Am J Physiol Cell Physiol 286: C55–C64, 2004. First published September 3, 2003; 10.1152/ajpcell.00131.2003. Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases Haruo Kutsuna,1,2 Kenichi Suzuki,1 Noriko Kamata,1 Takayuki Kato,1 Fumihiko Hato,1 Kensaku Mizuno,3 Hiromi Kobayashi,2 Masamitsu Ishii,2 and Seiichi Kitagawa1 1 Department of Physiology and 2Department of Dermatology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585; and 3Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Aramaki-aza-aoba, Aoba-ku, Sendai 980-8578, Japan Submitted 7 April 2003; accepted in final form 30 August 2003 Address for reprint requests and other correspondence: S. Kitagawa, Dept. of Physiology, Osaka City Univ. Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan (E-mail: kitagawas@med.osaka-cu.ac.jp). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. neutrophil; actin reorganization; cytokines; mitogen-activated protein kinase; tumor necrosis factor-␣; granulocyte-macrophage colony stimulating factor VARIOUS FUNCTIONS OF HUMAN http://www.ajpcell.org 0363-6143/04 $5.00 Copyright © 2004 the American Physiological Society C55 Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 neutrophils are activated by chemoattractants or inflammatory cytokines, such as tumor necrosis factor-␣ (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF) (16, 41, 42). Some functions such as superoxide anion (O⫺ 2 ) release and adherence appear to be similarly activated by chemoattractants and cytokines, whereas some functions such as locomotion and morphological changes may be differentially affected by these stimuli. For example, chemoattractants such as N-formyl-methionyl-leucyl-phenylalanine (FMLP) and interleukin-8 induce neutrophil chemotaxis, whereas TNF, GM-CSF, and G-CSF inhibit neutrophil chemotaxis (6, 13, 17, 28, 38). Polarization, a morphological change closely associated with neutrophil chemotaxis and actin polymerization, is induced by chemoattractants but not by TNF (2). These findings suggest that cytokines regulate actin reorganization in human neutrophils in a different way from chemoattractants. It is well known that chemoattractants induce an overall increase in F-actin content and actin polymerization, although the precise mechanisms for chemoattractant-induced actin reorganization remain to be elucidated (13, 28). On the other hand, actin reorganization induced by inflammatory cytokines is largely unknown. A major signaling system activated in human neutrophils stimulated by cytokines is the mitogen-activated protein kinase (MAPK) cascade (31, 32). In mammalian cells, there are at least three MAPK subtypes, i.e., extracellular signalregulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase. Each MAPK subtype is activated by phosphorylation on threonine and tyrosine residues by upstream dual-specificity kinases, such as MAPK/ERK kinase (MEK), MAPK kinase-3 or -6 (MKK3/6), and MKK4/7. Activation of the distinct MAPK subtype cascade is dependent on the types of cells and the stimuli used, and the functional role of each MAPK subtype may be different according to the types of cells (27, 39). Our recent studies with human neutrophils show that distinct MAPK subtype cascades are activated in a cytokine-specific manner and that the MEK-ERK and MKK3/6-p38 MAPK cascades play an important role in cytokine-induced activation of human neutrophil functions such as O⫺ 2 release, adherence, and upregulation of ␤2-integrin (30, 31, 32). In this study, we investigated actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF, and the role of MAPKs in these responses. The results show that in contrast to a chemotactic peptide FMLP, TNF, GM-CSF, and G-CSF induce an overall decrease in F-actin content (actin depolymerization) and concomitant morphological changes in human neutrophils and suggest that activation of ERK and p38 MAPK plays a critical role in these responses. Kutsuna, Haruo, Kenichi Suzuki, Noriko Kamata, Takayuki Kato, Fumihiko Hato, Kensaku Mizuno, Hiromi Kobayashi, Masamitsu Ishii, and Seiichi Kitagawa. Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases. Am J Physiol Cell Physiol 286: C55–C64, 2004. First published September 3, 2003; 10.1152/ajpcell.00131.2003.—Stimulation of human neutrophils with tumor necrosis factor-␣ (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF. Cytokine-induced actin depolymerization was sustained for at least 30 min after stimulation. In contrast, N-formyl-methionyl-leucylphenylalanine (FMLP) rapidly (within 45 s) induced an increase in the fluorescence intensity of FITC-phalloidin (actin polymerization) and morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK, and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished by PD-98059 and were unaffected by SB-203580. