Independent Mechanisms Chemotaxis and Exocytosis through Two ...

The Journal of Immunology

Heat Shock Protein 27 Regulates Neutrophil Chemotaxis and Exocytosis through Two Independent Mechanisms1 Neelakshi R. Jog,* Venkatakrishna R. Jala,§ Richard A. Ward,† Madhavi J. Rane,† Bodduluri Haribabu,*§ and Kenneth R. McLeish2†‡¶ The targets of the p38 MAPK pathway responsible for regulation of neutrophil chemotaxis and exocytosis are unknown. One target of this pathway is the actin-binding protein, heat shock protein 27 (Hsp27). Therefore, we tested the hypothesis that Hsp27 mediates p38 MAPK-dependent chemotaxis and exocytosis in human neutrophils through regulation of actin reorganization. Sequestration of Hsp27 by introduction of anti-Hsp27 Ab, but not an isotype Ab, inhibited fMLP-stimulated chemotaxis, increased cortical F-actin in the absence of fMLP stimulation, and inhibited fMLP-stimulated exocytosis. Pretreatment with latrunculin A prevented actin reorganization and the changes in fMLP-stimulated exocytosis induced by Hsp27 sequestration. To determine the role of Hsp27 phosphorylation, wild-type, phosphorylation-resistant, or phosphorylation-mimicking recombinant Hsp27 was introduced into neutrophils by electroporation. The phosphorylation-resistant mutant significantly reduced migration toward fMLP, whereas none of the Hsp27 proteins affected fMLP-stimulated or TNF-␣-stimulated exocytosis or actin polymerization. Endogenous Hsp27 colocalized with F-actin in unstimulated and fMLP-stimulated neutrophils, whereas phosphorylated Hsp27 showed cytosolic localization in addition to colocalization with F-actin. Our results suggest that Hsp27 regulates neutrophil chemotaxis and exocytosis in an actin-dependent, phosphorylation-independent manner. Phosphorylation of Hsp27 regulates chemotaxis, but not exocytosis, independent of regulation of actin reorganization. The Journal of Immunology, 2007, 178: 2421–2428.

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eutrophils are the primary cellular component of innate immunity. To participate in infection and inflammation, neutrophils undergo a series of highly coordinated responses, including adhesion to vascular endothelium, chemotaxis, granule exocytosis, and production of reactive oxygen metabolites. A number of reports indicate that one of the MAPKs, p38 MAPK, is required for many of these responses, including chemotaxis and exocytosis (1– 8). Additionally, inhibition of p38 MAPK activation in vivo blocks neutrophil accumulation at sites of inflammation (9, 10). We reported previously that MAPK-activated protein kinase-2 (MAPKAPK2),3 a serine-threonine kinase activated by p38 MAPK, mediates p38 MAPK-dependent chemotaxis and exocytosis (7). The molecular basis for MAPKAPK2 regulation of these neutrophil functions has not been established. Stimulated neutrophils exhibit dynamic assembly, disassembly, and reorganization of the actin cytoskeleton (11, 12) and actin reorganization is necessary for both chemotaxis and exocytosis (13–17). The first step in chemotaxis is an increase in actin poly-

*Department of Microbiology and Immunology, †Department of Medicine, ‡Department of Biochemistry and Molecular Biology, and §James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202; and ¶ Veterans Affairs Medical Center, Louisville, KY 40206 Received for publication March 27, 2006. Accepted for publication November 28, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the National Institutes of Health (DK62389, to R.A.W. and K.R.M.), the Department of Veterans Affairs (Merit Review, to K.R.M.), an American Heart Association Scientist Development Grant (0335278N, to M.J.R.), and the Ohio Valley affiliate of the American Heart Association (Predoctoral Fellowship Grant, to N.R.J.). 2 Address correspondence and reprint requests to Dr. Kenneth R. McLeish, Baxter I Research Building, 570 South Preston Street, Louisville, KY 40202. E-mail address: [email protected] 3 Abbreviations used in this paper: MAPKAPK2, MAPK-activated protein kinase-2; Hsp27, heat shock protein 27.

