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Prepublished online as a Blood First Edition Paper on October 3, 2002; DOI 10.1182/blood-2002-05-1435. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-05-1435v1 101/3/1181    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Chodniewicz, D. Articles by Zhelev, D. V. Search for Related Content PubMed PubMed Citation Articles by Chodniewicz, D. Articles by Zhelev, D. V. Related Collections Phagocytes Cytoskeleton Signal Transduction Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 February 2003, Vol. 101, No. 3, pp. 1181-1184 PHAGOCYTES Brief report Chemoattractant receptor-stimulated F-actin polymerization in the human neutrophil is signaled by 2 distinct pathways David Chodniewicz and Doncho V. Zhelev From the Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC.     Abstract Top Abstract Introduction Study design Results and discussion References We characterized the overall rate of F-actin polymerization in the pseudopod region by measuring the rate of extension of single pseudopods stimulated by f-Met-Leu-Phe. The rate of pseudopod extension was measured in the presence of inhibitors for signaling molecules that are known to be involved in motility. Our data show the existence of 2 distinct signaling pathways of actin polymerization in the pseudopod region: a phosphoinositide 3-kinase  (PI3K)-dependent and -independent pathway. The PI3K dependent signaling of F-actin polymerization also depends on protein kinase C  and protein kinase B (Akt/PKB). The PI3K-independent pathway depends on GTPase RhoA, the RhoA ROCK kinase, Src family tyrosine kinases, and NADPH, and is modulated by cAMP. (Blood. 2003;101:1181-1184) © 2003 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Neutrophils are cells of the innate immune system, which are recruited to sites of acute inflammation by sensing and crawling along chemoattractant gradients. The crawling of the cell is a result of highly synchronized processes of F-actin polymerization, cytoskeleton contraction, and adhesion to the surrounding tissue. These processes are signaled by G-protein-coupled chemokine receptors1 that after chemokine ligation bind the Gi subunit of the trimeric G proteins2 and release the G subunit. The released G subunit activates phosphoinositide 3-kinase 3 (PI3K) and phospholipase C- (PLC-).4 PI3K produces phosphatidylinositol 3,4,5-trisphosphate,4 and phosphatidylinositol 3,4-bisphosphate,5 whereas PLC- produces diacylglycerol and inositol 1,4,5-trisphosphate.4 The release of these lipid products signals a variety of responses in the activated cell, including motility. Apparently, the signaling of motility is strongly dependent on PI3K activation but not on PLC-.4 However, there is a PI3K-independent motility that has been demonstrated studying the migration of neutrophils from PI3K-null mice.4 Cell migration is intimately related to cytoskeleton dynamics,6 suggesting that many of the factors affecting migration may also affect cytoskeleton dynamics. Here we use a micropipet assay to measure the rate of extension of single pseudopods from nonadherent human neutrophils during chemoattractant stimulation. The pseudopods are filled with newly polymerized F-actin (Figure 1B), and we use the rate of pseudopod extension as a measure of the overall rate of F-actin polymerization in the pseudopod region. View larger version (36K): [in this window] [in a new window]   Figure 1. (A) Rate of pseudopod extension for cells incubated in the presence of increasing concentrations of wortmannin. The rate of extension is independent of wortmannin concentration above 500 nM. (B) Transmitted light and actin-stained fluorescent images of: (i-ii) passive neutrophil; (iii-iv) neutrophil with a pseudopod stimulated with 107 M fMLP; (v-vi) neutrophil with fMLP-stimulated pseudopod in buffer containing 1 µM wortmannin; and (vii-viii) neutrophil with fMLP-stimulated pseudopod in buffer with 20 µM PP2. Bar = 5 µm. (C) Average rates of pseudopod extension for cells incubated with different inhibitors and combinations of inhibitors. Each rate is the average from at least 10 cells. The inhibitors used were 1 µg/mL pertussis toxin; 1 µM wortmannin (WTM); 10 µM chelerythrine chloride; 40 µM Akt-inhibitor; 10 µM diphenyleneiodonium chloride (DPI); 20 µM PP2; 200 µM dibutyryl cyclic-AMP (dBcAMP); 20 µg/mL Clostridium botulinum C3 exoenzyme (C3); 10 µM Y-27632. The statistical significance for all measurements is P < .01, compared with control, calculated using the one-way analysis of variance test.     