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils. Phosphorylated ERK and p38 MAPK were not colocalized with F-actin in neutrophils stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF induce actin depolymerization and morphological changes through activation of ERK and/or p38 MAPK and that cytokine-induced actin reorganization may be partly responsible for the inhibitory effect of these cytokines on neutrophil chemotaxis. C56 ACTIN REORGANIZATION AND MAP KINASES MATERIALS AND METHODS AJP-Cell Physiol • VOL RESULTS TNF, GM-CSF, and G-CSF induce an overall decrease in F-actin content in neutrophils. The F-actin content in human neutrophils was analyzed using FITC-phalloidin. As shown in Fig. 1, stimulation of neutrophils with FMLP resulted in rapid increase in the fluorescence intensity, which reflects actin Fig. 1. Effects of tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and N-formylmethionyl-leucyl-phenylalanine (FMLP) on F-actin content in neutrophils. Neutrophils were stimulated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for 10 min, G-CSF (50 ng/ml) for 20 min, or FMLP (10⫺7 M) for 45 s at 37°C. F-actin content was measured by flow cytometry using FITC-phalloidin. Dotted line and shaded area represent the profiles of unstimulated control cells and unstained cells, respectively. Results shown are representative of 7 independent experiments. 286 • JANUARY 2004 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 Reagents. Highly purified recombinant human G-CSF, GM-CSF, and TNF produced by Escherichia coli were provided by Kirin Brewery (Tokyo, Japan), Schering-Plough (Osaka, Japan), and Dainippon Pharmaceutical (Osaka, Japan), respectively. The specific activity of TNF was 3 ⫻ 106 U/mg protein. Endotoxin contamination of each preparation was ⬍100 pg/mg protein. SB-203580 (p38 MAPK inhibitor) was purchased from Calbiochem (California, CA). FMLP and FITC-conjugated phalloidin were purchased from Sigma Chemical (St. Louis, MO). Conray was purchased from Mallinckrodt (St. Louis, MO). Ficoll and the enhanced chemiluminescence (ECL) Western blotting system were purchased from Amersham Pharmacia Biotech (Buckinghamshire, England). PD-98059 (MEK inhibitor) and rabbit polyclonal antibodies against ERK1/2, Thr202/Tyr204-phosphorylated ERK1/2, p38 MAPK, and Thr180/Tyr182-phosphorylated p38 MAPK were purchased from Cell Signaling Technology (Beverley, MA). Alexa Fluor 568-labeled goat anti-rabbit IgG antibody was purchased from Molecular Probes (Eugene, OR). Rabbit polyclonal antibodies against cofilin and Ser3-phosphorylated cofilin were prepared as described previously (3, 37). Preparation of cells. Human neutrophils were prepared from healthy adult donors as described previously (31), using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminated erythrocytes. Neutrophil fractions contained ⬎98% neutrophils. Cells were suspended in Hanks’ balanced salt solution (HBSS) containing 10 mM N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid (HEPES) (pH 7.4). Determination of actin reorganization and immunofluorescence. Actin reorganization was analyzed using flow cytometry and confocal laser scanning microscopy. Neutrophils (1 ⫻ 107/ml, 50 ␮l) suspended in HBSS were stimulated with TNF, GM-CSF, G-CSF, or FMLP for indicated periods at 37°C. When required, cells were pretreated with PD-98059 (50 ␮M) or SB-203580 (10 ␮M) for 20 min at 37°C before stimulation with each agent. The reactions were performed in polystyrene tubes (Falcon 2054; BD Labware) precoated with fetal calf serum (FCS) to prevent strong adhesive interaction between cytokine-stimulated neutrophils and polystyrene surface, which could affect actin reorganization. After incubation with each agent, cells were fixed with IntraPrep Reagent 1 (Immunotech, Marseille, France) for 15 min at room temperature. After being washed with phosphate-buffered saline (PBS), cells were permeabilized with IntraPrep Reagent 2 (Immunotech) for 5 min at room temperature and thereafter incubated with FITC-phalloidin (5 ng/ml) in the dark for 15 min at room temperature. Cells were washed with PBS, resuspended in PBS containing 0.5% paraformaldehyde, and analyzed by flow cytometry with FACS Calibur (Beckton Dickinson, Mountain View, CA) using CellQuest analysis software or by confocal laser scanning microscopy (Zeiss LSM510, Welwyn, Garden City, UK). For indirect immunofluorescence, cells were similarly processed as described above, and phospho-ERK1/2 or phospho-p38 MAPK antibody was added with