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merization at the leading edge of the cell which pushes the leading edge forward by protrusion of two F-actin-rich structures, lamellipodia and filopodia. Sequential coordinated polymerization and depolymerization of the actin cytoskeleton is necessary to maintain directed migration (18, 19). Previous studies in other cell types identified actin reorganization as a regulator of exocytosis. In chromaffin cells, the transport of cytoplasmic secretory granules to the plasma membrane depends upon reorganization of the cortical actin barrier underlying the plasma membrane (15, 20, 21). In human neutrophils and other cells, MAPKAPK2 phosphorylates heat shock protein 27 (Hsp27) on Ser15, Ser78, and Ser82 residues (22, 23). Hsp27 exists in several forms, including oligomers, tetramers, dimers, and monomers. Monomeric nonphosphorylated Hsp27 binds to the barbed end of actin filaments in vitro, preventing the addition of globular actin (24, 25). Phosphorylation of actin-associated Hsp27 results in its release from barbed ends, allowing actin polymerization to proceed (25, 26), and phosphorylation of Hsp27 by the p38 MAPK pathway is required for actin reorganization (27–32). In contrast, Zhu et al. (33) reported that phosphorylation of Hsp27 induced its translocation from the cytosol to the actin cytoskeleton in platelets. Pichon et al. (34) reported that phosphorylated Hsp27 localized at the base of lamellipodia of aortic smooth muscle cells and was excluded from the leading edge. That report and other studies suggested that phosphorylation of Hsp27 resulted in stabilization of the actin cytoskeleton (34 –36). Taken together, these studies suggest that MAPKAPK2 phosphorylation of Hsp27 regulates actin reorganization, although the mechanism by which this regulation occurs remains to be determined. The present study was conducted to test the hypothesis that phosphorylation of Hsp27 by the p38 MAPK pathway regulates chemotaxis and exocytosis in human neutrophils by regulating actin reorganization. Hsp27 activity was disrupted by the introduction of anti-Hsp27 Ab or mutant forms of Hsp27 into isolated human neutrophils. The results suggest that Hsp27 regulates these

2422 functions by both phosphorylation-dependent and phosphorylation-independent mechanisms.

Materials and Methods Neutrophil isolation Neutrophils were isolated from healthy donors using plasma-Percoll gradients, as previously described (37). After isolation, neutrophils were washed and resuspended in LPS-free Krebs-Ringer phosphate buffer (pH 7.2) containing 0.2% dextrose (Krebs). Microscopic evaluation of isolated cells showed that ⬎97% of cells were neutrophils. Trypan-blue exclusion indicated that ⬎97% cells were viable. The Human Studies Committee of the University of Louisville (Louisville, KY) approved the use of human donors.

Introduction of Abs into neutrophils Anti-Hsp27 Ab was introduced into neutrophils via an endocytic pathway, as described previously for neuronal cells and neutrophils (38, 39). Briefly, 8 ⫻ 106 neutrophils/100 ␮l of RPMI 1640 were incubated with murine monoclonal anti-Hsp27 Ab (2 ␮g) (Stressgen Biotechnology) or isotype control Ab (2 ␮g; Santa Cruz Biotechnology) at 37°C and 5% CO2 for 2 h. Neutrophils were washed three times and then resuspended in 1 ml of Krebs-Ringer phosphate buffer supplemented with 1.2 mM Mg2⫹ and 0.5 mM Ca2⫹ (Krebs⫹). The entry of Abs into ⬎90% of neutrophils was confirmed by confocal microscopy following incubation with fluoresceinlabeled Abs (data not shown).

Expression and purification of recombinant proteins Hsp27-wt, Hsp27-3A, and Hsp27-3D in pcDNA 3.1 were obtained from Dr. R. Benndorf (University of Michigan, Ann Arbor, MI). These genes were cloned into pRSET B using BamHI and XhoI restriction sites. The plasmids were transformed into BL-21-pLysS (Invitrogen Life Technologies) competent cells. The cultures were grown overnight and recombinant proteins were purified by the Ni-NTA Purification System (Invitrogen Life Technologies).

