Filamin-A Regulates Neutrophil Uropod Retraction through RhoA during Chemotaxis

Filamin-A (FLNa) has been shown to be a key cross-linker of actin filaments in the leading edge of a motile melanoma cell line, however its role in neutrophils undergoing chemotaxis is unknown. Using a murine transgenic model in which FLNa is selectively deleted in granulocytes, we report that, while neutrophils lacking FLNa show normal polarization and pseudopod extension, they exhibit obvious defects in uropod retraction. This uropod retraction defect was found to be a direct result of reduced FLNa mediated activation of the small GTPase RhoA and myosin mediated actin contraction in the FLNa null cells. This results in a neutrophil recruitment defect in FLNa null mice. The compensatory increase in FLNb levels that was observed in the FLNa null neutrophils may be sufficient to compensate for the lack of FLNa at the leading edge allowing for normal polarization, however this compensation is unable to regulate RhoA activated tail retraction at the rear of the cell.     

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma661), which generates PtdIns(4,5)P(2), is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKIgamma661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P(2) at the uropod during chemotaxis. Overexpression of kinase-dead PIPKIgamma661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P(2) synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKIgamma661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.     

Ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) regulates neutrophil chemotaxis by hydrolyzing released ATP to adenosine

Polymorphonuclear neutrophils release ATP in response to stimulation by chemoattractants, such as the peptide N-formyl-methionyl-leucyl-phenylalanine. Released ATP and the hydrolytic product adenosine regulate chemotaxis of neutrophils by sequentially activating purinergic nucleotide and adenosine receptors, respectively. Here we show that that ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) is a critical enzyme for hydrolysis of released ATP by neutrophils and for cell migration in response to multiple agonists (N-formyl-methionyl-leucyl-phenylalanine, interleukin-8, and C5a). Upon stimulation of human neutrophils or differentiated HL-60 cells in a chemotactic gradient, E-NTPDase1 tightly associates with the leading edge of polarized cells during chemotaxis. Inhibition of E-NTPDase1 reduces the migration speed of neutrophils but not their ability to detect the orientation of the gradient field. Studies of neutrophils from E-NTPDase1 knock-out mice reveal similar impairments of chemotaxis in vitro and in vivo. Thus, E-NTPDase1 plays an important role in regulating neutrophil chemotaxis by facilitating the hydrolysis of extracellular ATP.     

Guanylyl cyclase protein and cGMP product independently control front and back of chemotaxing Dictyostelium cells

Chemotaxis of amoeboid cells is driven by actin filaments in leading pseudopodia and actin-myosin filaments in the back and at the side of the cell to suppress pseudopodia. In Dictyostelium, cGMP plays an important role during chemotaxis and is produced predominantly by a soluble guanylyl cyclase (sGC). The sGC protein is enriched in extending pseudopodia at the leading edge of the cell during chemotaxis. We show here that the sGC protein and the cGMP product have different functions during chemotaxis, using two mutants that lose either catalytic activity (sGCDelta cat) or localization to the leading edge (sGCDeltaN). Cells expressing sGCDeltaN exhibit excellent cGMP formation and myosin localization in the back of the cell, but they exhibit poor orientation at the leading edge. Cells expressing the catalytically dead sGCDelta cat mutant show poor myosin localization at the back, but excellent localization of the sGC protein at the leading edge, where it enhances the probability that a new pseudopod is made in proximity to previous pseudopodia, resulting in a decrease of the degree of turning. Thus cGMP suppresses pseudopod formation in the back of the cell, whereas the sGC protein refines pseudopod formation at the leading edge.     

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.     