FITC-phalloidin after permeabilization with IntraPrep Reagent 2. After incubation in the dark for 15 min at room temperature, cells were washed with PBS and stained with Alexa Fluor 568-labeled goat anti-rabbit IgG antibody in the dark for 15 min at room temperature. Cells were washed with PBS and resuspended in PBS containing 0.5% paraformaldehyde. Fluorescence images were photographed with a confocal laser scanning microscope. Western blotting. Neutrophils (1 ⫻ 107/ml) suspended in HBSS were prewarmed for 10 min at 37°C and thereafter stimulated with TNF (100 U/ml), GM-CSF (5 ng/ml), or G-CSF (50 ng/ml) for 10 min at 37°C. When required, cells were pretreated with PD-98059 (50 ␮M) or SB-203580 (10 ␮M) for 20 min at 37°C. The reactions were terminated by rapid centrifugation, and the pellets were frozen in liquid nitrogen, resuspended in ice-cold extraction buffer containing 50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovana- date, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 100 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin, and lysed for 10 min at 4°C. After rapid centrifugation, the supernatant was mixed 1:1 with 2⫻ sample buffer [4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% mercaptoethanol, and a trace amount of bromophenol blue dye in 125 mM Tris䡠HCl, pH 6.8], heated at 100°C for 5 min and then frozen at ⫺80°C until use. Samples were subjected to 10% SDS gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol at 2 mA/cm2 for 1.5 h at 25°C. Residual binding sites on the membrane were blocked by incubating the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25°C. The blots were washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with appropriate antibody overnight at 4°C. After being washed, the membrane was incubated with anti-rabbit IgG antibody conjugated with horseradish peroxidase, and the antibody complexes were visualized by the ECL detection system as directed by the manufacturer. Immunoreactive bands were quantified by a National Institutes of Health (NIH) Image program on a Macintosh computer. Kinase assay of p38 MAPK. The kinase activity of p38 MAPK was determined by using a nonradioactive immunoprecipitation kinase assay kit (Cell Signaling Technology) according to the manufacturer’s instructions. Phosphorylated p38 MAPK was immunoprecipitated by using immobilized monoclonal antibody against Thr180/Tyr182-phosphorylated p38 MAPK. The resulting immunoprecipitates were incubated with GST-ATF-2 fusion protein in the presence of ATP. Phosphorylation of ATF-2 was determined by Western blotting using antibody against Thr71-phosphorylated ATF-2. When required, PD98059 (50 ␮M) and/or SB-203580 (1 or 10 ␮M) were added to the reaction mixture. Statistical analysis. Student’s t-test was used to determine statistical significance. ACTIN REORGANIZATION AND MAP KINASES polymerization (13, 28). The maximal change was detected at 45 s after stimulation, and the increased fluorescence intensity returned to the basal level within 5 min (Fig. 2A). In contrast to FMLP, stimulation of neutrophils with TNF, GM-CSF, or AJP-Cell Physiol • VOL G-CSF resulted in decreased fluorescence intensity, which may reflect actin depolymerization (Fig. 1). The maximal change in the fluorescence intensity was detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF, respectively (Fig. 2A). No significant change was detected at early time points (within 5 min) after stimulation with each cytokine. The decreased fluorescence intensity induced by these cytokines was sustained at least for 30 min after stimulation. The effect of TNF, GM-CSF, and G-CSF on the changes in fluorescence intensity was dependent on the concentration of cytokines used as stimuli (Fig. 2B). Significant effect was obtained at 1 U/ml TNF, 0.5 ng/ml GM-CSF, and 50 ng/ml G-CSF, respectively. To further confirm that these cytokines induced actin depolymerization, cells were analyzed with confocal microscopy. As shown in Fig. 3, confocal microscopy revealed that control cells showed diffuse distribution of F-actin and round shape. Stimulation of cells with TNF, GM-CSF, or G-CSF resulted in an overall decrease in F-actin content with redistribution of F-actin and concomitant morphological changes. In contrast, FMLP induced an overall increase in F-actin content with concomitant morphological changes. Typical polarization induced by FMLP was not detected at this early time point (28) (Fig. 3, see also Fig. 6). Involvement of ERK and p38 MAPK in actin depolymerization and morphological changes induced by TNF, GM-CSF, or G-CSF. A major signaling system activated in human neutrophils in response to