a change in superoxide production per se is not strictly correlated with microbicidal activity. However, fMLP-stimulated chemokinesis was markedly inhibited, while random migration and fMLP-stimulated chemotaxis were partially inhibited, by PD98059. These data demonstrate, for the first time, that the ERK cascade plays only a minor role in the microbicidal activity of neutrophils and that the ERK cascade is involved primarily in regulating neutrophil migration in response to fMLP.

Neutrophil Apoptosis and Dysfunction in Uremia

The high prevalence of bacterial infections among patients with end-stage renal disease suggests that "professional" phagocytes such as neutrophils are functionally impaired. This dysfunction has been ascribed to uremic toxins, malnutrition, and dialysis. The aim of this study was to investigate the contribution of apoptosis to neutrophil dysfunction in uremia. Neutrophils harvested from uremic patients (n = 6) and age-/gender-matched healthy control subjects (n = 6) were incubated with either 50% autologous plasma or 10% fetal calf serum. After 24-h incubation, apoptosis was quantified by flow cytometry by using propidium iodide nuclear staining. Neutrophils from healthy volunteers were also incubated with either 50% heterologous normal or uremic plasma. After 24-h incubation, apoptosis was quantified by flow cytometry and transmission electron microscopy. In addition, superoxide production was determined by measuring the capacity to reduce ferri- to ferro-cytochrome C by using 4-beta-phorbol 12-beta-myristate 13-alpha-acetate or N-formyl methionyl-leucyl-phenylalanine (fMLP) for stimulus. Phagocytosis was determined by the uptake of 14C-labeled heat-killed Staphylococcus aureus. Compared with normal neutrophils, uremic neutrophils demonstrated greater apoptosis in the presence of autologous plasma (9 +/- 4 versus 19 +/- 6%, P = 0.01) as well as 10% fetal calf serum (19 +/- 7 versus 31 +/- 6%, P = 0.03). Furthermore, compared with normal neutrophils exposed to heterologous normal plasma, those exposed to heterologous uremic plasma exhibited higher apoptosis rates (19 +/- 3 versus 40 +/- 5%, P = 0.002), lower tMLP-stimulated superoxide production (22.6 +/- 2.5 versus 15.5 +/- 1.1 nmol O2*-/3.12 x 10(5) cells/30 min, P = 0.01), and a lower phagocytosis index (38 +/- 3% versus 27 +/- 5%, P = 0.04). Apoptosis correlated inversely with fMLP-stimulated superoxide production (r = -0.60, P = 0.04) and phagocytosis (r = -0.57, P = 0.05). These results suggest that uremic neutrophils undergo accelerated in vitro apoptosis. Furthermore, uremic plasma accelerates apoptosis of normal neutrophils, resulting in a dysfunctional pattern that is similar to that observed in uremia.

A High Affinity Binding Site for [3H]-Dofetilide on Human Leukocytes

Certain Class III anti-arrhythmic agents have been shown to interact with human leukocytes and after antigenic and mitogenic activation. We hypothesized that a binding site for the Class III anti-arrhythmic agent, dofetilide, would exist on human leukocytes. Analysis of binding isotherms defined the presence of a single high affinity binding site on mononuclear cells and neutrophils: Kd26±4 nm, Bmax61±14 fmol/106cells and Kd33±14 nm, Bmax163±45 fmol/106cells, respectively. Other Class III drugs inhibited [3H]-dofetilide binding at physiologically relevant concentrations, but the IC50values of E4031 and quinidine were significantly higher for leukocytes than for cardiac myocytes. Interestingly, verapamil inhibited [3H]-dofetilide binding to leukocytes, but not to cardiac myocytes at physiologic concentrations (10μm). Charybdotoxin and tetraethlyammonium inhibited [3H]-dofetilide binding to leukocytes atμmand mmconcentrations, respectively, however, apamin did not inhibit binding even at 1μmconcentrations. These data suggest that a Ca2+-activated K+channel, like K(Ca) mini (apamin-insensitive isoform), is a candidate for the leukocyte [3H]-dofetilide binding site. To assess the functional significance of defetilide binding to leukocyte biology, we evaluated fMLP-stimulated superoxide production in the presence or absence of dofetilide. Dofetilide, at 30 nmsuppressed of superoxide production. In conclusion, dofetilide binds to human leukocytes at physiologic concentrations and this binding alters leukocyte function possibly through interaction with a Ca2+-activated K+channel.

Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase.

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.