A Highlights from MBoC Selection: Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton

Chemotaxis allows neutrophils to seek out sites of infection and inflammation. The asymmetric accumulation of filamentous actin (F-actin) at the leading edge provides the driving force for protrusion and is essential for the development and maintenance of neutrophil polarity. The mechanism that governs actin cytoskeleton dynamics and assembly in neutrophils has been extensively explored and is still not fully understood. By using neutrophil-like HL-60 cells, we describe a pivotal role for Rictor, a component of mammalian target of rapamycin complex 2 (mTORC2), in regulating assembly of the actin cytoskeleton during neutrophil chemotaxis. Depletion of mTOR and Rictor, but not Raptor, impairs actin polymerization, leading-edge establishment, and directional migration in neutrophils stimulated with chemoattractants. Of interest, depletion of mSin1, an integral component of mTORC2, causes no detectable defects in neutrophil polarity and chemotaxis. In addition, experiments with chemical inhibition and kinase-dead mutants indicate that mTOR kinase activity and AKT phosphorylation are dispensable for chemotaxis. Instead, our results suggest that the small Rho GTPases Rac and Cdc42 serve as downstream effectors of Rictor to regulate actin assembly and organization in neutrophils. Together our findings reveal an mTORC2- and mTOR kinase–independent function and mechanism of Rictor in the regulation of neutrophil chemotaxis.     

Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis

We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, we show that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis.     

A requirement of MAPKAPK2 in the uropod localization of PTEN during FMLP-induced neutrophil chemotaxis

The directionality control in chemotaxis is the result of a reciprocal regulation of PI3-kinase and PTEN subcellular localization. MK2(-/-) neutrophils have a directionality loss in fMLP-induced chemotaxis. We found that in polarized WT neutrophils PTEN was localized in the uropod region. However, MK2(-/-) neutrophils or p38 MAPK inhibitor-SB203580-pretreated WT neutrophils showed a disrupted PTEN subcellular localization. Some PTEN was localized at the leading edge of the polarized neutrophils, which may lower the concentration of PI3-kinase lipid product PtdIns(3,4,5)P3 required for directionality sensing. FMLP-stimulated MK2(-/-) neutrophils or SB203580-pretreated WT neutrophils also had disrupted F-actin polarization. F-actin polymerization inhibitor lantrunculin-B disrupted the polarization of PTEN, but not PtdIns(3,4,5)P3. The results suggest that PTEN uropod polarization is F-actin polymerization-dependent and may be through the effect of MK2 on F-actin polarization.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation.     

Syk-mediated translocation of PI3Kdelta to the leading edge controls lamellipodium formation and migration of leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.     

Plasma membrane organization is essential for balancing competing pseudopod- and uropod-promoting signals during neutrophil polarization and migration

Exposure of neutrophils to chemoattractant induces cell polarization and migration. These behaviors require the asymmetric activation of distinct signaling pathways and cytoskeletal elements in the protruding pseudopod at the front of cells and the retracting uropod at the rear. An important outstanding question is, how does the organization of the plasma membrane participate in establishing asymmetry during polarization and migration? To answer this question, we investigated the function of cholesterol, a lipid known to influence membrane organization. Using controlled cholesterol depletion, we found that a cholesterol-dependent membrane organization enabled cell polarization and migration by promoting uropod function and suppressing ectopic pseudopod formation. At a mechanistic level, we showed that cholesterol was directly required for suppressing inappropriate activation of the pseudopod-promoting Gi/PI3-kinase signaling pathway. Furthermore, cholesterol was required for dampening Gi-dependent negative feedback on the RhoA signaling pathway, thus enabling RhoA activation and uropod function. Our findings suggest a model in which a cholesterol-dependent membrane organization plays an essential role in the establishment of cellular asymmetry by balancing the activation and segregating the localization of competing pseudopod- and uropod-inducing signaling pathways during neutrophil polarization and migration.     

Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration

Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) production, a polarized distribution of PIP(3) was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP(3) or induce cell polarization. Thus, the increase in PIP(3) concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.     