stimulation with TNF, GM-CSF, or G-CSF is the MAPK cascades; specifically, the ERK and p38 MAPK cascades (31). Our recent studies show that ERK and p38 MAPK play a critical role in cytokine-induced activation of human neutrophil functions such as O⫺ 2 release, adherence, and upregulation of ␤2-integrin (30, 31, 32). Then, possible involvement of the MAPK cascades in cytokine-induced actin depolymerization and morphological changes was explored using PD-98059 (MEK inhibitor) (1) and SB-203580 (p38 MAPK inhibitor) (18). As shown in Fig. 4, pretreatment of cells with PD-98059 (50 ␮M) significantly and partially inhibited actin depolymerization induced by TNF or GM-CSF and almost completely abolished actin depolymerization induced by G-CSF. Similarly, pretreatment of cells with SB-203580 (10 ␮M) also significantly and partially inhibited actin depolymerization induced by TNF or GM-CSF but not by G-CSF. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils; i.e., ERK is activated by TNF, GM-CSF, and G-CSF, whereas p38 MAPK is activated by TNF and GM-CSF, but not by, or very weakly by, G-CSF (31) (see also Fig. 9A). When PD-98059 (50 ␮M) and SB-203580 (10 ␮M) were used in combination, TNF- or GM-CSF-induced actin depolymerization was almost completely abolished. G-CSF-induced actin depolymerization was almost completely abolished by PD98059 alone. In contrast, neither PD-98059 nor SB-203580 affected FMLP-induced actin polymerization, in agreement with the previous report (9) (Fig. 4). These inhibitors alone or in combination also did not affect the rate of actin depolymerization (a “falling” phase of actin polymerization), which followed rapid actin polymerization in response to FMLP (Fig. 4C, the data obtained with each inhibitor alone are not shown). These findings obtained by flow cytometry were confirmed by confocal microscopy. As shown in Fig. 3, TNF- or GM- 286 • JANUARY 2004 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 Fig. 2. Effects of TNF, GM-CSF, G-CSF, and FMLP on F-actin content in neutrophils. A: neutrophils were stimulated with TNF (100 U/ml), GM-CSF (5 ng/ml), G-CSF (50 ng/ml), or FMLP (10⫺7 M) for indicated times at 37°C, and then F-actin content was measured by flow cytometry. The change in F-actin content is expressed as a relative fold change in the mean fluorescence intensity, and the fluorescence intensity at time 0 is expressed as 1. Data are expressed as means ⫾ SD of 3–6 experiments. *Significantly decreased compared with unstimulated control cells (P ⬍ 0.01). †Significantly increased compared with unstimulated control cells (P ⬍ 0.01). B: neutrophils were stimulated with indicated concentrations of TNF or GM-CSF for 10 min or G-CSF for 20 min at 37°C, and then F-actin content was measured by flow cytometry. The change in F-actin content is expressed as a relative fold change in the mean fluorescence intensity as described in A. Data are expressed as means ⫾ SD of 3 experiments. *Significantly decreased compared with unstimulated control cells (P ⬍ 0.01). C57 C58 ACTIN REORGANIZATION AND MAP KINASES CSF-induced actin depolymerization as well as morphological changes was partially inhibited by pretreatment of cells with PD-98059 or SB-203580. When PD-98059 and SB-203580 were used in combination, TNF- or GM-CSF-induced actin depolymerization as well as morphological changes was almost completely abolished, and these cells showed apparently similar profiles to control cells (Fig. 3, A and B). On the other hand, G-CSF-induced actin depolymerization as well as morphological changes was almost completely abolished by pretreatment of cells with PD-98059 and was unaffected by SB-203580 (Fig. 3C). In contrast, neither PD-98059 nor SB203580 affected FMLP-induced actin polymerization and morphological changes (Fig. 3D). FMLP-induced polarization and actin polymerization are prevented by cytokine pretreatment. The results depicted in Figs. 1–4 show that actin reorganization is differentially regulated by cytokines and FMLP, suggesting that FMLP-induced polarization and actin polymerization might be affected by cytokine pretreatment. To explore this possibility, neutrophils were pretreated with each cytokine for 10 or 20 min and AJP-Cell Physiol • VOL thereafter challenged with FMLP. As shown in Fig. 5, later challenge with FMLP caused significant actin polymerization in TNF-, GM-CSF-, or G-CSF-pretreated neutrophils when analyzed at 45 s after FMLP stimulation. However, the magnitude of FMLP-induced actin polymerization in cytokinepretreated cells was always less than polymerization that was apparent in cells treated with FMLP alone (P ⬍ 0.05 for all cytokines, n ⫽ 4). Confocal