Actin Polymerization In Neutrophils Research Articles

A Mathematical Model for Ligand/Receptor/G-Protein Dynamics and Actin Polymerization in Human Neutrophils

A mathematical model is proposed for describing the dynamics of the chemotactic peptide-stimulated actin polymerization response in human neutrophils. The response pathway utilizes the guanine nucleotide binding protein (G-protein) signal transduction cascade common to many receptor systems and allows adaptation in the continued presence of ligand. The development of such a model is an important first step toward understanding, predicting, and ultimately manipulating neutrophil responses.The model is divided into two parts, ligand/receptor/G-protein dynamics and the actin polymerization mechanisms. Fast (receptor precoupled to G-protein) and slow (free receptor) signaling pathways involving ligand/receptor/G-protein interactions produce an activated signaling molecule. The actin polymerization mechanism utilizes an actin binding protein which complexes with actin monomer and inhibits polymerization in an unstimulated cell. During stimulation, the activated signaling molecule enhances the dissociation of the monomer/binding protein complexes, allowing the actin polymerization response to occur. The fast and slow signaling pathways are predicted to have different roles in controlling the time course of this actin polymerization. Additionally, precoupled receptors are predicted to have a larger ligand association rate constant than non-precoupled (free) receptors.Model simulations agree with many of the experimentally observed characteristics of both the stimulated F-actin response and ligand/receptor binding kinetics for both the fluorescent peptide ligand CHO-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine-fluorescein (CHO-NLFNTK-fl) and the non-fluorescent peptide ligand CHO-methionyl-leucyl-phenylalanine (CHO-MLF).

Threshold and graded response behavior in human neutrophils: effect of varying G-protein or ligand concentrations.

Observing the qualitative characteristics of response behavior as key variables in the signal transduction cascade are changed can provide insight into the fundamental roles of these interactions in producing cellular responses. Using flow cytometric assays and pertussis toxin (PT) treatment of human neutrophils, we have shown that actin polymerization stimulated with the chemoattractants N-formyl-Met-Leu-Phe, leukotriene B4, and interleukin-8 exhibits threshold behavior in terms of G-protein number. Partial PT treatment resulted in both responding and nonresponding populations of cells upon stimulation. As PT treatment was increased, the responding population of cells continued to respond maximally, while the number of cells responding decreased. We also showed that N-formyl peptide-stimulated oxidant production exhibits threshold behavior in terms of G-protein number, and the threshold for oxidant production is significantly greater than that for actin polymerization. The threshold behavior observed with PT treatment contrasted with the graded response behavior seen when cells were stimulated with different doses of ligand. For actin polymerization, only one population of cells was observed at submaximal ligand concentrations, and as ligand concentration was decreased the whole population responded submaximally. For oxidant production, as ligand concentration was decreased there were two populations of cells, but the responding cells responded submaximally. A mathematical model incorporating receptor/ligand binding and G-protein activation was developed to account for these differences in response behavior. Our results predict that an early signal transduction event in addition to, and not initiated by G-protein activation, is necessary to account for actin polymerization and oxidant production in neutrophils.

The Role of Wiskott-Aldrich Syndrome Protein in Signaling Cytoskeletal Remodeling • 793

Analysis of Wiskott-Aldrich Syndrome Protein (WASP) has revealed several domains, including domains homologous to actin regulating proteins. It has also been shown that expression of WASP in cells induces actin polymerization and that this is dependent upon WASP binding CDC42, a small GTP-binding protein involved in cytoskeletal signaling. These observations have led to the hypothesis that WASP plays a role in signaling to the actin cytoskeleton and that an underlying defect in this pathway due to mutations in WASP leads to the clinical manifestations associated with the classic Wiskott-Aldrich Syndrome (WAS) or the milder form, X-linked thrombocytopenia (XLT). To test this theory we have examined the kinetics of actin polymerization in neutrophils (PMN's), monocytes, lymphocytes and platelets in response to a variety of stimuli in WAS and XLT patients. In addition, we obtained scanning electron microscopic (SEM) images of platelets during actin remodeling in order to examine the ability of these cells to produce cytoarchitectural features associated with stimulation (microspikes, filopodia). PMN's were stimulated through signal transduction pathways important to phagocytosis (3G8 antibody), chemotaxis (fMLP), and adhesion (IB4 antibody). Stimulation of these pathways in PMN's isolated from WAS and XLT patients resulted in an actin polymerization response that was kinetically and quantitatively indistinguishable from control cells. Stimulation of monocytes from both WAS and XLT patients with fMLP also resulted in a normal actin polymerization response. As platelets and lymphocytes are thought to be the most severely affected cells in this disorder, these cells were examined for their ability to dynamically remodel their actin cytoskeleton. Lymphocytes isolated from WAS as well as XLT patients were able to produce a normal actin remodeling response to stimulation with phorbol ester and Bryostatin. SEM analysis of platelets isolated from WAS and XLT patients showed that they were able to produce robust microspikes and filopodia upon activation with PGF2α. These cells also produced an actin polymerization response to ADP and PGF2α that was indistinguishable from control cells. These observations present comprehensive evidence that signaling and functioning of the actin cytoskeleton is normal in peripheral blood cells isolated from WAS as well as XLT patients. These data call into question the hypothesis that WASP plays a critical role in signaling to the actin cytoskeleton and that an underlying defect in this pathway leads to the clinical manifestations seen in WAS.