Study design Top Abstract Introduction Study design Results and discussion References Cell isolation Venous blood was drawn from consenting, healthy adult donors into vacutainers anticoagulated with EDTA (ethylenediaminetetraacetic acid). Neutrophils were isolated by a one-step density gradient centrifugation on Polymorphoprep (Nycomed, Oslo, Norway), at 500g for 35 minutes at 23°C. The cells were washed once with Ca2+/Mg2+-containing Hanks balanced salt solution (HBSS; Sigma Chemical, St Louis, MO) and resuspended in a 25% autologous plasma/HBSS solution. The cells were added to the manipulation chamber and used in the experiments. Micromanipulation A single, passive neutrophil at 37°C was held in a supporting pipet with a 5 µm internal diameter. Another pipet with an internal diameter of 1 µm was filled with f-Met-Leu-Phe (fMLP) and positioned 1 µm or less from the cell surface. The chemoattractant solution was blown over the cell, which initiated the formation and extension of a single pseudopod. Pseudopod extension was observed using an inverted Nikon microscope equipped with a 60× oil immersion objective (numerical aperture 1.4). The microscope images were recorded using a CCD camera (Cohu, San Diego, CA). A real-time counter and the chamber temperature were multiplexed onto the recorded images using a multiplexer (Vista Electronics, La Mesa, CA). The recorded images were analyzed with Metamorph Imaging software (Universal Imaging, Downingtown, PA) and the rate of pseudopod extension was calculated from the measured distances and time lapses. For further details on calculation of the rates of pseudopod extension, see supplementary information included with online article. Cell incubation with inhibitors The used inhibitors were initially diluted in either ethanol or dimethyl sulfoxide at a maximum final concentration of 0.8% or 0.2%, respectively. The presence of ethanol or dimethyl sulfoxide had no effect on the measured rates of pseudopod extension (data not shown). The cells were incubated with the inhibitors at room temperature (23°C) using the following concentrations (except where otherwise indicated): pertussis toxin (1 µg/mL 60 minutes); wortmannin (WTM; 10 nM to 1000 nM, 15 minutes); dibutyryl cyclic-AMP (dBcAMP; 200 µM, 30 minutes) (Sigma); chelerythrine chloride (10 µM, 30 minutes); diphenyleneiodonium chloride (DPI; 10 µM, 30 minutes); PP2 (20 µM, 20 minutes); piceatannol (50 µM, 30 minutes) (Biomol Research Laboratories, Plymouth Meeting, PA); 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Akt-inhibitor; 10 µM to 40 µM, 30 minutes) (Calbiochem, San Diego, CA); Clostridium botulinum C3 exoenzyme (C3; 20 µg/mL1, 3 hours); Y-27632 (10 µM, 30 minutes); U-73122 (5 µM and 25 µM, 30 minutes) (Biomol). Control measurements on cells not incubated with inhibitors were performed before and after the measurements on cells with inhibitors. The rates of pseudopod extension from the 2 groups of control measurements were not statistically different. Labeling of filamentous actin Single cells were fixed and stained in the experimental chamber. Each cell was manipulated to produce a single pseudopod and was fixed with 4% paraformaldehyde during pseudopod extension. The fixed cell was transferred into solution containing 50 mg/mL monooleoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) and 100 µM Alexa Fluor 488-conjugated phalloidin (Molecular Probes, Eugene, OR) for 10 minutes at room temperature. Finally, the cell was transferred into HBSS and its fluorescence was observed with a SIT camera (Hamamatsu, Bridgewater, NJ). Fluorescent images were taken in successive 0.50 µm planes and the out-of-focus light was removed using the Metamorph Imaging 3D deconvolution algorithm.     