microscopy revealed that FMLPinduced polarization, which was detected at 10 min after stimulation, was prevented by pretreatment of cells with TNF, GM-CSF, or G-CSF (Fig. 6). Prevention or attenuation of FMLP-induced actin polymerization by cytokine pretreatment was also confirmed by confocal microscopy. Phosphorylated ERK and p38 MAPK are localized separately from F-actin. The results described above indicate a close relationship between actin depolymerization and activation of ERK and p38 MAPK. If activation of ERK and p38 MAPK is actually involved in actin depolymerization in human neutrophils, it would be expected that phosphorylated ERK and p38 MAPK are localized separately from F-actin and are not 286 • JANUARY 2004 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 Fig. 3. Analysis with confocal microscopy. Neutrophils were stimulated with TNF (100 U/ml) (A) or GM-CSF (5 ng/ml) (B) for 10 min, G-CSF (50 ng/ml) (C) for 20 min, or FMLP (10⫺7 M) (D) for 45 s at 37°C. When required, cells were pretreated with PD-98059 (PD, 50 ␮M) and/or SB-203580 (SB, 10 ␮M) for 20 min before stimulation with TNF, GM-CSF, G-CSF, or FMLP. As a control, FMLP-induced actin polymerization is shown in A, B, and C using the same cell preparations. F-actin distribution and morphological changes were analyzed with confocal microscopy using FITC-phalloidin. Results shown are representative of 7 independent experiments. ACTIN REORGANIZATION AND MAP KINASES C59 Fig. 4. Effects of PD-98059 and SB-203580 on F-actin content in neutrophils stimulated by TNF, GM-CSF, G-CSF, or FMLP. Neutrophils were pretreated with PD-98059 (PD, 50 ␮M) and/or SB-203580 (SB, 10 ␮M) for 20 min at 37°C. Thereafter, cells were stimulated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for 10 min, G-CSF (50 ng/ml) for 20 min, or FMLP (10⫺7 M) for 45 s at 37°C. F-actin content was measured by flow cytometry. A: profiles of changes in FITC-phalloidin fluorescence intensity. In each column, dotted and solid lines represent unstimulated control cells and cells stimulated with each agonist, respectively. Bold line represents the profile obtained in the presence of PD-98059 and/or SB-203580. The results shown are representative of 5 independent experiments. B: the change in F-actin content is expressed as a relative fold change in the mean fluorescence intensity, and the fluorescence intensity of unstimulated control cells is expressed as 1. Data are expressed as means ⫾ SD of 3–7 experiments. *Significantly decreased by stimulation with TNF, GM-CSF, or G-CSF (P ⬍ 0.01). #Significantly increased by stimulation with FMLP (P ⬍ 0.01). ⫹,†Cytokine-induced decrease in F-actin content was significantly inhibited by PD-98059 and/or SB-203580 (⫹P ⬍ 0.01, †P ⬍ 0.05). C: neutrophils were pretreated with PD-98059 (50 ␮M) and SB-203580 (10 ␮M) for 20 min at 37°C. Thereafter, cells were stimulated with FMLP (10⫺7 M) for indicated times at 37°C, and then F-actin content was measured by flow cytometry. The change in F-actin content is expressed as a relative fold change in the mean fluorescence intensity, and the fluorescence intensity at time 0 is expressed as 1. Data are expressed as means ⫾ SD of 6 experiments. FMLP-induced change in F-actin content was unaffected by pretreatment with PD-98059 and SB-203580. AJP-Cell Physiol • VOL colocalized with F-actin, because depolymerization of the surrounding F-actin by activated ERK or p38 MAPK could cause the spatial separation of remaining F-actin from phosphorylated ERK or p38 MAPK. As shown in Fig. 7, in unstimulated control neutrophils F-actin was diffusely distributed in the cytoplasm with no or negligible staining for phosphorylated ERK and p38 MAPK. In neutrophils stimulated by TNF for 10 min, phosphorylated ERK and p38 MAPK were strongly stained in the cytoplasm, whereas F-actin was markedly decreased and residual F-actin was primarily localized at the periphery with uneven distribution. It should be noted that phosphorylated ERK and p38 MAPK are localized separately from F-actin in TNF-stimulated cells (Fig. 7). Essentially, the similar results were obtained for neutrophils stimulated by GM-CSF or G-CSF for 10 min. Phosphorylated ERK and p38 MAPK were strongly stained in the cytoplasm in GM-CSFstimulated cells, and both molecules were localized separately from F-actin. In G-CSF-stimulated cells, phosphorylated ERK was strongly stained in the cytoplasm with negligible staining for phosphorylated p38 MAPK, and it was localized separately from F-actin (Fig. 7). In neutrophils stimulated by FMLP for 45 s, increased F-actin was primarily localized at the periphery, and phosphorylated ERK and p38 