Introduction of recombinant proteins into neutrophils Neutrophils were resuspended in electroporation buffer (140 mM KCl, 10 mM HEPES, 10 mM D glucose, 1 mM MgCl2, 0.193 mM CaCl2, and 1 mM EGTA (pH 7.2)) containing recombinant protein and exposed to one pulse of 50 ␮F capacitance at 800V (Gene Pulser II; Bio-Rad). To determine the efficiency of protein transduction, recombinant proteins were labeled with fluorescein using normal human serum-fluorescein (Pierce Biotechnology) before electroporation. Fluorescein-labeled proteins were introduced into neutrophils as described above and cells were analyzed by confocal microscopy (Zeiss Axiovert 100 M microscope using LSM510 software).

Chemotaxis Chemotaxis was assayed using transwell chambers, as described previously (40). Neutrophils were resuspended at 1 ⫻ 106 cells/ml in KRPB, and added to the upper chamber of polypropylene transwells. fMLP (3 ⫻ 10⫺7 M final concentration) was added to the lower chambers of the transwells. Transwells were incubated at 37°C with 5% CO2 for 30 min. Following incubation, the polyester membranes were fixed and stained with H&E and dried at room temperature overnight. Membranes were cut and fixed on glass slides, keeping the bottom surface upright, and viewed by light microscopy using ⫻100 magnification. Cells within the scale that entered the membrane or passed through the membrane were counted. Results are expressed as the mean ⫾ SEM number of cells migrating across a 6.5-mmdiameter circle of the membrane.

Exocytosis Exocytosis of secretory vesicles and specific granules was assayed by measuring plasma membrane expression of CD35 and CD66b, respectively, as previously described (6). Briefly, neutrophils were suspended in KRPB⫹, stimulated with fMLP for 3 min, and incubated at 4°C for 30 min with FITC-conjugated monoclonal anti-CD35 (BD Pharmingen) or FITC-conjugated monoclonal anti-CD66b (Accurate Chemical). FITC-conjugated mouse IgG1 (BD Pharmingen) was used as an isotype control. To examine the effect of disrupting actin on exocytosis, cells were incubated with 1 ⫻ 10⫺6 M latrunculin A (Molecular Probes) for 1 h at 37°C before stimulation. Fluorescence intensity was measured by flow cytometry (Coulter Epics XL Flow Cytometer). Exocytosis of gelatinase granules was determined using an ELISA for matrix metalloproteinase 9 (R&D Systems) according to the manufacturer’s instructions.

Hsp27 REGULATES CHEMOTAXIS AND EXOCYTOSIS Determination of F-actin content in neutrophils Neutrophils were fixed and permeabilized with 3.7% paraformaldehyde and 2% saponin, respectively. Cells were stained for F-actin using 1.5 U of fluorescein-phalloidin (Molecular Probes) for 30 min at 4°C. The fluorescence intensity was measured by flow cytometry (Coulter Epics XL Flow Cytometer).

Confocal microscopy Following stimulation, neutrophils were stained for F-actin, as described above. For colocalization studies, after fixation and permeabilization, cells were blocked with 2% normal goat serum and 2% BSA in PBS (blocking buffer), and incubated with 1 ␮g anti-Hsp27 mouse mAb (Stressgen Biotechnology) or anti-phospho-Hsp27 Ser82 rabbit polyclonal Ab (Cell Signaling) in blocking buffer overnight at 4°C. Cells were washed twice with PBS and incubated with rhodamine-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Molecular Probes) and 1.5 U of fluorescein-phalloidin for 1 h at 4°C. Cells were washed twice and imaging was performed with a Zeiss Axiovert 100 M microscope using LSM510 software.

Statistical analysis ANOVA was performed using GraphPad Instat (GraphPad Software). Differences among groups were determined using the Tukey-Kramer post hoc test; statistical significance was defined as p ⬍ 0.05.

