Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.     

Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting.Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils.An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.     

Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils

The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl- phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor- mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.     

Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.     

Human immunoglobulin G potentiates superoxide production induced by chemotactic peptides and causes degranulation in isolated human neutrophils

Neutrophils are major cellular mediators of host defense and inflammation. They can be activated to produce superoxide and to release the contents of their granules to the extracellular space. We observed that monomeric human immunoglobulin G (IgG) sensitizes these cells to the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLP). In cells submaximally stimulated by fMLP this enhancement was especially prominent. With saturating fMLP concentrations, the rate of O2- production was still about twice that in the control. No synergy with other activators (phorbol myristate acetate, concanavalin A) was observed. Binding of fMLP to the cells was decreased by IgG, resembling the effect of cytochalasin B. IgG did not induce O2- production on its own, but it stimulated degranulation of the neutrophils.     

Diacylglycerol kinase inhibitors R59022 and dioctanoylethylene glycol potentiate the respiratory burst of neutrophils by raising cytosolic Ca2+

The diacylglycerol kinase inhibitors, R59022 and dioctanoylethylene glycol (diC8-eg), potentiate stimulation of the respiratory burst by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. However, in contrast to the potentiation observed in intact cells, neither R59022 nor diC8-eg enhanced the effect of fMLP on O2 consumption in electropermeabilized neutrophils, under conditions where cytosolic [Ca2+] was held constant using EGTA. In unstimulated, intact cells treatment with the diacylglycerol kinase inhibitors elicited an increase in cytosolic Ca2+ ([Ca2+]i). The results suggest that enhancement of the respiratory burst by diC8-eg and R59022 is mediated by a rise in [Ca2+]i, rather than by inhibition of diacylglycerol kinase.     

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.     

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.     

Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils

Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils.     

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.     

The major outer sheath protein of Treponema denticola selectively inhibits Rac1 activation in murine neutrophils

Treponema denticola major outer sheath protein (Msp) inhibits neutrophil chemotaxis in vitro , but key regulatory mechanisms have not been identified. Because the Rac small GTPases regulate directional migration in response to chemoattractants, the objective was to analyse the effects of Msp on formyl -methionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil polarization and Rac activation in murine neutrophils. Msp pretreatment of neutrophils inhibited both polarization and chemotactic migration in response to fMLP. Activation of small GTPases was measured by p21 binding domain (PBD) pulldown assays, followed by Western analysis, using monoclonal anti-Rac1, anti-Rac2, anti-cdc42 and anti-RhoA antibodies. Enriched native Msp selectively inhibited fMLP-stimulated Rac1 activation in a concentration-dependent manner, but did not affect Rac2, cdc42 or RhoA activation. Murine neutrophils transfected with vectors expressing fluorescent probes PAK-PBD-YFP and PH-AKT-RFP were used to determine the effects of Msp on the localization of activated Rac and PI3 kinase products. Real-time confocal images showed that Msp inhibited the polarized accumulation of activated Rac and PI3-kinase products upon exposure to fMLP. The findings indicate that T. denticola Msp inhibition of neutrophil polarity may be due to the selective suppression of the Rac1 pathway.     

Cross-linking of beta2 integrins caused diminished responses of neutrophils to priming agents like lipopolysaccharide or tumor necrosis factor-alpha: possible involvement of tyrosine kinase Syk

Neutrophils up-regulate beta2 integrins like CD11b/CD18 in response to lipopolysaccharide (LPS). Up-regulation of beta2 integrins causes neutrophils to adhere to surfaces, and to release superoxide anion (O2-). When neutrophils are exposed to LPS plus plasma under conditions not favorable for adherence (absence of Mg2+), the cells do not spontaneously release O2-, but instead they are primed for enhanced release of O2- after subsequent triggering by fMLP. In the presence of Mg2+, neutrophils adhere in response to LPS but fMLP-triggered O2- release by LPS-primed neutrophils is diminished. To understand why adherence interferes with the response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (fMLP), beta2 integrins were cross-linked by mouse monoclonal antibodies that had been immobilized by surface-bound anti-mouse antibody. When unprimed neutrophils were trapped on the surface by these cross-linked monoclonal antibodies, O2- release was triggered, and priming by LPS for fMLP-triggered O2- release was diminished, indicating that this cross-linking of beta2 integrins mimicked adherence. Alkaline phosphatase is up-regulated by LPS or tumor necrosis factor-alpha, and this response was also diminished by the cross-linking antibodies. The diminished alkaline phosphatase up-regulation was reversed by genistein, a general inhibitor of tyrosine kinases, and by piceatannol, an inhibitor for Syk kinase. Piceatannol also inhibited the phosphorylation of Syk caused by cross-linking of beta2 integrins. These results suggested that adherence-induced triggering and Syk kinase activation might be responsible for the diminished response of LPS-primed neutrophils to fMLP when neutrophils were adherent.     

Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils

Human neutrophils exposed to protein-coated polystyrene or cultured endothelial monolayers produce large quantities of H2O2 in response to soluble stimuli that elicit little or no secretion of reactive oxygen species from cells in suspension. To characterize the mechanisms involved in this adherence-dependent respiratory burst, we have investigated the possible role of one integrin known to participate in the adhesion of neutrophils to endothelial cells, CD11b/CD18 (Mac-1). H2O2 production was examined with chemotactic factor-stimulated human and canine neutrophils exposed to protein-coated surfaces and cultured human and canine endothelial cells. The two protein-coated surfaces used were type I collagen-coated glass or plastic, a surface to which neither human nor canine neutrophils adhered, and keyhole limpet hemocyanin (KLH)-coated glass or plastic, a surface to which human and canine neutrophils adhered only after chemotactic stimulation. FMLP-stimulated human neutrophils and platelet activating factor-stimulated canine neutrophils failed to produce detectable H2O2 when in contact with type I collagen, but secreted large amounts of H2O2 when adherent to KLH or endothelial cell monolayers. FMLP-stimulated neutrophils from patients with CD18-deficiency failed to adhere to any of these surfaces and failed to produce H2O2 under these conditions. mAb reactive with CD18 and CD11b were equally effective in markedly inhibiting the adhesion of normal human neutrophils to these surfaces and markedly inhibited the production of H2O2. A mAb reactive with CD18 blocked adhesion of stimulated canine neutrophils, and mAb directed against both CD18 and CD11b blocked H2O2 production by canine neutrophils on KLH and endothelium. A nonbinding mAb and a mAb reactive with CD11a did not inhibit H2O2 production of human cells on KLH or endothelial monolayers, and nonbinding and binding control mAb did not inhibit H2O2 production by canine neutrophils. These results indicate that Mac-1 (CD11b/CD18) can mediate adhesion-dependent H2O2 production by human and canine neutrophils exposed to chemotactic factors.     

Immunomodulating action of low intensity millimeter waves on primed neutrophils

Comparative investigation of the susceptibility of intact and primed neutrophils of the NMRI strain mice to low intensity millimeter wave (mm wave) irradiation (41.95 GHz) was performed. The specific absorption rate was 0.45 W/kg. Isolated neutrophils were primed by a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) at a subthreshold concentration of 10 nM for 20 min, and then the cells were activated by 1 microM fMLP. Production of the reactive oxygen species (ROS) was estimated by the luminol dependent chemiluminescence technique. It was found that the preliminary mm wave irradiation of the resting cells at 20 degrees C did not act on the ROS production induced by the chemotactic peptide. The exposure of the primed cells results in a subsequent increase in the fMLP response. Therefore, the primed neutrophils are susceptible to the mm waves. Specific inhibitors of the protein kinases abolished the mm wave effect on the primed cells. The data indicate that protein kinases actively participate in transduction of the mm wave signal to effector molecules involved in neutrophil respiratory burst.     

Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.     

In vitro effect of actinomycin D on human neutrophil function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.     

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.     

Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation

We attempted to apply the particle counting method that employs laser-light scattering technique to quantify the change in numbers of neutrophil homotypic aggregates of 3 graded-sizes (small, medium and large). Ex vivo activation of human neutrophils by a chemotactic peptide, fMLP, predominantly produced small-sized aggregates (< 15 cells), and also, transiently, medium-sized aggregates (16-130 cells). Co-treatment of neutrophils with fMLP and cytochalasin B mainly produced medium-sized aggregates, with very few large-sized aggregates (> 130 cells). Interestingly, when protein kinase C was activated with phorbol 12-myristate 13-acetate small-, medium- and even large-sized aggregates of neutrophils were formed. Presence of extracellular calcium was required to produce these neutrophil aggregations. Both prostaglandin E2 (PGE2) and wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI-3K), inhibited neutrophil aggregation, whereas dbcAMP, a cell permeable analog of cyclic AMP, did not, confirming that PGE2 causes neutrophil aggregation probably through PI-3K inhibition rather than activation of adenylate cyclase. These results suggest that the application of the light scattering technique to characterize human neutrophil aggregates by both size and numbers, has advantages over conventional optical turbulent aggregometry, in that it discriminates neutrophil aggregations produced by different mechanisms of intracellular signaling.     

Cl⁻/HCO₃⁻ exchange activity in fMLP-stimulated human neutrophils

It is well known that chemotactic agents active Na(+)/H(+) exchanger, increasing intracellular pH of neutrophils, but their effect on bicarbonate transporters have not been established yet. To study the effect of fMLP on the activity of Cl(-)/HCO(3)(-) exchange, the rate of pH recovery after acute Cl(-) readmission in cell subjected to an alkaline load by CO(2) washout in a Cl-free medium was measured. The activity of the exchanger was reduced to 72% of control when cells were pre-incubated for 5 min with 0.1 μM fMLP and reached 48% of control in steady state after acute exposure. After extracellular bicarbonate or TMA addition the rate recovery of intracellular pH was reduce at 72% and at 84%, respectively. The inhibitory effect on the intracellular pH recovery was not affected by blockers of Na(+)/H(+) exchange. We conclude from these studies that an increase of pH(i) produced for this chemotactic agent is facilitated by the simultaneous activation of Na(+)/H(+) exchange and inhibition of Cl(-)/HCO(3)(-) exchange in neutrophils.