MAPK were primarily stained in the central region and were localized separately from F-actin (Fig. 7). At 10 min after stimulation with FMLP, F-actin was primarily localized in the polarized portion, and phosphorylated ERK was still localized separately from F- 286 • JANUARY 2004 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 Fig. 5. Effect of FMLP on F-actin content in cytokine-pretreated neutrophils. Neutrophils were stimulated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for 10 min, G-CSF (50 ng/ml) for 20 min, or FMLP (10⫺7 M) for 45 s at 37°C. When required, TNF-, GM-CSF-, or G-CSF-stimulated cells were further challenged with FMLP (10⫺7 M) for 45 s. F-actin content was measured by flow cytometry. Results shown are representative of 4 independent experiments. C60 ACTIN REORGANIZATION AND MAP KINASES Fig. 6. Effect of cytokine pretreatment on FMLP-induced polarization and actin polymerization in neutrophils. Neutrophils were pretreated with TNF (100 U/ml) or GM-CSF (5 ng/ml) for 10 min, or G-CSF (50 ng/ml) for 20 min and thereafter stimulated with FMLP (10⫺7 M) for 10 min at 37°C. F-actin distribution and morphological changes were analyzed with confocal microscopy using FITC-phalloidin. The results shown are representative of 4 independent experiments. AJP-Cell Physiol • VOL of p38 MAPK but not MKK3/6. Then, the effects of PD-98059 and SB-203580 on p38 MAPK kinase activity were analyzed using immunoprecipitated p38 MAPK obtained from TNFstimulated neutrophils. Consistent with increased phosphorylation of p38 MAPK, the kinase activity of p38 MAPK was increased by stimulation with TNF when the kinase activity was determined using ATF-2 as a substrate (Fig. 9C). The kinase activity of p38 MAPK was markedly inhibited by SB-203580 (1 and 10 ␮M) but unaffected by PD-98059 (50 ␮M). SB-203580 (1 and 10 ␮M) and PD-98059 (50 ␮M) in combination caused essentially the same effect as SB-203580 (1 and 10 ␮M) alone (Fig. 9C). These findings indicate that the concentrations of PD-98059 and SB-203580 used in the present experiments are sufficient for obtaining significant inhibition of ERK and/or p38 MAPK activity and that PD98059 and SB-203580 may inhibit cytokine-induced actin depolymerization and morphological changes through inhibiting the distinct MAPK subtype cascades. DISCUSSION The present experiments show that stimulation of human neutrophils with cytokines (TNF, GM-CSF, and G-CSF) results in an overall decrease in F-actin content (actin depolymerization) with concomitant morphological changes. Actin depolymerization induced by these cytokines is detected at 10–20 min after stimulation and is apparently preceded by activation of ERK and p38 MAPK, which is detectable within 3 min after stimulation with these cytokines (31). In addition, cytokine-induced actin depolymerization and morphological changes were inhibited by PD-98059 and SB-203580 in a cytokine-specific manner. The cell responses induced by TNF and GM-CSF, which activate ERK and p38 MAPK, were partially inhibited by either PD-98059 or SB-203580 and were almost completely abolished by these inhibitors in combination. On the other hand, the cell responses induced by G-CSF, which primarily activates ERK, was almost completely abolished by PD-98059 but not inhibited by SB-203580. Furthermore, phosphorylated ERK and p38 MAPK are localized 286 • JANUARY 2004 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 actin. Staining for phosphorylated p38 MAPK became much weaker at this time point. These findings obtained with staining for phosphorylated ERK and p38 MAPK in control and stimulated neutrophils were consistent with those obtained with immunoblotting for these molecules (see Fig. 9) (data not shown for FMLP-stimulated cells) (31). The studies with confocal microscopy reveal that phosphorylated ERK and p38 MAPK are localized separately from F-actin in neutrophils stimulated by FMLP as well as cytokines. Cofilin was dephosphorylated by stimulation with FMLP but not with cytokines. Cofilin is a major actin-depolymerizing protein, being active in the dephosphorylated form (4). To explore the possible participation of cofilin in cytokine-induced depolymerization in neutrophils, the level of phosphorylated cofilin was analyzed using antibody against Ser3-phosphorylated cofilin. As shown in Fig. 8, cofilin was rapidly (within 45 s) dephosphorylated by stimulation with FMLP, in agreement with the previous report (11). The increased dephosphorylation of cofilin was sustained for at least 10 min after stimulation with FMLP. On the other hand, the level of phosphorylated cofilin was unaltered by stimulation with TNF, GM-CSF or