Adenosine inhibits actin dynamics in human neutrophils: Evidence for the involvement of cAMP

The mechanisms by which adenosine regulates the inflammatory reaction are poorly characterized. In this study, we investigated the effects of adenosine on neutrophil actin polymerization elicited by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or IgG-opsonized yeast particles. We used bodipy-phallacidin staining in combination with flow cytometry and found that adenosine markedly reduced actin polymerization triggered by IgG-yeast, whereas the effect on the fMLP-response was less pronounced. Similar or even more pronounced effects were obtained with the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), suggesting an A2 receptor-mediated mechanism. The following observations indicate that the A2 receptor-induced effects involve the cAMP-protein kinase A (PKA) signaling pathway: (1) a combination of NECA and the cAMP-specific phosphodiesterase (PDE) inhibitor Ro 20-1724 raised the cAMP content in both unstimulated and stimulated neutrophils and also further inhibited the actin dynamics; (2) the PKA inhibitor H89 reversed the inhibitory effects of NECA on the actin dynamics; (3) Ro 20-1724, isoproterenol and dibutyryl cAMP (DBcAMP) reduced actin polymerization in almost the same way as NECA did. NECA together with Ro 20-1724 impaired the fMLP-induced shape changes and cortical accumulation of actin filaments. In contrast, H89 potentiated the fMLP-induced formation of a submembranous ring of actin filaments. Neutrophils phagocytosing yeast particles in the presence of NECA and Ro 20-1724 were predominantly round in shape, and their ability to extend actin-rich pseudopods around the prey was reduced. These effects were partly antagonized by H89. In correlation with the effects on actin polymerization, NECA more effectively diminished IgG-induced upregulation of the beta2 integrin CD11b/CD18 than such upregulation induced by fMLP. The inhibitory effects of A2-receptor activation on actin dynamics and beta2 integrin expression in neutrophils exposed to IgG-yeast were also associated with a cAMP-dependent reduction of the phagocytic capacity. In conclusion, we show that adenosine inhibits actin dynamics and shape changes in neutrophils via a cAMP-dependent pathway. This finding further characterizes the mechanisms by which adenosine functions as an important modulator of the inflammatory response.

Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis

Phospholipids mediate important effects as extracellular messengers in diverse biological systems. We investigated the effects of phosphatidic acid, a biologically active phospholipid potentially involved in the inflammatory process, on calcium mobilization and actin polymerization in human neutrophils and correlated these effects with induction of chemotactic migration. Intermediate-chain length phosphatidic acid (DiC10-PA) induced a biphasic increase in intracellular Ca 2+ characterized by a rapid rise commencing immediately upon addition of stimulus followed by a secondary increase which, unlike the initial response, was eliminated by chelation of extracellular Ca 2+. Neither of these responses were induced by C10-lysophosphatidic acid or diacylglycerol. The tyrosine kinase inhibitor herbimycin-A (5–10 μg/ml) completely blunted the initial but not the delayed response effected by DiC10-PA. Long-chain phosphatidic acid (DiC18:1) induced only an initial rapid increase in intracellular Ca 2+ and this response was similarly markedly attenuated by herbimycin-A. Among several physiologically relevant phospholipids, only phosphatidic acid was able to induce Ca 2+ mobilization; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol — used individually or in mixed liposomes — were without effect. Phosphatidic acid conferred calcium-mobilizing activity upon inactive liposome preparations and phosphatidic acid-enriched cellular plasma membranes possessed similar calcium-mobilizing activity. Both DiC10-PA and DiC18:1-PA induced actin polymerization in neutrophils at rates which mirrored the influence of each agent on Ca 2+ mobilization. Herbimycin-A blunted the initial increase in actin polymerization effected by phosphatidic acid but had no effect on the delayed, EGTA-sensitive phase. DiC10-PA and DiC18:1-PA also induced neutrophil migration along a concentration gradient. Phospholipids that failed to induce a calcium transient, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, likewise failed to induce either actin polymerization or chemotactic migration. Unlike chemotaxis induced by zymosan-activated human serum, phosphatidate-induced chemotaxis was strongly inhibited by pretreatment of cells with herbimycin-A. Consistent with these observations, phosphatidic acid induced the tyrosine phosphorylation of several proteins as early as 10 s after stimulation. Phosphorylation of two distinct proteins with approximate molecular sizes of 72 and 82 kDa was inhibited by levels of herbimycin A used to effectively inhibit calcium mobilization, actin polymerization and chemotaxis. Thus, in neutrophilic leukocytes, extracellular phosphatidic acid induces a unique tyrosine kinase-based signalling pathway that results in calcium mobilization and actin polymerization. These processes may promote directed cellular migration as a consequence of the interaction of phosphatidic acid with neutrophil plasma membranes.

Effects of 5-oxo-6,8,11,14-eicosatetraenoic acid on expression of CD11b, actin polymerization, and adherence in human neutrophils.

Human neutrophils contain a highly specific dehydrogenase that converts 5-hydroxy-6,8,11,14-eicosatetraenoic acid to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-Oxo-ETE is a potent stimulator of calcium mobilization, chemotaxis, and aggregation in these cells and has similar effects on eosinophils. The primary objectives of the current study were to determine whether this compound could increase the surface expression of integrins and stimulate actin polymerization in neutrophils. 5-Oxo-ETE stimulated the expression of CD11b and, to a lesser extent, CD11c, on neutrophils, but had no significant effects on the expression of CD11a, CD16 (Fc gammaRIII), or CD32 (Fc gammaRII). Surface expression of CD11b in response to 5-oxo-ETE was maximal after 12 min and remained constant thereafter. The EC50 for this response (50 nM) was lowered to 20 nM by preincubation of neutrophils with PMA. 5-Oxo-ETE (EC50, 10 nM) also rapidly stimulated actin polymerization in neutrophils, with a maximal response at 20 s. This response was blocked by pretreatment of neutrophils with the Gi protein inhibitor, pertussis toxin, and by homologous desensitization due to preincubation with 5-oxo-ETE. However, preincubation with leukotriene B4 or platelet-activating factor had no effect on the response of neutrophils to subsequent addition of 5-oxo-ETE. The adherence of neutrophils to plasma-coated plastic was also stimulated by 5-oxo-ETE with a time course similar to that for the surface expression of CD11b. Low concentrations of PMA (0.3 nM) enhanced this response. These results raise the possibility that 5-oxo-ETE could contribute to the infiltration of neutrophils into inflammatory sites.

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes—A flow cytometric study

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10 −8 M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

Effect of major surgery on neutrophil chemotaxis and actin polymerization in neonates and children

The authors have examined the effect of major surgery in neonates and older children on neutrophil (PMN) chemotaxis and on actin polymerization, an essential early step in PMN movement. Isolated PMNs from the following subjects were studied: healthy adult volunteers (n = 28), healthy newborns (n = 21), newborns undergoing major surgery (n = 7), and older infants and children undergoing major surgery (n = 14). Chemotaxis was measured by a micropore filter assay, and actin polymerization was measured by flow cytometry. Blood samples from surgical patients were obtained preoperatively, hourly during the procedure, immediately postoperatively, and 48 hours after surgery. Mean preoperative newborn PMN chemotaxis was similar to that of healthy newborn PMN, and mean preoperative PMN chemotaxis in children was similar to that of healthy adults. There were no significant alterations in PMN chemotaxis during or after major surgery in neonates or children. Peak PMN actin polymerization, after stimulation with formyl methionyl leucyl phenylalanine (FMLP) (10 nm), was significantly diminished in healthy neonates compared with adults ( P < .005). Preoperative surgical neonates had similar peak PMN actin polymerization levels to those of healthy newborns, and older preoperative children had similar levels to adults. PMN actin polymerization did not significantly change during or after major surgery. Despite reductions in PMN chemotaxis and actin polymerization in healthy neonates, there is no further impairment of these PMN functions during or after major surgery. Our data suggest that PMN chemotactic function is resistant to the stress of uncomplicated major surgery in neonates and children.

