F-actin Content Of Neutrophils Research Articles

PKD regulates actin polymerization, neutrophil deformability, and transendothelial migration in response to fMLP and trauma.

Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F-actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil-like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma.

Myristoylated alanine-rich C-kinase substrate (MARCKS) protein regulation of human neutrophil migration.

Neutrophil migration into infected tissues is essential for host defense, but products of activated neutrophils can be quite damaging to host cells. Neutrophil influx into the lung and airways and resultant inflammation characterizes diseases such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis. To migrate, neutrophils must reorganize the actin cytoskeleton to establish a leading edge pseudopod and a trailing edge uropod. The actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) has been shown to bind and cross-link actin in a variety of cell types and to co-localize with F-actin in the leading edge lamellipodium of migrating fibroblasts. The hypothesis that MARCKS has a role in the regulation of neutrophil migration was tested using a cell-permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with MANS significantly inhibited both their migration and beta2 integrin-dependent adhesion in response to N-formyl-methionyl-leucyl-phenylalanine (fMLF), IL-8, or leukotriene (LT)B(4). The IC(50) for fMLF-induced migration and adhesion was 17.1 microM and 12.5 microM, respectively. MANS significantly reduced the F-actin content in neutrophils 30 seconds after fMLF stimulation, although the peptide did not alter the ability of cells to polarize or spread. MANS did not alter fMLF-induced increases in surface beta2 integrin expression. These results suggest that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.

Impairment of leukocyte deformability in patients undergoing esophagectomy

Impaired deformability might contribute to the accumulation of activated leukocytes within pulmonary microcapillaries, leading to acute lung injury. The purpose of our study was to investigate changes in leukocyte deformability during periods of inflammation after esophagectomy. The study group comprised 20 patients who underwent esophagectomy. Changes in leukocyte deformability were investigated by examining filtration through a silicon microchannel, which simulated human pulmonary microcapillaries. Changes in the neutrophil cytoskeleton were investigated by measuring neutrophil F-actin assembly. The severity of patient clinical outcome was evaluated by the lung injury score. Leukocyte filtration through the microchannel was significantly weaker in esophagectomy patients than in healthy subjects (p<0.01). After esophagectomy, filtration was further impaired compared with preoperative values (p<0.05). The neutrophil F-actin content was higher in patients than in controls (p<0.01), and increased after esophagectomy compared with preoperative values (p<0.01). We concluded that circulating leukocytes showed reduced deformability and appeared to be sequestered within microcapillaries after esophagectomy. Changes in neutrophil cytoskeleton were considered to be responsible for the reduced deformability. Leukocyte accumulation within pulmonary microcapillaries might be related to the pathogenesis of lung injury after esophagectomy.

Role of the actin cytoskeleton during respiratory burst in chemoattractant‐stimulated neutrophils

The aim of this study was to clarify the role of the actin cytoskeleton during chemotactic peptide fMet-Leu-Phe (fMLF)-stimulated respiratory burst in human neutrophil granulocytes. Reactive oxygen species (ROS) was measured as luminol-amplified chemiluminescence (CL) and F-actin content as bodipy phallacidin fluorescence in neutrophils treated with latrunculin B or jasplakinolide, an inhibitor and activator of actin polymerization, respectively. Latrunculin B markedly decreased, whereas jasplakinolide increased, the F-actin content in neutrophils, unstimulated or stimulated with fMLF. Latrunculin B enhanced the fMLF-triggered ROS-production more than tenfold. Jasplakinolide initially inhibited the fMLF-induced CL-response, however, caused a potent second sustained phase (>400% of control). Both actin drugs triggered a substantial CL-response when added 5-25 min after fMLF. This was also valid for chemotactic doses of fMLF, where latrunculin B and jasplakinolide amplified the ROS-production 5-10 times. By using specific signal transduction inhibitors, we found that the NADPH oxidase activation triggered by destabilization of the actin cytoskeleton occurs downstream of phospholipase C and protein kinase C but is mediated by Rho GTPases and tyrosine phosphorylation. In conclusion, rearrangements of the actin cytoskeleton are a prerequisite in connecting ligand/receptor activation, generation of second messengers and assembly of the NADPH oxidase in neutrophil granulocytes.