G-CSF for 5–20 min (Fig. 8). These findings indicate that cytokine-induced actin depolymerization is unlikely to be ascribed to increased level of dephosphorylated cofilin. Effects of PD-98059 and SB-203580 on the activation of ERK and activity of p38 MAPK. To determine the specificity of the effects of PD-98059 and SB-203580 on cytokine-induced actin depolymerization and morphological changes in neutrophils, the effects of these inhibitors on activation of ERK and activity of p38 MAPK were analyzed. As shown in Fig. 9B, TNF-, GM-CSF-, or G-CSF-induced phosphorylation of ERK was markedly inhibited by PD-98059 (50 ␮M) but unaffected by SB-203580 (10 ␮M). PD-98059 (50 ␮M) and SB-203580 (10 ␮M) in combination caused essentially the same effect as PD-98059 (50 ␮M) alone. On the other hand, TNF- or GMCSF-induced phosphorylation of p38 MAPK was neither affected by PD-98059 (50 ␮M) nor SB-203580 (10 ␮M) as expected (data not shown), because SB-203580 is an inhibitor ACTIN REORGANIZATION AND MAP KINASES C61 separately from F-actin in neutrophils stimulated by these cytokines. These findings indicate that TNF-, GM-CSF-, and G-CSF-induced actin depolymerization and morphological changes may be mediated through activation of ERK and/or p38 MAPK. The cell responses induced by these cytokines show remarkable contrast to the responses provoked by a chemotactic peptide FMLP, which stimulates an overall increase in F-actin content (actin polymerization) with concomitant morphological changes (13, 28), and FMLP-induced responses are not affected by PD-98059 nor SB-203580 (9). It should be noted that phosphorylated ERK and p38 MAPK are also localized separately from F-actin even in neutrophils AJP-Cell Physiol • VOL stimulated by FMLP, suggesting that activation of ERK and p38 MAPK is associated with actin depolymerization even in neutrophils stimulated by FMLP. On the other hand, a “falling” phase of FMLP-induced actin polymerization was unaffected by MAPK inhibitors, suggesting that this phase is primarily regulated by other molecules than MAPKs. The present experiments also show that morphological changes are accompanied with actin depolymerization (an overall decrease in F-actin content), as well as polymerization. Accumulated evidence indicates that the MAPK cascades play an important role in various neutrophil functions. It has been demonstrated that ERK is involved in cytokine (TNF and 286 • JANUARY 2004 • www.ajpcell.org Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 Fig. 7. Phosphorylated ERK and p38 MAPK are localized separately from F-actin. Neutrophils were stimulated with TNF (100 U/ml), GM-CSF (5 ng/ml), or G-CSF (50 ng/ml) for 10 min or with FMLP (10⫺7 M) for 45 s or 10 min at 37°C. Localization of phosphorylated ERK (red color in upper panel for each set) and phosphorylated p38 MAPK (red color in lower panel for each set) was analyzed with indirect immunofluorescence using phospho-ERK1/2 or phospho-p38 MAPK antibody and Alexa Fluor 568-labeled goat anti-rabbit IgG antibody. F-actin distribution (green color for each set) was analyzed using FITC-phalloidin. Left panels: double staining for F-actin and phosphorylated ERK or p38 MAPK. Right panels: Nomarski images. Control cells were incubated for 10 min at 37°C in the absence of any stimuli. Control cells in B were stained with phospho-ERK1/2 (upper panels) or phospho-p38 MAPK (lower panels) antibody, whereas control cells in A were stained with isotype-matched control antibody. Results shown are representative of 4 independent experiments. C62 ACTIN REORGANIZATION AND MAP KINASES this possible adhesive interaction, the reactions were performed in FCS-precoated tubes in the present experiments. Possible involvement of MAPKs in actin reorganization has been reported in several cell types. However, the precise mechanisms by which MAPKs regulate actin reorganization remain to be determined. In endothelial cells, p38 MAPK is Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on May 7, 2017 Fig. 8. Cofilin is dephosphorylated by stimulation with FMLP but not with cytokines. Neutrophils were stimulated with TNF (100 U/ml), GM-CSF (5 ng/ml), G-CSF (50 ng/ml), or FMLP (10⫺7 M) for indicated times at 37°C. Western blotting was performed using antibodies against phosphorylated and nonphosphorylated forms of cofilin. The ratio of phosphorylated cofilin to total cofilin band intensity was calculated, and the ratio at time 0 is expressed as 1 (lower panel). Results shown are representative of 3 independent experiments. GM-CSF)-induced O⫺ 2 release and adherence, Fc␥ receptor (Fc␥R) IIa-mediated phagocytosis and activation of myosin light chain