Results Hsp27 sequestration enhances actin reorganization To determine whether Hsp27 has a role in neutrophil chemotaxis and exocytosis, Hsp27 was sequestered by introducing an antiHsp27 mAb (38, 39). A previous study showed that introduction of higher concentrations anti-Hsp27 Ab into neutrophils removed Hsp27 from the Akt-signaling complex, blocked Akt activation, and induced apoptosis (39). The lower concentration of Ab (2 ␮g/ 8 ⫻ 106 cells in 100 ␮l) used in the present study, however, did not induce apoptosis (data not shown). Additionally, viability of cell signaling was maintained, as fMLP-stimulated p38 MAPK phosphorylation did not differ among control cells and cells incubated with anti-Hsp27 Ab or isotype Ab (data not shown). The ability of introduction of anti-Hsp27 Ab to sequester Hsp27, alter Hsp27 phosphorylation, and alter actin polymerization in neutrophils was determined by confocal microscopy. Fig. 1A shows diffuse cytosolic distribution of F-actin and Hsp27 and minimal staining for phosphorylated Hsp27 in cells incubated with isotype control Ab. Upon stimulation with 1 ⫻ 10⫺7 M fMLP for 1 min, cortical polymerization of F-actin was observed. The majority of Hsp27 redistributed with the F-actin, and the amount of phosphorylated Hsp27 increased dramatically, and both colocalized with F-actin and appeared in the cytosol. Introduction of anti-Hsp27 Ab induced cortical polymerization of F-actin that was not further enhanced on subsequent stimulation with fMLP (Fig. 1B). The amount of Hsp27 and phosphorylated Hsp27 detected was markedly reduced compared with cells exposed to isotype Ab. Confocal microscopy demonstrated that internalization of anti-Hsp27 Ab and associated actin polymerization occurred in ⬎90% of neutrophils under the experimental conditions used (data not shown). These results indicate that introduction of a concentration of antiHsp27 that does not induce apoptosis resulted in sequestration of Hsp27, prevented phosphorylation of Hsp27 upon fMLP stimulation, and increased actin polymerization. Hsp27 sequestration inhibits fMLP-stimulated chemotaxis and exocytosis As chemotaxis requires coordinated reorganization of the actin cytoskeleton, we next examined the ability of fMLP to stimulate chemotaxis following sequestration of Hsp27. The data in Fig. 2 show that pretreatment with anti-Hsp27 Ab, but not pretreatment with isotype-matched Ab, significantly reduced the number of cells migrating across the transwell chamber under both stimulated


2423

FIGURE 2. Hsp27 sequestration inhibits fMLP stimulated chemotaxis. Anti-Hsp27 and isotype control antisera were introduced into neutrophils as described in Materials and Methods. After incubation with Abs for 2 h, cells were washed with KRBP⫹ and chemotaxis toward fMLP was measured as the number of cells migrating across transwells. Introduction of 2 ␮g of anti-Hsp27 Ab, but not 2 ␮g of isotype control Ab, significantly decreased migration both in the presence and absence of fMLP. Pretreatment of cells with 3 ␮M SB203580 for 1 h partially inhibited migration toward fMLP. ⴱ, p ⬍ 0.001 when compared with migration under basal conditions; #, p ⬍ 0.05 when compared with basal migration of control cells; §, p ⬍ 0.01 when compared with migration toward fMLP in control cells. The data represent mean ⫾ SEM of 10 independent experiments.