Serial Changes in Leukocyte Deformability and Whole Blood Rheology in Patients With Sepsis or Trauma

The objective of this study was to investigate serial changes in leukocyte deformability and rheologic properties of whole blood in patients with sepsis or trauma. Seventeen sepsis patients and 22 trauma patients were enrolled. Leukocyte deformability and rheologic properties of whole blood were determined with the use of a microchannel array etched on a single-crystal silicon tip, simulating the microvasculature. The number of obstructed microchannels (NOM) was used as a measure of leukocyte deformability. Transit time (TT), i.e., the time taken for 100 microL of whole blood to pass through the microchannel array was also used as rheologic measure. Oxidative activity and F-actin content of neutrophils was measured in patients with sepsis. NOM and TT significantly increased in patients when sepsis was diagnosed. In survivors, NOM and TT decreased at the time of recovery from sepsis, but in non-survivors values remained high. Oxidative activity and F-actin content of neutrophils increased significantly as leukocyte deformability decreased. In patients with severe trauma, NOM and TT increased after injury and decreased by the time of recovery from the critical stage. We conclude that leukocyte deformability decreases in patients with sepsis or severe trauma and that this change negatively affects rheologic properties of whole blood.

Dialysis neutropenia: The role of the cytoskeleton

Dialysis neutropenia is the result of pulmonary sequestration of neutrophils after complement activation by the dialyzer membrane. Increased expression of neutrophil adhesion receptors, such as CD11b/CD18, suggests that neutrophil adhesion to the capillary endothelium is a possible mechanism. An alternative hypothesis is that the complement fragment C5a modulates neutrophil mechanical properties via the cytoskeleton-largely filamentous actin (F-actin)-stiffening them and thereby slowing their passage through the pulmonary capillaries. To investigate this hypothesis, we developed an assay to measure the F-actin content of neutrophils in whole blood using flow cytometry and the stain NBD-phallacidin. We measured neutrophil F-actin content during hemodialysis of patients with polysulfone (N = 6), Hemophan (N = 6), and Cuprophan membranes sterilized with either ethylene oxide (N = 5) or steam (N = 6). Cell counts, neutrophil and monocyte CD11b expression and plasma C5a concentrations were also measured. The results confirm the strong relationship between the degree of neutropenia, increases in CD11b expression and plasma C5a levels reported by previous researchers. Modulation of the F-actin content of neutrophils was also strongly related to C5a levels, indicating that the neutrophil cytoskeleton is active during dialysis. Modeling of cell counts suggests that with Cuprophan a substantial fraction of neutrophils and monocytes are sequestered before they even pass through the dialyzer, suggesting some form of systemic activation of these cells. Evidence for systemic activation was also seen in measurements of F-actin content, but not CD11b expression, a finding that strengthens the case for the involvement of the cytoskeleton in dialysis neutropenia.

Whole-blood incubation method to study neutrophil cytoskeletal dynamics

To reduce artifactual effects in the study of filamentous (F)-actin dynamics in neutrophils, we have developed a whole-blood incubation method. Neutrophils in whole blood contained significantly less basal F-actin than did separated neutrophils. Although the peak relative F-actin content of neutrophils in whole blood after formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation was significantly higher than that of separated neutrophils at 10 −9 to 10 −6 M fMLP concentrations ( p<0.05), there was no significant difference in increase in mean fluorescence intensity and the EC 50 (concentration of stimulant giving a half-maximum response). On the other hand, the EC 50 of platelet-activating factor (PAF) between separated neutrophils and whole-blood-incubated neutrophils differed significantly (1.6±1.1×10 −9 M in separated neutrophils and 2.0±0.7×10 −8 M in whole-blood-incubated neutrophils, p<0.05). The whole-blood incubation method described presently reduces the sample volume, cost and time needed to separate neutrophils, prevents neutrophil activation during separation, and reserves all blood components that may affect neutrophil function. For these reasons, the conditions adopted in the present method are thought to simulate well neutrophils circulating in vivo and the method would be preferable to other neutrophil function tests performed to study actin dynamics.

Effect of maternal labor and mode of delivery on neutrophil actin response to N-formylmethionyl-leucyl-phenylalanine in healthy neonates.

Diminished actin response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) has been used to explain the impaired chemotaxis of neonatal neutrophils, but the effect of labor on this response has not been evaluated before. Therefore, we tested the hypothesis that labor stress may have an effect on actin response of cord blood neutrophils to FMLP. This response is compared by phallacidin stain of neutrophil F-actin and flow cytometry analysis among 3 groups of healthy neonates: group I, 16 vaginally delivered neonates; group II, 16 neonates delivered by elective cesarean section without labor; and group III, 16 neonates delivered by cesarean section after labor. In the group III, 10 infants were delivered by an emergency cesarean section because of fetal heart rate deceleration indicating fetal distress (one minute Apgar score < 7 in 5 cases [50%]), and the other 6 infants were delivered because of failure of progression of labor (one minute Apgar score < 7 in 2 cases [33.3%]). Stimulated relative F-actin content of neutrophils in group III was increased compared with groups I and II. There was no such difference between neutrophils from infants of groups I and II. The results of this study indicate that fetal distress may enhance neutrophil actin response to FMLP, while labor or mode of delivery might not have such effect.