kinase, and Fc␥R-mediated actin polymerization (7, 9, 21, 30, 31, 32). On the other hand, p38 MAPK is reported to be involved in chemotaxis, LPS-induced activation of NF-␬B and synthesis of TNF, Fc␥RIIIb-mediated H2O2 production, TGF-␤1-induced actin polymerization, and cytokine (TNF, GM-CSF, and IL-1␤)-induced O⫺ 2 release, adherence, and upregulation of ␤2 integrin (7, 8, 10, 23, 24, 30, 31, 32, 35). The results presented here show that both ERK and p38 MAPK play a critical role in cytokine-induced actin depolymerization and morphological changes in human neutrophils. The physiological role of sustained actin depolymerization induced by cytokines remains to be determined. One possible role is that sustained strong actin depolymerization might inhibit neutrophil chemotaxis by preventing coordinated polymerization, depolymerization, and redistribution of actin cytoskeleton, which is crucial for directed cell motility (6, 13, 28, 29, 38). In fact, FMLP-induced polarization was prevented by cytokine pretreatment, and the extent of FMLP-induced actin polymerization in cytokine-pretreated neutrophils was less than that in control cells unexposed to cytokines. Consistent with this is the findings that neutrophil chemotaxis is impaired by preexposure of cells with TNF, GM-CSF, or G-CSF (6, 13, 17, 28, 38). It is conceivable that cytokines may partly contribute to accumulation of neutrophils to the inflammatory sites by preventing neutrophils from leaving there, where a lot of cytokines are produced. It has been reported that TNF induces a slight increase in F-actin content in human neutrophils (5), in contrast to our present experiments. The reason for this discrepancy is unknown. One possible reason is that strong adhesive interaction between TNF-stimulated neutrophils and plastic surface might affect actin reorganization. To prevent AJP-Cell Physiol • VOL Fig. 9. Effects of PD-98059 and SB-203580 on activation of ERK and activity of p38 MAPK. A: neutrophils were stimulated with TNF (100 U/ml), GM-CSF (5 ng/ml), or G-CSF (50 ng/ml) for 10 min at 37°C. Western blotting was performed using antibodies against phosphorylated (upper and middle panels) and nonphosphorylated (lower panel) forms of ERK1/2 and p38 MAPK. B: neutrophils were pretreated with PD-98059 (PD, 50 ␮M) and/or SB-203580 (SB, 10 ␮M) for 20 min at 37°C and thereafter stimulated with TNF (100 U/ml), GM-CSF (5 ng/ml), or G-CSF (50 ng/ml) for 10 min at 37°C. Western blotting was performed using antibodies against phosphorylated (upper panel) and nonphosphorylated (lower panel) forms of ERK1/2. C: neutrophils were stimulated with TNF (100 U/ml) for 10 min at 37°C. Phosphorylated p38 MAPK was immunoprecipitated with antibody against the phosphorylated form of p38 MAPK. In vitro kinase assay was then performed using GSTATF-2 fusion protein as a substrate. When required, PD-98059 (PD, 50 ␮M) and/or SB-203580 (SB, 1 or 10 ␮M) were added to the reaction mixture. Phosphorylation of ATF-2 was determined by immunoblotting using antibody against phosphorylated ATF-2. The cell lysates equivalent to 6 ⫻ 106 cells were loaded onto each lane. Although two bands were detected by this method, an upper band was identified as phosphorylated ATF-2 on a basis of molecular weight. Results shown are representative of 3 independent experiments. 286 • JANUARY 2004 • www.ajpcell.org ACTIN REORGANIZATION AND MAP KINASES AJP-Cell Physiol • VOL apparent after a lag time of 5–10 min. By contrast, cofilin was rapidly dephosphorylated by FMLP stimulation, which might partly contribute to actin depolymerization (a “falling” phase of actin polymerization) after rapid actin polymerization in response to FMLP. It is also possible that other molecules such as WASP (Wiskott-Aldrich syndrome protein) may be involved in actin depolymerization in neutrophils stimulated by cytokines or FMLP (34). Although the precise mechanisms for cytokine-induced actin depolymerization and morphological changes in human neutrophils remain to be elucidated, the present experiments demonstrate that ERK and p38 MAPK play a critical role in these responses, as well as cytokineinduced O⫺ 2 release and adherence. GRANTS This work was supported by Grant-in-Aid for Scientific Research, Japan 13770467. REFERENCES 1. Alessi DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489–27494, 1995. 2. Alonso-Lebrero JL, Serrador JM, Dominguez-Jimenez C, Barrerio O, Luque A, del Pozo MA, Snapp K, Kansas G, Schwartz-Albeiz R, Furthmayr H, Lozano F, and Sanchez-Madrid F. Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells. Blood 95: 2413–2419, 2000. 3. 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