The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites.

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.     

Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.     

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI-/-) mice. We demonstrate that murine neutrophils expressed cGKIalpha. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI-/- bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI-/- than in WT. Enhanced chemotaxis was also observed with cGKI-/- peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI-/- vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI-/- than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI-/- (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI-/- (approximately 60%) than in WT (approximately 90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIalpha down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIalpha on the secretagogue responses are complex, depending on the granule subtype.     

Granulocyte-macrophage colony-stimulating factor primes neutrophils by activating a pertussis toxin-sensitive G protein not associated with phosphatidylinositol turnover

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine which produces diverse biological effects in target cells of myeloid origin. GM-CSF enhances the production of superoxide anion (O2-) by mature neutrophils in response to chemotactic peptides such as formyl-methionyl-leucyl-phenylalanine (fMLP), but alone it has no effect on this system. This process has been termed "priming." fMLP activates neutrophils via a pertussis toxin-sensitive GTP-binding protein, leading to the rapid production of the second messengers diacylglycerol (DAG) and inositol trisphosphate, via phosphatidylinositol turnover, and arachidonic acid (AA) by a presumptive phospholipase A2-mediated mechanism. All three second messengers may lead to the generation of O2-. We investigated the effect of priming of GM-CSF on these systems. GM-CSF had no effect on fMLP-stimulated DAG and inositol trisphosphate levels, nor did it amplify the response to exogenously added phorbol ester (to mimic the action of DAG) or calcium ionophore. Neutrophils primed with the cytokine showed a small, but significant, enhancement of fMLP-stimulated AA release. Compared with unprimed controls, primed neutrophils also showed a significant increase in O2- production when stimulated with either AA or the nonhydrolyzable GTP analogue, GTP-gamma-S. The magnitude of enhanced O2- production was similar to that observed after fMLP treatment of primed cells. All of these effects, including the increased sensitivity to AA treatment, were inhibited by pertussis toxin. These data show that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein coupled to a metabolic pathway that mobilizes and utilizes arachidonic acid.     

Effect of arachidonic acid reacylation on leukotriene biosynthesis in human neutrophils stimulated with granulocyte-macrophage colony-stimulating factor and formyl-methionyl-leucyl-phenylalanine

Priming of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) followed by treatment with formyl-methionyl-leucyl-phenylalanine (fMLP) stimulates cells in a physiologically relevant manner with modest 5-lipoxygenase activation and formation of leukotrienes. However, pretreatment of neutrophils with thimerosal, an organomercury thiosalicylic acid derivative, led to a dramatic increase (>50-fold) in the production of leukotriene B(4) and 5-hydroxyeicosatetraenoic acid, significantly higher than that observed after stimulation with calcium ionophore A23187. Little or no effect was observed with thimerosal alone or in combination with either GM-CSF or fMLP. Elevation of [Ca(2+)](i) induced by thimerosal in neutrophils stimulated with GM-CSF/fMLP was similar but more sustained compared with samples where thimerosal was absent. However, [Ca(2+)](i) was significantly lower compared with calcium ionophore-treated cells, suggesting that a sustained calcium rise was necessary but not sufficient to explain the effects of this compound on the GM-CSF/fMLP-stimulated neutrophil. Thimerosal was found to directly inhibit neutrophil lysophospholipid:acyl-CoA acyltransferase activity at the doses that stimulate leukotriene production, and analysis of lysates from neutrophil preparations stimulated in the presence of thimerosal showed a marked increase in free arachidonic acid, supporting the inhibition of the reincorporation of this fatty acid into the membrane phospholipids as a mechanism of action for this compound. The dramatic increase in production of leukotrienes by neutrophils when a physiological stimulus such as GM-CSF/fMLP is employed in the presence of thimerosal suggests a critical regulatory role of arachidonate reacylation that limits leukotriene biosynthesis in concert with 5-lipoxygenase and cytosolic phospholipase A(2)alpha activation.     

Mycobacterial cord factor enhances migration of neutrophil‐like HL‐60 cells by prolonging AKT phosphorylation

Trehalose 6,6′‐dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno‐stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N‐formylmethionyl‐leucyl‐phenylalanine (fMLP)‐induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage‐inducible C‐type lectin, a receptor for TDM, and induced secretion of pro‐inflammatory cytokines and chemokines in differentiated HL‐60 cells. In 2‐ and 3‐D neutrophil migration assays, TDM‐stimulated neutrophils showed increased fMLP‐induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM‐activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Molecular cloning of the fMet-Leu-Phe receptor from neutrophils.