The actin regulatory protein HS1 interacts with Arp2/3 and mediates efficient neutrophil chemotaxis

HS1 is an actin regulatory protein and cortactin homolog that is expressed in hematopoietic cells. Antigen receptor stimulation induces HS1 phosphorylation, and HS1 is essential for T cell activation. HS1 is also expressed in neutrophils; however, the function of HS1 in neutrophils is not known. Here we show that HS1 localizes to the neutrophil leading edge, and is phosphorylated in response to the chemoattractant formyl-Met-Leu-Phe (fMLP) in adherent cells. Using live imaging in microchannels, we show that depletion of endogenous HS1 in the neutrophil-like PLB-985 cell line impairs chemotaxis. We also find that HS1 is necessary for chemoattractant-induced activation of Rac GTPase signaling and Vav1 phosphorylation, suggesting that HS1-mediated Rac activation is necessary for efficient neutrophil chemotaxis. We identify specific phosphorylation sites that mediate HS1-dependent neutrophil motility. Expression of HS1 Y378F, Y397F is sufficient to rescue migration of HS1-deficient neutrophils, however, a triple phospho-mutant Y222F, Y378F, Y397F did not rescue migration of HS1-deficient neutrophils. Moreover, HS1 phosphorylation on Y222, Y378, and Y397 regulates its interaction with Arp2/3. Collectively, our findings identify a novel role for HS1 and its phosphorylation during neutrophil directed migration.     

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.     

Anaphylatoxin signaling in human neutrophils. A key role for sphingosine kinase

Anaphylatoxins activate immune cells to trigger the release of proinflammatory mediators that can lead to the pathology of several immune-inflammatory diseases. However, the intracellular signaling pathways triggered by anaphylatoxins are not well understood. Here we report for the first time that sphingosine kinase (SPHK) plays a key role in C5a-triggered signaling, leading to physiological responses of human neutrophils. We demonstrate that C5a rapidly stimulates SPHK activity in neutrophils and differentiated HL-60 cells. Using the SPHK inhibitor N,N-dimethylsphingosine (DMS), we show that inhibition of SPHK abolishes the Ca2+ release from internal stores without inhibiting phospholipase C or protein kinase C activation triggered by C5a but has no effect on calcium signals triggered by other stimuli (FcgammaRII). We also show that DMS inhibits degranulation, activation of the NADPH oxidase, and chemotaxis triggered by C5a. Moreover, an antisense oligonucleotide against SPHK1, in neutrophil-differentiated HL-60 cells, had similar inhibitory properties as DMS, suggesting that the SPHK utilized by C5a is SPHK1. Our data indicate that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate. Exogenously added sphingosine has a dual effect on C5a-stimulated oxidative burst: it has a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations; however, C5a-triggered protein kinase C activity was only reduced at high concentration of sphingosine. In contrast, C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. Exogenous sphingosine-1-phosphate, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and oxidative burst and had a priming effect, enhancing all the C5a-triggered responses. Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins in human neutrophils and point out SPHK as a potential therapeutic target for the treatment of diseases associated with neutrophil hyperactivation.     

mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion

We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA-signaling axis, independently of actin reorganization during neutrophil chemotaxis.     

A PLCβ/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis

Neutrophils, in response to a chemoattractant gradient, undergo dynamic F-actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLCβ-PKC and PI3Kγ-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis.     

Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration

A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (μ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extra-cellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-met-leu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.     

Cytosolic free Ca(2+) changes and calpain activation are required for beta integrin-accelerated phagocytosis by human neutrophils

Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca(2+) imaging, the temporal relationship between changes in cytosolic free Ca(2+) concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca(2+) changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca(2+) which resulted from Ca(2+) influx. This latter rise in cytosolic free Ca(2+) concentration also liberated a fraction of beta2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized beta2 integrin labelling. Inhibitors of calpain activation prevented both the Ca(2+)-induced liberation of beta2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca(2+) signal. Therefore, we propose that Ca(2+) activation of calpain causes beta2 integrin liberation, and that this signal plays a key role in the acceleration of beta2 integrin-mediated phagocytosis.