FIGURE 1. Hsp27 sequestration enhances actin reorganization. AntiHsp27 and isotype control antisera were introduced into neutrophils as described in Materials and Methods. Following incubation with Abs, cells were washed with KRBP⫹, and incubated with or without 1 ⫻ 10⫺7 M fMLP for 60 s at 37°C. Cells were fixed and permeabilized, stained for F-actin using fluorescein-phalloidin, and stained with rabbit anti-Hsp27 or rabbit anti-phosphorylated Hsp27 (phosHsp27) using rhodamine-labeled goat anti-rabbit Ab as the secondary Ab. Cells were imaged at room temperature with a Zeiss Axiovert 100 M microscope with a Zeiss PlanNeofluar ⫻100/1.3 oil immersion lens, using LSM510 (version 3.2) software. In cells incubated with isotype control antisera (A), F-actin and Hsp27 showed a diffuse cytoplasmic distribution, while low levels of phosphorylated Hsp27 were observed. Stimulation with fMLP resulted in enhanced cortical F-actin, to which Hsp27 colocalized. A marked increase in phosphorylated Hsp27 was observed in cytoplasm and colocalized with cortical actin. Cells incubated with anti-Hsp27 Ab demonstrated increased cortical F-actin in the absence of fMLP (B), which was not altered by subsequent addition of fMLP. The amount of Hsp27 and phosphorylated Hsp27 observed were markedly reduced in both basal and fMLP-stimulated cells.

and unstimulated conditions. Pharmacological inhibition of p38 MAPK by 3 ␮M SB203580, a concentration that we have previously reported blocks Hsp27 phosphorylation (7), significantly reduced fMLP-stimulated chemotaxis. Neutrophil granule exocytosis is enhanced by disruption of the actin cytoskeleton (41– 44). To determine whether exocytosis was also dependent on Hsp27, cells were pretreated with anti-Hsp27 Ab before stimulation with 1 ⫻ 10⫺7 M fMLP for 3 min. Secretory vesicle exocytosis was measured as increased plasma membrane expression of CD35 and specific granule exocytosis was measured as increased plasma membrane expression of CD66b. Pretreatment with anti-Hsp27 Ab inhibited plasma membrane expression of CD35 in both unstimulated and fMLP-stimulated cells, while isotype control had no effect (Fig. 3A). Pretreatment with SB203580 did not inhibit fMLP-stimulated secretory vesicle exocytosis. In contrast to CD35, pretreatment with anti-Hsp27 Ab stimulated increased CD66b expression in absence of fMLP and subsequent

stimulation with fMLP failed to further increase CD66b expression (Fig. 3B). Introduction of isotype control Ab and pretreatment with SB203580 failed to alter basal or fMLP-stimulated CD66b expression.

FIGURE 3. Hsp27 sequestration alters fMLP-stimulated exocytosis. Following introduction of anti-Hsp27 Ab or isotype Ab, neutrophils were stimulated with 1 ⫻ 10⫺7 M fMLP for 3 min at 37°C. Exocytosis of secretory vesicles (A) or specific granules (B) was measured as mean fluorescence intensity of CD35 or CD66b expression, respectively, by flow cytometry. Introduction of anti-Hsp27 Ab inhibited secretory vesicle exocytosis in the presence or absence of fMLP, enhanced specific granule exocytosis in the absence of fMLP, and inhibited fMLP-stimulated specific granule exocytosis. Inhibition of p38 MAPK by pretreatment with 3 ␮M SB203580 for 1 h had no effect on exocytosis of either granule subtype. ⴱ, p ⬍ 0.001 compared with basal expression; #, p ⬍ 0.05 compared with basal expression in control cells; §, p ⬍ 0.001 when compared with fMLPstimulated expression in control cells. The data are presented as mean ⫾ SEM of four to five independent experiments.

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Hsp27 REGULATES CHEMOTAXIS AND EXOCYTOSIS

FIGURE 4. Disruption of actin cytoskeleton prevents the effect of Hsp27 sequestration on actin reorganization. To demonstrate the effect of latrunculin A on actin polymerization, neutrophils were incubated with 1 ⫻ 10⫺6 M latrunculin A for the last 1 h of incubation with anti-Hsp27 Ab or isotype-matched Ab. Cells were stained for F-actin with fluoresceinlabeled phalloidin and imaged by confocal microscopy. Treatment with latrunculin A prevented actin polymerization stimulated by anti-Hsp27 Ab and by fMLP.