Doxycycline reduction of F-actin content of human neutrophils and fibroblasts.

We and others have shown that tetracyclines inhibit leukocyte adherence, migration, and phagocytosis, functions presumed to involve actin microfilament metabolism. In this study we investigated the influence of a tetracycline (doxycycline, Dc) on neutrophil and fibroblast actin metabolism. Human neutrophils and fibroblasts were pretreated with Dc or cytochalasin B (cB), stimulated with either the chemotactic peptide FMLP or phorbol myristate acetate (PMA), and the changes in phalloidin conjugate, associated F-actin were followed microscopically or quantified fluorometrically. Doxycycline suppressed, in a dose-related manner, the rise in F-actin content of neutrophils that normally follows their activation with either FMLP or PMA. Cytochalasin B had a similar affect on actin microfilament synthesis. Incubation of fibroblasts in Dc led to a loss of actin microfilaments and caused flattened adherent cells to round up and detach. Both Dc and cB also inhibited Con A-induced acceptor capping on neutrophils, a phenomenon known to be dependent on the presence of intact actin microfilaments. The data show that both Dc and cB influence actin metabolism and suggest they do so by differing mechanisms.

Beta 2 integrin engagement triggers actin polymerization and phosphatidylinositol trisphosphate formation in non-adherent human neutrophils.

Beta 2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way beta 2 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of beta 2 integrins. Antibody engagement of beta 2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the beta 2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca2+ only marginally affected the beta 2 integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca2+ has a modulatory effect it is not an absolute requirement for beta 2 integrin-induced actin polymerization. beta 2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in beta 2 integrin-induced actin assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Signal Pathway Regulation of Interleukin-8-Induced Actin Polymerization in Neutrophils

Abstract Interleukin-8 (IL-8), a recently described peptide cytokine, is a neutrophil chemoattractant and activator that exerts effects similar to fMLP, yet their receptors and their roles in pathophysiology differ. The effect of IL-8 on the neutrophil cytoskeleton has not been well studied; therefore, we compared and contrasted the effects of IL-8 and fMLP on neutrophil actin conformation and on the signal pathway regulation of actin responses. IL-8 caused a rapid, dose-dependent increase in neutrophil F-actin content within 30 seconds. The maximum increase was twofold. These changes were accompanied by the development of F-actin-rich pseudopods, as noted with fluorescence microscopy and scanning electron microscopy. Selected biochemical inhibitors were used to study the regulation of the IL-8-induced actin changes. Incubation of neutrophils with 2 micrograms/mL pertussis toxin resulted in a 67% inhibition of the IL-8-induced F-actin increase. The protein kinase C (PKC) inhibitors, staurosporine and H7, did not inhibit the increase in F-actin caused by IL-8. IL-8 caused a rapid increase in neutrophil intracellular calcium that could be completely inhibited by the chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA). However, BAPTA-treated neutrophils retained the ability to increase F-actin in response to IL-8. Similar results were seen with fMLP, indicating that, similar to fMLP, the IL-8-induced actin response is mediated through pertussis-toxin-sensitive G-proteins but is neither dependent on PKC nor increases in cytosolic calcium. Thus, although IL- 8 and fMLP exert their effects on neutrophils through different receptors, the signal transduction pathways used and the effects on actin conformation and pseudopod formation are similar.

Signal pathway regulation of interleukin-8-induced actin polymerization in neutrophils

Interleukin-8 (IL-8), a recently described peptide cytokine, is a neutrophil chemoattractant and activator that exerts effects similar to fMLP, yet their receptors and their roles in pathophysiology differ. The effect of IL-8 on the neutrophil cytoskeleton has not been well studied; therefore, we compared and contrasted the effects of IL-8 and fMLP on neutrophil actin conformation and on the signal pathway regulation of actin responses. IL-8 caused a rapid, dose-dependent increase in neutrophil F-actin content within 30 seconds. The maximum increase was twofold. These changes were accompanied by the development of F-actin-rich pseudopods, as noted with fluorescence microscopy and scanning electron microscopy. Selected biochemical inhibitors were used to study the regulation of the IL-8-induced actin changes. Incubation of neutrophils with 2 micrograms/mL pertussis toxin resulted in a 67% inhibition of the IL-8-induced F-actin increase. The protein kinase C (PKC) inhibitors, staurosporine and H7, did not inhibit the increase in F-actin caused by IL-8. IL-8 caused a rapid increase in neutrophil intracellular calcium that could be completely inhibited by the chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPTA). However, BAPTA-treated neutrophils retained the ability to increase F-actin in response to IL-8. Similar results were seen with fMLP, indicating that, similar to fMLP, the IL-8-induced actin response is mediated through pertussis-toxin-sensitive G-proteins but is neither dependent on PKC nor increases in cytosolic calcium. Thus, although IL-8 and fMLP exert their effects on neutrophils through different receptors, the signal transduction pathways used and the effects on actin conformation and pseudopod formation are similar.