Results and discussion Top Abstract Introduction Study design Results and discussion References The measured rate of pseudopod extension from the surface of initially passive neutrophils is almost constant7 and depends on chemoattractant concentration.8 The pseudopods extend and retract in the continuous presence of chemoattractant.7 We studied the ability of inhibitors for some of the key signaling molecules of migration to modulate the initial rate of pseudopod extension. We used the specific PI3K inhibitor wortmannin,9 which was shown to reduce neutrophil migration to the level of that observed for PI3K-null neutrophils.4 We stimulated pseudopod extension with 107 M fMLP in human neutrophils incubated with increasing concentrations of wortmannin (Figure 1A). Wortmannin decreased the rate of pseudopod extension by up to 80% compared with control. This result demonstrates that the polymerization of 80% of the F-actin in the pseudopod region is PI3K dependent, while the remaining 20% is not. Pertussis toxin completely abolished fMLP-stimulated pseudopod extension, demonstrating that pseudopod extension is signaled by only the G-protein-coupled receptors. We incubated neutrophils with the PLC- inhibitor U73122 to test the possibility for PLC- dependence on the rate of pseudopod extension. The incubation of the cells in 5 µM U73122 had no affect on the rate of pseudopod extension; however, it completely inhibited the release of intracellular calcium transients monitored with the calcium probe fluo-3 (data not shown). When the cells were incubated in 25 µM U73122, the rate of pseudopod extension was reduced by 20%. However, this reduction could have been a result of nonspecific binding of U73122 at high concentrations. It has been shown that at concentrations above 20 µM, U73122 significantly reduces the intracellular superoxide anion concentration.10 Therefore, there was a possibility that the decreased rate of pseudopod extension observed for the cells in the presence of 25 µM U73122 was a result of decreased superoxide anion concentration. We tested this possibility by incubating cells with the NADPH oxidase inhibitor DPI. The rate of pseudopod extension in the presence of 10 µM DPI decreased by 20%, and the incubation of cells with the combination of 10 µM DPI and 1 µM wortmannin abolished pseudopod formation. These results demonstrate that the PI3K-independent pseudopod extension in the neutrophil is dependent on NADPH oxidase. The activation of the neutrophil by chemoattractants involves NADPH oxidase activation; however, the involvement of NADPH oxidase activation in F-actin polymerization is unclear. Downstream from the activated chemokine receptors the polymerization of F-actin in the neutrophil is signaled by the GTPases Cdc42 and Rac,11 which are also key signaling molecules in motility.12 Cdc42 binds to the Wiskott-Aldrich syndrome protein WASp, which recruits phosphatidylinositol 4,5-bisphosphate, profilin, and the Arp2/3 complex, to form new barbed ends for actin polymerization.13 Similarly, Rac binds the WASp family protein WAVE, which recruits profilin and Arp2/3 to form new barbed ends.13 RhoA is another GTPase involved in motility.12 In fibroblasts and other cell types it signals stress fiber formation14; however, in the neutrophil RhoA is known to regulate only integrin detachment.15 The guanine exchange factors that activate Cdc42, Rac, and RhoA are not well characterized; however, it is well documented that GTPase activation is PI3K dependent.16 It is also known that the rearrangement of the cellular cytoskeleton by the Rho GTPases RhoA, Cdc42, and Rac, is dependent on the activation of the Src family tyrosine kinases,17 which are key molecules in the signaling of F-actin polymerization by the integrin receptors.18 Src activity is dependent on chemokine receptor activation similar to Rho GTPase activation19 and is regulated by superoxide anion production.20 These findings suggest that tyrosine kinase