Divalent ions and actin polymerization in human neutrophils

Divalent ions and actin polymerization in human neutrophils

Divalent ions and actin polymerization in human neutrophils

Divalent ions and actin polymerization in human neutrophils

108 Defective actin polymerization in neutrophils from patients with myelodysplastic syndromes

108 Defective actin polymerization in neutrophils from patients with myelodysplastic syndromes

Impaired actin polymerization and depolymerization in neutrophils from patients with thermal injury

Acquired neutrophil dysfunction is considered an important cause of increased susceptibility to infection in patients with burns. In the early postinjury phase, large amounts of circulating chemoattractants, cytokines and endotoxins induce strong systemic activation of neutrophils which may impair their motile functions. Actin is the most prevalent component of the microfilament lattice that generates force for the neutrophil motile responses, and in the present study we examined the dynamics of actin polymerization and depolymerization in neutrophils from 11 patients with large burns. At admission, the amount of polymerized actin in unstimulated neutrophils was 39.9 per cent higher than that of parallel controls. In addition, there was a positive correlation between the amount of polymerized actin and the total body surface area (TBSA) burn. The time course of patient neutrophil actin polymerization in response to FMLP, C5a, (Ser-IL-8) 72, (Ala-IL-8) 77 and crosslinking of surface FcγRII was similar to that of controls, and the maximal amount of neutrophil F-actin was demonstrated after 30 s stimulation. At the peak of actin polymerization, however, patient neutrophils contained 27.3, 24.0, 24.7 and 25.6 per cent more polymerized actin than control cells stimulated with FMLP, (Ser-IL,-8) 72, (Ala-8) 77 and FcγRII crosslinking, respectively. However, the relative increase of neutrophil F-actin following stimulation was significantly lower in patients than in controls. Moreover, the rate of patient neutrophil actin depolymerization was 39.0, 23.5, 63.3 and 51.7 per cent lower than that of controls after stimulation with FMLP, C5a (Ser-IL-8) 72 and FcγRII crosslinking, respectively. At discharge, the dynamics of neutrophil actin polymerization and depolymerization were similar to that of controls. The results demonstrate that in neutrophils during the early postburn phase, there are increased basal levels of polymerized actin, a lower responsiveness to stimulation and a reduced rate of actin depolymerization. As periodic polymerization and depolymerization of actin is essential for all neutrophil motile responses, it is probable that the alterations observed may contribute significantly to the overall neutrophil dysfunction following thermal injury.

Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils.

At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.

Fluid shear stress induces actin polymerization in human neutrophils

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterize the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca2+. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca2+. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca2+]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions. © 1996 Wiley-Liss, Inc.

Fluid shear stress induces actin polymerization in human neutrophils

We have previously reported that a physiological range of shear stress induces neutrophil homotypic aggregation mediated by lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-3 (ICAM-3) interactions. To further characterize the homotypic aggregation, actin polymerization was investigated in neutrophils stimulated by shear stress in comparison with formyl-methionyl-leucyl-phenylalanine (fMLP). In fMLP-stimulated neutrophils, actin polymerization was localized in the pseudopods, and this reaction was not mediated by a cytosolic level of Ca2+. In contrast to fMLP stimulation, the actin polymerization induced by shear stress in a cone-plate viscometer was localized in cell-cell contact regions, and this polymerization required the increase of intracellular Ca2+. This shear stress-induced actin polymerization was not observed when neutrophils were pretreated with anti-LFA-1 or anti-ICAM-3 antibody. In conclusion, LFA-1 and ICAM-3 interaction mediated by the increase of [Ca2+]i generated the intercellular signal in order to accumulate F-actin in the cell-cell contact regions. © 1996 Wiley-Liss, Inc.

