Diacylglycerol accumulation and superoxide anion production in stimulated human neutrophils

Exogenous diacylglycerols stimulate neutrophil superoxide anion production, suggesting that endogenous diacylglycerols may function as second messengers for this biological response. We have measured the diacylglycerol mass in human neutrophils stimulated by fMet-Leu-Phe, ionomycin, and concanavalin A and have correlated the kinetics and magnitude of the diacylglycerol response with those for superoxide anion production. For each stimulus, no increase in diacylglycerol mass was detected prior to the onset of superoxide anion generation. However, large sustained increases in diacylglycerol concentration (260-2000% of basal levels) occurred in parallel with the rise in superoxide anion. The cessation or continuation of diacylglycerol accumulation and superoxide anion production also correlated. The diacylglycerol response was proportional to the stimulus concentration and correlated with the concentration dependence for superoxide anion. Pretreatment of neutrophils with cytochalasin B enhanced both superoxide anion and diacylglycerol responses with all three stimuli. These data support the hypothesis that diacylglycerol functions as a modulator of superoxide anion generation causing a sustained or augmented respiratory burst.     

Diacylglycerol mass measurements in stimulated HL-60 phagocytes

The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to [32P]-labeled phosphatidic acid catalyzed by E. coli diacylglycerol kinase. The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min. Diacylglycerol returned toward basal levels by 15 min. The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36). Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively. t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase. Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase. The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol. These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C.     

Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.

Relatively high levels of propranolol (170 microM) markedly attenuated the generation of 1,2 diacylglycerol in neutrophils stimulated with either FMLP plus cytochalasin B or with 20.0 mM NaF. This effect resulted from inhibition of phosphatidic acid phosphohydrolase as it was accompanied by a corresponding increase in the recovery of phosphatidic acid in organic extracts of stimulated cells. Although propranolol enhanced phosphatidic acid levels in neutrophils treated with FMLP alone, the drug had only a slight inhibitory influence on diglyceride generation in these cells. The effect of propranolol on enhancement of PA levels in neutrophils treated with FMLP alone strongly correlated with enhancement of FMLP-induced O2- generation. However, propranolol induced a similar dose-dependent inhibition of O2- generation in neutrophils stimulated with either FMLP + cytochalasin B or with 20.0 mM NaF. These results are consistent with the hypothesis that both phosphatidic acid and diacylglycerol are required for optimal initiation of neutrophil O2- release.     

Leukotriene B4 triggers highly characteristic and specific functional responses in neutrophils: studies of stimulus specific mechanisms

By using human neutrophils we studied the on-off phenomenon for leukotriene B4 (LTB4) -induced functional responses compared with fMetLeuPhe (fMLP). LTB4 induced rapidly appearing and disappearing neutrophil chemiluminescent (CL), superoxide anion formation, aggregatory and membrane depolarizing responses, whereas fMLP responses were slower both in onset and termination. Increases of intracellular calcium concentrations (as reflected by quin2 and fura-2 fluorescence) were of similar magnitude for both stimuli; however, LTB4 responses were more rapidly terminated and fMLP responses were biphasic. When intracellular calcium fluxes, calmodulin or protein kinase C activities were inhibited by quin2, trifluoperazine, verapamil or 3,4,5-trimethoxybenzoic acid 8-diethylamino)octyl ester (TMB-8), profound changes were noted for chemiluminescent and aggregation kinetics induced by fMLP, whereas kinetics of LTB4 responses were less affected. When drugs were used to modulate cAMP levels, or to inhibit cyclo- and lipoxygenase metabolites of arachidonic acid, no effects on response kinetics were observed. Cytochalasin B both amplified and delayed responses although chemiluminescent responses to fMLP were amplified more than those to LTB4. Despite those effects cytochalasin B did not enhance peak fura-2 or quin2 responses to either fMLP or LTB4. Thus, LTB4 rapidly initiates functional responses in neutrophils, and stimulus-specific response patterns are already discernable during the mobilization of calcium, and can be modulated by interference with calcium-dependent reactions.     