activation by chemoattractant receptors may provide an alternative signaling pathway for F-actin polymerization. We inhibited the Src family tyrosine kinases with PP2 and the Syk family tyrosine kinases with piceatannol. The inhibition of Syk with 50 µM piceatannol had no affect on the rate of pseudopod extension (data not shown); however, the inhibition of Src with 20 µM PP2 reduced the rate of pseudopod extension by 20%. The simultaneous incubation of cells with 20 µM PP2 and 1 µM wortmannin abolished pseudopod extension, while the incubation of cells with 20 µM PP2 and 10 µM DPI decreased the rate of pseudopod extension by 30% (Figure 1C). These results show that the Src family protein tyrosine kinases are involved in the PI3K-independent signaling of F-actin polymerization. Since the Src signaling of F-actin polymerization is PI3K independent, while the activation of Cdc42 and Rac is dependent on PI3K,16 we hypothesized that Src activation was mediated by RhoA. To test this hypothesis we incubated cells with the RhoA inhibitor C3 and found that the rate of pseudopod extension decreased by 25% (Figure 1C). Similarly, the inhibition of the RhoA kinase ROCK with 10 µM Y-27632 resulted in the decrease of the rate of pseudopod extension by 20%. The simultaneous incubation of cells with C3 or Y-27632 and 1 µM wortmannin abolished pseudopod extension. These results show that RhoA is involved in the PI3K-independent signaling of F-actin polymerization. We also studied the atypical PKC,21 Akt/PKB,22 and cAMP,23 which are known to affect neutrophil migration, for their ability to modulate the rate of pseudopod extension. We measured the rate of pseudopod extension in the presence of 10 µM chelerythrine chloride (Figure 1C) and found that the rate of pseudopod extension decreased by 80%. The simultaneous incubation of cells with 10 µM chelerythrine chloride and 1 µM wortmannin also reduced the rate of pseudopod extension by 80%, whereas the incubation of cells with 10 µM chelerythrine chloride and 20 µM PP2 abolished pseudopod extension. Therefore, PKC is part of the PI3K-dependent F-actin polymerization. Akt/PKB is involved in cell polarization22 and therefore was expected to be involved in the signaling of F-actin polarization. The incubation of cells with increasing concentrations of the Akt-inhibitor24 reduced the rate of pseudopod extension by up to 80% (see supplementary information included with online article). The incubation of cells with both 40 µM Akt-inhibitor and 20 µM PP2 abolished pseudopod extension. Thus, Akt/PKB is part of the PI3K-dependent signaling pathway. The elevation of the intracellular cAMP has been shown to modulate neutrophil migration.25 The effect of cAMP on migration is most probably manifested through protein kinase A activation and RhoA phosphorylation. Based on the results presented here we expected that the elevation of the cAMP would decrease the rate of pseudopod extension. Indeed, the rate of pseudopod extension for cells incubated with 200 µM dibutyryl cAMP decreased by 20%. Our results demonstrate the existence of 2 distinct pathways for F-actin polymerization during chemoattractant-stimulated lamella extension in the human neutrophil. One pathway is dependent on PI3K activation and downstream is dependent on PKC and Akt/PKB (Figure 2). This pathway controls the formation of 70% to 80% of the F-actin in the lamella region. The alternative pathway of F-actin polymerization controls 20% to 30% of the newly formed F-actin in the lamella region. This pathway is dependent on the activation of RhoA, ROCK, Src family tyrosine kinases, and NADPH, and is modulated by cAMP (Figure 2). The exact relation between the latter set of signaling molecules is unknown. However, the reduction of the rate of pseudopod extension by 30% for cells incubated with the combined C3, PP2, DPI, and dBcAMP (Figure 1C, last column) suggests that RhoA, ROCK, Src tyrosine kinase, NADPH oxidase, and cAMP belong to the same signaling pathway. View larger version (24K): [in this window] [in a new window]   Figure 2. Schematic diagram showing the groups of molecules involved in the PI3K-dependent and -independent signaling of F-actin polymerization during pseudopod extension. The PI3K-dependent F-actin polymerization is dependent also on PKC and Akt/PKB, while the PI3K-independent F-actin polymerization is dependent on RhoA, ROCK, Src family tyrosine kinases, NADPH, and cAMP.     