The bacterial chemotactic peptide fMet-Leu-Phe (fMLP) activates neutrophils upon binding to surface receptors. In a previous communication we reported the functional reconstitution of the fMLP receptor in Xenopus laevis oocytes (Coats, W. D., and Navarro, J. (1990) J. Biol. Chem. 265, 5964-5966). In this work we report the isolation of the cDNA encoding the fMLP receptor from neutrophils. A rabbit neutrophil cDNA library was screened with an oligonucleotide probe deduced from the nucleotide sequence of G-protein-coupled receptors, and a cDNA encoding the fMLP receptor was isolated. This cDNA was characterized according to the following criteria: 1) Analysis of the deduced amino acid sequence revealed that the clone belongs to a G-protein-coupled receptor. 2) Tissue distribution analysis of the mRNA indicated that the message is only found in neutrophils. 3) In vitro translation of the message revealed a protein corresponding in size to the deglycosylated fMLP receptor. 4) X. laevis oocytes injected with the fMLP receptor message exhibited fMLP-dependent calcium mobilization and specific binding to the fMLP analog 125I-labeled fNle-Leu-Phe-Nle-Tyr-Lys (where Nle is norleucine and fNle is formylnorleucine). The molecular cloning of the fMLP receptor should provide the framework to analyze the relationship between structure, function, and regulation of this receptor.     

Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.

The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.     

Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate.

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.     

TNF is a key mediator of septic encephalopathy acting through its receptor, TNF receptor-1

In this study, we demonstrate that mice deficient in TNFR1 (TNFR1(-/-)) were resistant to LPS-induced encephalopathy. Systemic administration of lipopolysaccharide (LPS) induces a widespread inflammatory response similar to that observed in sepsis. Following LPS administration TNFR1(-/-) mice had less caspase-dependent apoptosis in brain cells and fewer neutrophils infiltrating the brain (p<0.039), compared to control C57Bl6 (TNFR1(+/+)) mice. TNFR1-dependent increase in aquaporin (AQP)-4 mRNA and protein expression was observed with a concomitant increase in water content, in brain (18% increase in C57Bl6 mice treated with LPS versus those treated with saline), similar to cerebral edema observed in sepsis. Furthermore, absence of TNFR1 partially but significantly reduced the activation of astrocytes, as shown by immunofluorescence and markedly inhibited iNOS mRNA expression (p<0.01). Septic encephalopathy is a devastating complication of sepsis. Although, considerable work has been done to identify the mechanism causing the pathological alterations in this setting, the culprit still remains an enigma. Our results demonstrate for the first time that endotoxemia leads to inflammation in brain, with alteration in blood-brain barrier, up-regulation of AQP4 and associated edema, neutrophil infiltration, astrocytosis, as well as apoptotic cellular death, all of which appear to be mediated by TNF-alpha signaling through TNFR1.     

Neutrophil integrin assay for clinical studies

The level of expression of neutrophil adhesion molecules may be a useful marker for neutrophil activation in clinical studies. We therefore determined neutrophil integrin expression under various experimental conditions using a Fluorescence Activated Cell Sorter (FACS) after the cells had been labelled with fluorescent conjugated antibodies to the integrin subunits CD11a, CD11b and CD18. Levels of labelled CD11b and CD18 increased after activation with the chemotactic peptide formyl-methionyl-leucyl phenylalanine (fMLP) in a dose- and time-dependent manner, but CD11a did not, indicating that CD11a would not be a useful marker of neutrophil activation. The baseline expression of CD11b and CD18 on unstimulated neutrophils was similar in heparin and EDTA anti-coagulated blood but the response to activation with fMLP was significantly less for the EDTA anti-coagulated samples (p < 0·01 in paired t-test). The labelling of integrins was significantly higher in unfixed whole blood samples compared to samples fixed with 1 per cent paraformaldehyde. However, the increase in labelling induced by fMLP was similar whether or not the samples were fixed after activation. Labelling of CD11b and CD18 was greater for preparations of isolated neutrophils than for neutrophils in whole blood, and the response to fMLP stimulation tended to be lower for the isolated cells. Our results indicate that heparin should be used as anti-coagulant in clinical studies utilizing whole blood if subsequent activation of neutrophils is planned (e.g. to detect in vivo priming), although EDTA may be used if baseline expression alone is to be measured. Fixation of blood samples should not affect the ability to detect neutrophil activation.     

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.     

CD38 cleavage in fMLP- and IL-8-induced chemotaxis is dependent on p38 MAP kinase but independent of p44/42 MAP kinase

In this study, we examined the mechanism by which CD38 cleavage is regulated through the mitogen-activated protein (MAP) kinases after stimulation by fMLP and interleukin-8 (IL-8) in neutrophils. Both fMLP and IL-8 increased chemotaxis and decreased CD38 protein in neutrophils, but did not change CD38 mRNA levels. Both fMLP and IL-8 increased CD38 in supernatants, which was inhibitable with PMSF. fMLP stimulation resulted in phosphorylation of p38 MAP kinase and p42/44 MAP kinase (ERK). SB20358, a p38 MAP kinase inhibitor, down-regulated neutrophil chemotaxis. Conversely, PD98059, an ERK inhibitor, did not influence chemotaxis to either agonist. The addition of SB20358 blocked the decrease of CD38 on neutrophils and the increase in supernatants induced by fMLP or IL-8, whereas PD98059 did not. These findings suggest that CD38-mediated chemotaxis to fMLP or IL-8 is characterized by proteolytic cleavage of CD38 and signaling through p38 MAP kinase. Activation of the protease for cleavage appears to be a postreceptor event that is dependent on p38 MAP kinase signaling.     

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.     

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response.