Effects of conventional peritoneal dialysates on leukocyte adhesion and CD11b, CD18 and CD14 expression

Bacterial peritonitis is the most important complication of peritoneal dialysis (PD), limiting its widespread application. Conventional glucose-based peritoneal dialysates (G-PDS) depress oxygen consumption, chemiluminescence, superoxide production, phagocytosis, bacterial killing and actin polymerization in neutrophils (PMN) in vitro. Expression of adhesion receptors is critical to leukocyte activation, adhesion, migration and phagocytosis. The effects of G-PDS on basal and stimulated leukocyte adhesion molecule expression and leukocyte adhering capacity is unknown. We examined the effect of a five minutes incubation of whole blood in either HEPES-buffered saline or G-PDS containing 1.5% (83 mM), 2.5% (139 mM) or 4.25% (236 mM) glucose, at pH = 5.2, and pH = 7.4. PMN intracellular pH was measured spectrofluorometrically. Leukocyte CD11b, CD18 and CD14 were measured by flow cytometry using monoclonal antibodies in otherwise unstimulated cells or 60 minutes after lipopolysaccharide (LPS) stimulation. In addition, leukocyte adhering capacity to nylon wool was tested. In an attempt to dissect the effect of high glucose concentrations from that of the attendant hyperosmolality, the experiments were repeated with dialysates in which glucose was substituted by sodium chloride (NaCl-PDS) to attain identical osmolalities. G-PDS, as well as the mixtures of spent and fresh G-PDS, significantly depressed the basal PMN expression of adhesion receptors CD11b and CD18 and monocyte expression of CD14, and substantially mitigated the LPS-mediated up-regulation of CD11b and CD18. Likewise, G-PDS significantly inhibited leukocyte adhering capacity without affecting cell viability. Similar results were observed with NaCl-PDS. The observed abnormalities were primarily osmolality-dependent, and largely intra- and extracellular pH-independent. Impaired adhesion receptor expression and cell adhesion capacity shown here reveal another dimension of the G-PDS-induced leukocyte abnormalities.

Platelets enhance Fcγ receptor-mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization

The interaction of platelets with neutrophil granulocytes is considered to play an important role in the inflammatory process, and the present study was focused on platelet-induced modulation of Fcgamma receptor-mediated functions in neutrophils. We found that phagocytosis and the respiratory burst (measured as luminol-enhanced chemiluminescence), triggered in neutrophils by immunoglobulin G (IgG)-opsonized yeast particles, were potentiated by platelets and that maximal enhancement was achieved at a physiological neutrophil/platelet ratio of about 1:50 to 1:100. Platelets both increased the intra- and extracellular generation of oxygen radicals as well as the release of myeloperoxidase from stimulated neutrophils. The presence of platelets also induced a cortical actin polymerization in neutrophils, which might explain the increased phagocytic capacity. Platelets appear to affect neutrophil function in a contact-independent manner that most likely involves ATP, indicated by the following: (1) platelet supernatants, but not fixed platelets, affected neutrophil function in the same way as viable platelets; (2) platelets raised the extracellular ATP level four- to fivefold; (3) exogenous ATP mimicked the effects of platelets on actin polymerization, phagocytosis, and the respiratory burst in neutrophils; (4) hydrolysis of extracellular ATP with apyrase or blocking of ATP receptors with suramin reversed the platelet-induced enhancement of neutrophil function. An increased accumulation of extracellular adenosine, induced by inhibiting endogenous adenosine deaminase or adding exogenous adenosine, reversed the effects of platelets. The platelet-induced potentiation of the respiratory burst was inhibited by the tyrosine kinase inhibitor genistein, suggesting that tyrosine phosphorylation is involved. However, platelets did not significantly affect the Fcgamma receptor-triggered calcium response in neutrophils. In conclusion, we show that platelets, through an ATP-dependent mechanism, potentiate IgG-mediated ingestion and production of oxygen metabolites in neutrophils.

Actin polymerization in human eosinophils, unlike human neutrophils, depends on intracellular calcium mobilization.

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca2+]i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca2+ -depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes.

Actin polymerization in human eosinophils, unlike human neutrophils, depends on intracellular calcium mobilization

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca2+]i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca2+ -depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes.

Actin polymerization in human eosinophils, unlike human neutrophils, depends on intracellular calcium mobilization

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca2+]i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca2+-depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes. © 1996 Wiley-Liss, Inc.