Footnotes Submitted May 17, 2002; accepted September 4, 2002. Prepublished online as Blood First Edition Paper, October 3, 2002; DOI 10.1182/blood-2002-05-1435. Supported by grant HL57629 to D.Z. from the National Institutes of Health (NIH). D.C. is a recipient of a fellowship from the NIH Research Training Grant GM08555. Blood drawing was supported by grant M01-RR-30 from the NIH to the General Clinical Research Centers Program at Duke University. The online version of the article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Doncho V. Zhelev, Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708-0300; e-mail: dvzh{at}duke.edu.     References Top Abstract Introduction Study design Results and discussion References 1. Locati M, Murphy PM. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu Rev Med. 1999;50:425-440[CrossRef][Medline] [Order article via Infotrieve]. 2. Arai H, Charo IF. Differential regulation of Gprotein-mediated signaling by chemokine receptors. J Biol Chem. 1996;271:21814-21819[Abstract/Free Full Text]. 3. Hirsch E, Katanaev VL, Garlanda C, et al. Central role for G protein-coupled phosphoinositide 3kinase  in inflammation. Science. 2000;287:1049-1053[Abstract/Free Full Text]. 4. Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D. Roles of PLC-2 and -3 and PI3K in chemoattractant-mediated signal transduction. Science. 2000;287:1046-1049[Abstract/Free Full Text]. 5. Payrastre B, Missy K, Giuriato S, Bodin S, Plantavid M, Gratacap M-P. Phosphoinositides: key players in cell signalling, in time and space. Cell Signal. 2001;13:377-387[CrossRef][Medline] [Order article via Infotrieve]. 6. Zigmond SH. Recent quantitative studies of actin filament turnover during cell locomotion. Cell Motil Cytoskel. 1993;25:309-316[Medline] [Order article via Infotrieve]. 7. Zhelev DV, Alteraifi AM, Hochmuth RM. F-actin network formation in tethers and in pseudopods stimulated by chemoattractant. Cell Motil Cytoskel. 1996;35:331-344[CrossRef][Medline] [Order article via Infotrieve]. 8. Alteraifi AM, Zhelev DV. Transient increase of free cytosolic calcium during neutrophil motility responses. J Cell Sci. 1997;110:1967-1977[Abstract/Free Full Text]. 9. Davies SP, Reedy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000;351:95-105[CrossRef][Medline] [Order article via Infotrieve]. 10. Ferretti ME, Nalli M, Biondi C, et al. Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues. Cell Signal. 2001;13:233-240[CrossRef][Medline] [Order article via Infotrieve]. 11. 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Evidence of  protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis. J Biol Chem. 1998;273:30306-30315[Abstract/Free Full Text]. 22. Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 2000;287:1037-1040[Abstract/Free Full Text]. 23. Harvath L, Robbins JD, Russell AA, Seamon KB. cAMP and human neutrophil chemotaxis: elevation of cAMP differentially affects chemotactic responsiveness. J Immunol. 1991;146:224-232[Abstract/Free Full Text]. 24. Hu Y, Qiao L, Wang S, et al. 3(hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth. J Med Chem. 2000;43:3045-3051[CrossRef][Medline] [Order article via Infotrieve]. 25. Ydrenius L, Molony L, Ng-Sikorski J, Andersson T. Dual action of cAMP-dependent protein kinase on granulocyte movement. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-05-1435v1 101/3/1181    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Chodniewicz, D. Articles by Zhelev, D. V. Search for Related Content PubMed PubMed Citation Articles by Chodniewicz, D. Articles by Zhelev, D. V. Related Collections Phagocytes Cytoskeleton Signal Transduction Brief Reports Social Bookmarking                   What's this?

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