On the biological occurrence and regulation of 1-acyl and 1-O-alkyl-diradylglycerols in human neutrophils. Selective destruction of diacyl species using Rhizopus lipase

The occurrence and regulation of 1-ether-linked diradylglycerol in human neutrophils were investigated using a sensitive and practical analytical mass method which distinguishes 1-O-alkyl- (EAG) versus 1-acyl (DAG) diglycerides. After phosphorylation of diglycerides to the corresponding [32P]phosphatidic acids using [gamma-32P]ATP and diglyceride kinase (Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600), lipase from Rhizopus arrhizus selectively degraded the 1-acyl-containing species (DAG), but the ether lipid (EAG) was resistant and was identified and quantified after thin layer chromatography separation. By using this method, unstimulated neutrophils were demonstrated to contain both DAG and EAG (100-180 and 40-95 pmol/10(7) cells, respectively). The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) caused a rapid (30 s) and transient increase (1.6-fold) in DAG, but no increase in EAG. Opsonized zymosan produced a 6-8-fold sustained increase in DAG peaking at 2 to 3 min, but only a small (1.7-fold) increase in EAG which was not seen until later times (10 min). Thus, under these stimulation conditions, the major diglyceride was DAG. However, in neutrophils "primed" with cytochalasin B or phorbol ester, formyl-methionyl-leucyl-phenylalanine caused a significant increase in EAG. Neutrophils pretreated with cytochalasin B and then stimulated by fMLP showed a rapid (15-60 s) increase (more than 3-fold) in total diglycerides which was sustained beyond 5 min. At the earliest time points (15-30 s), the increase was due almost entirely to DAG (3-fold), but at 1 min and beyond, EAG comprised as much as 40% of the total (up to a 5-fold increase in EAG). Neutrophils pretreated with phorbol ester prior to fMLP stimulation showed a rapid (around 30 s) more than 2-fold increase in both DAG and EAG. Thus, priming conditions (in particular cytochalasin B) may alter either the access of phospholipase(s) C and/or D to membrane phospholipids or may affect their activities, allowing hydrolysis of 1-O-alkyl-containing lipids to generate 1-O-alkyl-containing diglycerides.     

Activation of neutrophils: measurement of actin conformational changes by flow cytometry

Actin, which comprises approximately 10% of the weight of cytoplasmic protein of neutrophils, is the principal component of the cytoplasmic microfilament lattice. It can exist in either of two physical states, G-actin, which is monomeric, or F-actin, which is polymeric or filamentous. Actin microfilaments support many forms of cell movement. Continuous remodeling of the microfilament lattice, which seems integral to sustained movement, is possible in part because of the ability of actin to change rapidly between its monomeric G-state and its filamentous F-state. Changes in the G- and F-actin equilibrium may be studied by flow analysis using a fluorescent probe which is specific for F-actin, 7-nitrobenz-2-oxa-1,3-diazole-(NBD)-phallacidin. Alterations in neutrophil F-actin have been measured in response to chemotactic agents (e.g., formyl peptides and leukotriene B4), inhibitors of cell movement (e.g., N-ethylmaleimide and cytochalasin B), agents that promote the oxidative burst (e.g., formyl peptides and phorbol esters), and priming agents [e.g., tumor necrosis factor (TNF)]. Measurements may be taken at intervals of a few seconds, allowing comparison of rapid changes in the F-actin content to other rapidly occurring changes, such as altered membrane ion permeability and activation of cellular enzymes. The use of metabolic inhibitors has allowed dissection of some of the biochemical pathways involved in actin assembly in living cells. Although clinical studies are few thus far, the technique has also been used to study basal and stimulated F-actin levels in circulating neutrophils in neonates and in family members of patients with neutrophil-actin dysfunction.     

Granulocyte/macrophage colony-stimulating factor primes human neutrophils for increased diacylglycerol generation in response to chemoattractant

Pretreatment of human neutrophils with granulocyte macrophage-colony stimulating factor (GM-CSF) augments several biological responses to chemoattractants (e.g. the respiratory burst, degranulation, and chemotaxis). However, little is known regarding the intracellular effects of priming with GM-CSF. In the present study, we have investigated the effects of GM-CSF on the generation of diacylglycerol (DAG), a proposed mediator of neutrophil responses. GM-CSF alone produced only a small increase in cellular DAG mass, which was most apparent after 30 min. GM-CSF pretreatment (60 min), however, caused a striking augmentation in DAG generation in response to the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), compared with neutrophils preincubated without GM-CSF. The augmentation in DAG generation correlated with an enhancement by GM-CSF of superoxide generation in response to fMLP. The data suggest that GM-CSF may exert some of its biological effects by enhancing DAG generation in response to a second agonist.     

Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

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.     

Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5-25 microg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 +/- 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 +/- 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.     

The FPR2-specific ligand MMK-1 activates the neutrophil NADPH-oxidase,but triggers no unique pathway for opening of plasma membrane calcium channels

Human neutrophils express formyl peptide receptor 1 and 2 (FPR1 and FPR2), two highly homologous G-protein-coupled cell surface receptors important for the cellular recognition of chemotactic peptides. They share many functional as well as signal transduction features, but some fundamental differences have been described. One such difference was recently presented when the FPR2-specific ligand MMK-1 was shown to trigger a unique signal in neutrophils [S. Partida-Sanchez, P. Iribarren, M.E. Moreno-Garcia, et al., Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose, J. Immunol. 172 (2004) 1896–1906]. This signal bypassed the emptying of the intracellular calcium stores, a route normally used to open the store-operated calcium channels present in the plasma membrane of neutrophils. Instead, the binding of MMK-1 to FPR2 was shown to trigger a direct opening of the plasma membrane channels. In this report, we add MMK-1 to a large number of FPR2 ligands that activate the neutrophil superoxide-generating NADPH-oxidase. In contrast to earlier findings we show that the transient rise in intracellular free calcium induced by MMK-1 involves both a release of calcium from intracellular stores and an opening of channels in the plasma membrane. The same pattern was obtained with another characterized FPR2 ligand, WKYMVM, and it is also obvious that the two formyl peptide receptor family members trigger the same type of calcium response in human neutrophils.     

A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP(106-126), and amyloid beta(1-42). Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer's, and prion disease.     

Features of the translocation of protein kinase C in neutrophils stimulated with the chemotactic peptide f-Met-Leu-Phe

The effects of f-Met-Leu-Phe (fMLP) on neutrophils, i.e. elevation of the levels of cytoplasmic Ca2+ and intramembranous diacylglycerol, would be expected to be accompanied by translocation of protein kinase C (PKC) to the plasmalemma. However, fMLP-induced PKC translocation could hitherto be demonstrated only when cells were additionally treated with cytochalasin B. We show here that treatment of guinea pig neutrophils with fMLP alone does lead to a significant PKC translocation which can be inhibited by pertussis toxin. The translocation can be detected only if the incubation is terminated within 30 sec after addition of fMLP, the termination is rapid, e.g. by application of a freeze clamp-technique, and the concentration of Ca2+ chelators in the buffer used for lysing the cells is low.     

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.     

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met-Leu-Phe

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met-Leu-Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP. MSUM, CPPD, and fMLP stimulated a significant adherence of neutrophils to hOB after a 1h incubation. This effect was not abrogated by pretreating the cells with an anti-CD18 mAb. MSUM stimulated more efficiently the adherence of neutrophils to non-preactivated hOB while CPPD were more efficient when hOB were preactivated. Crystal-free conditioned media from MSUM- or CPPD-stimulated hOB mobilized intracellular free calcium in human neutrophils. Thus, microcrystals were powerful promoters of neutrophil adherence to hOB via a CD18-independent mechanism. The bacterial peptide fMLP also stimulated the adherence of neutrophils to hOB. Functional neutrophil-hOB interactions could be important in bone pathophysiology of crystal- or infection-associated arthritis.     

Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.     

A formyl peptide substituted with a conformationally constrained phenylalanine residue evokes a selective immune response in human neutrophils

Formyl-Met-Leu-Phe-OH (fMLP) binds to formyl peptide receptors, FPR1 and FPR2, and evokes migration and superoxide anion production in human neutrophils. To obtain a more effective and selective ligand, fMLP analogs in which the Phe residue was substituted with four isomers of cyclopropanephenylalanine were synthesized. While Z-isomer peptides induced both migration and superoxide anion production, E-isomer peptides elicited only chemotaxis. Homologous receptor desensitization experiments revealed that E-isomer peptides bound to FPR2. Although a selective agonist of chemotaxis also binds to FPR2 without increasing intracellular calcium concentration, E-isomer peptide elevated the concentration to the same level as fMLP. Understanding of mechanisms responsible for the selectivity of the reported selective agonists and ?Phe-substituted analogs should prove useful for revealing the relationship between receptor–ligand interactions and biological responses of human neutrophils.     

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.     

Hexokinase translocation during neutrophil activation,chemotaxis, and phagocytosis: disruption by cytochalasin D,dexamethasone, and indomethacin

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.