Inhibition of human neutrophil aggregation by albumin. Relationship with cytoskeleton reorganization

Albumin, at concentration normally present in plasma (∼600 μM), signicantly inhibited leukotriene B 4 formation induced by a receptor mediated (fMet-Leu-Phe) and a receptor independent (calcium ionophore A23187) stimuli in human neutrophils. The inhibition of leukotriene B 4 synthesis was accompanied by a concomitant reduction of neutrophil aggregation. In addition, this plasma protein prevented the increase in F-actin content of neutrophils stimulated with fMet-Leu-Phe and A23187, thus suppressing actin polymerization. These data indicate that albumin profoundly affects biochemical and functional aspects of neutrophils suggesting, for this plasma protein, a regulatory role in the overall pattern of the inflammatory reaction.

Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor.

Tumor necrosis factor (TNF) is a 17,000-Da protein which is produced by mononuclear cells upon exposure to endotoxin. Increases in adherence, phagocytosis, hydrogen peroxide release, and lysozyme secretion have been demonstrated after prolonged incubation of human neutrophils with TNF. In this study, the ability of highly purified recombinant human TNF to modulate neutrophil responses to soluble stimuli was evaluated. Tumor necrosis factor alone (0.1 to 10,000 units/ml) failed to induce neutrophil superoxide anion (O2-) production, granule release, or aggregation when incubated for up to 25 min at 37 degrees C. TNF did, however, stimulate a significant time-, dose-, and temperature-dependent increase in neutrophil F-actin content. Although exposure of neutrophils to TNF alone caused no superoxide anion production, it enhanced the O2- production in response to the chemotactic peptide, f-methionyl-leucyl-phenylalanine (FMLP) or the tumor promotor, phorbol myristate acetate, by as much as 278%. The enhancement was time-, dose-, and temperature-dependent and was due to a more rapid initial rate of O2- production. The TNF enhancement of FMLP-induced O2- production was blocked when an anti-TNF monoclonal antibody 241-1H11, is present during the preincubation period. TNF preincubation also enhanced FMLP-induced lysozyme release, but had no effect on aggregation and actin polymerization by FMLP. The kinetics of NADPH oxidase activation by arachidonic acid was unaltered by TNF. These results indicate that brief exposures to recombinant human TNF are able to enhance or prime the neutrophil oxidative burst in response to a second stimulus.

The effects of sulfhydryl inhibitors and cytochalasin on the cytoplasmic and cytoskeletal actin of human neutrophils.

To better understand the changes that occur in cytoplasmic actin during cell movement, we studied the effect of inhibitors of cell movement on the molecular conformation of actin and its incorporation into the Triton-insoluble cytoskeleton of human neutrophils. The sulfhydryl reactive compound N-ethylmaleimide caused an increase in cellular F-actin as measured by uptake of the F-actin specific fluorescent probe 7-nitrobenz-2-oxadiazole-phallacidin. However, N-ethylmaleimide reduced the amount of actin associated with the Triton-insoluble cytoskeleton. Dithiobisnitrobenzoic acid, a sulfhydryl reagent that does not cross cell membranes efficiently, did not alter the F-actin content of neutrophils. The effect of N-ethylmaleimide was blocked by the presence of dithiothreitol, a donor of sulfhydryl groups. N-ethylmaleimide did not affect the polymerization of actin in a cell-free system. Cytochalasin B did not alter F-actin content of neutrophils but did decrease actin in cytoskeletons of resting neutrophils. Cytochalasin inhibited the increase in F-actin initiated by the chemoattractant N-formylmethionylleucylphenylalanine. We propose that N-ethylmaleimide blocks the stabilization of G-actin in cytoplasm, interferes with the incorporation of F-actin polymer into the cytoskeleton, and depolymerizes the cytoskeleton. In contrast cytochalasin stabilizes G-actin in the presence of chemotactic peptide. These data suggest that reversible conversion of G-actin to F-actin and incorporation of F-actin into the Triton-insoluble cytoskeleton are important for neutrophil movement.

Calcium ionophore, phorbol ester, and chemotactic peptide-induced cytoskeleton reorganization in human neutrophils.

Formyl-methionylleucylphenylalanine (fMLP) activation of neutrophils causes an increase in intracellular Ca2+, activation of protein kinase C and an increase in F-actin content. To examine the role of Ca2+ and protein kinase C activation as determinants of change in F-actin content of neutrophils, we used the NBD-phallacidin extraction assay to compare the kinetics and extent of change in F-actin content of cells activated with fMLP, the calcium ionophore A23187 or phorbol myristate acetate (PMA). All stimuli increase the F-actin content in a dose-dependent manner; however, the rate of increase is slower and the maximum F-actin content is less for calcium ionophore and PMA than for fMLP-activated cells. The A23187-induced increase in F-actin content, but not that of fMLP, depends upon external free [Ca2+]. In A23187-activated cells, F-actin content increases at [Ca2+]free greater than or equal to 5 microM, is maximal at [Ca2+]free greater than or equal to 10 microM and is negligible at physiologic free [Ca2+] (10(-7)-10(-6) M). Combinations of PMA with A23187 or fMLP inhibit the A23187, but not the fMLP, activated actin polymerization. Comparison and combination of these activators shows that neither Ca2+-dependent activation with A23187 nor activation with PMA alone or in combination mimic the fMLP-induced changes in cytoskeleton organization of neutrophils.

A method for quantifying F-actin in chemotactic peptide activated neutrophils: study of the effect of tBOC peptide.

The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F-actin content of fMLP-activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F-actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5-10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP-induced change in the F-actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP-induced increase followed by a decrease in F-actin content. The tBOC peptide inhibits fMLP-induced actin polymerization. Addition of tBOC during fMLP-induced polymerization or at times when F-actin content is maximal enhances F-actin depolymerization. The rate of F-actin depolymerization is greater than or equal to fourfold faster in the presence than in the absence of tBOC. The results show that The NBDphallacidin extraction assay is useful for studying the kinetics of change in F-actin content of nonmuscle cells; fMLP receptor occupancy is required for fMLP-dependent polymerization but not depolymerization; and both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F-actin content of fMLP-activated cells are discussed.

Chemotactic peptide modulation of actin assembly and locomotion in neutrophils.

To determine the relationship between the state of actin polymerization in neutrophils and the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced changes in the locomotive behavior of neutrophils, the mean rate of locomotion (mROL), the percent G-actin, and the relative F-actin content of neutrophils were determined. The mROL was quantified by analysis of the locomotion of individual cells; the percentage of total actin as G-actin was measured by DNase I inhibition; and the F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained neutrophils. Neutrophils stimulated with fMLP exhibit a change in their mROL that is biphasic and dose dependent. The mROL of neutrophils exposed to 10(-8) M fMLP, the KD, is 11.9 +/- 2.0 micron/min (baseline control 6.2 +/- 1.0 micron/min). At 10(-6) M fMLP, the mROL returns to baseline levels. Stimulation of neutrophils with fMLP also induces action polymerization. Evidence for actin polymerization includes a 26.5% reduction in G-actin and a twofold increase in the amount of NBD-phallacidin staining of cells as determined by FACS analysis. The NBD-phallacidin staining is not due to phagocytosis, is inhibited by phalloidin, requires cell permeabilization, and is saturable at NBD-phallacidin concentrations greater than 10(-7)M. The fMLP-induced increase in NBD-phallacidin staining occurs rapidly (less than 2 min), is temperature dependent, and is not due to cell aggregation. Since NBD-phallacidin binds specifically to F-actin, the increase in fluorescent staining of cells likely reflects an increase in the F-actin content of fMLP-stimulated cells. FACS analysis of NBD-phallacidin-stained cells shows that the relative F-actin content of neutrophils stimulated with 10(-11)-10(-8)M fMLP increases twofold and remains increased at concentrations greater than 10(-8)M fMLP. Therefore, the fMLP-induced increase in F-actin content of neutrophils as determined by FACS analysis of NBD-phallacidin-stained cells coincides with a decrease in G-actin and correlates with increased mROL of neutrophils under some (10(-11)-10(-8)M fMLP) but not all (greater than 10(-8)M fMLP) conditions of stimulation. Quantification of the F-actin content of nonmuscle cells by FACS analysis of NBD-phallacidin-stained cells may allow rapid assessment of the state of actin polymerization and correlation of that state with the motile behavior of nonmuscle cells.