Disruption of actin cytoskeleton prevents the effect of Hsp27 sequestration on actin reorganization and exocytosis To determine whether the effect of Hsp27 sequestration on exocytosis was due to enhanced actin reorganization, the actin cytoskeleton was disrupted by pretreatment with 1 ␮M latrunculin A, in conjunction with incubation with anti-Hsp27 Ab or isotype control Ab, before fMLP stimulation. Latrunculin A prevents actin polymerization by binding to globular actin monomers, making them unavailable to actin filaments (45, 46). Staining for F-actin showed that increased cortical F-actin induced by introduction of anti-Hsp27 Ab was inhibited by preincubation with latrunculin A (Fig. 4). Pretreatment with latrunculin A had no effect on introduction of anti-Hsp27 Ab into neutrophils (data not shown). Fig. 5A confirms that incubation with anti-Hsp27 Ab, but not isotype control Ab, stimulated specific granule exocytosis, while preventing subsequent fMLP-stimulated exocytosis. Pretreatment with latrunculin A enhanced fMLP-stimulated CD66b expression in control and isotype Ab-treated cells. Latrunculin A pretreatment of cells incubated with anti-Hsp27 Ab reduced basal exocytosis to the control levels and partially restored fMLP-stimulated exocytosis. Similar studies of CD35 expression could not be performed, as latrunculin A pretreatment resulted in a biphasic pattern of expression upon fMLP stimulation due to endocytosis of CD35 (data not shown). Therefore, the effect of Hsp27 sequestration, in the presence or absence of latrunculin A, on gelatinase granule exocytosis was determined. Fig. 5B shows that fMLP stimulated gelatinase release in control cells, cells incubated with latrunculin A alone, and cells incubated with isotype Ab in presence or absence of latrunculin A. Introduction of anti-Hsp27 Ab, but not isotype control Ab, stimulated gelatinase release and prevented fMLP-stimulated release. Pretreatment with latrunculin A restored both the basal and stimulated gelatinase release to normal. Taken together, these data indicate that Hsp27 sequestration stimulated exocytosis of gelatinase and specific granules by a mechanism dependent on increased actin polymerization and Hsp27 sequestration blocked fMLP-stimulated exocytosis.

FIGURE 5. Disruption of the actin cytoskeleton prevents the effect of Hsp27 sequestration on exocytosis. To determine whether the effect of Hsp27 sequestration was mediated by enhanced actin polymerization, neutrophils were incubated with anti-Hsp27 Ab or isotype Ab, with or without 1 ⫻ 10⫺6 M latrunculin A. Specific granule exocytosis was determined as mean fluorescence intensity of CD66b expression by flow cytometry, and gelatinase granule exocytosis was measured as gelatinase released using an ELISA. Introduction of anti-Hsp27 Ab increased specific granule (A) and gelatinase granule (B) exocytosis in absence of fMLP treatment, and this increase was inhibited by latrunculin A pretreatment. Latrunculin A pretreatment restored the ability of fMLP to stimulate exocytosis of both granule subtypes. ⴱ, p ⬍ 0.001 when compared with basal expression of CD66b or gelatinase release; #, p ⬍ 0.01 when compared with basal CD66b expression or gelatinase release in control cells; §, p ⬍ 0.001 when compared with fMLP-stimulated CD66b expression in control cells. The data are represented as mean ⫾ SEM of three to five independent experiments.

Hsp27 phosphorylation is required for neutrophil chemotaxis, but not exocytosis The Hsp27 sequestration experiments suggest that Hsp27 regulation of actin reorganization participates in both neutrophil chemotaxis and exocytosis. To examine whether phosphorylation of Hsp27 is required for regulation of these functions, recombinant wild-type Hsp27 (Hsp27-wt), a phosphorylation resistant mutant of Hsp27, where all three serine residues phosphorylated by MAPKAPK2 were mutated to alanine residues (Hsp27-3A), and a phosphorylation mimicking mutant of Hsp27, where all three serine residues were mutated to aspartic acid residues (Hsp273D), were introduced into neutrophils by electroporation. Preliminary studies showed that electroporation led to a protein transduction efficiency of 70 – 80% and similar levels of recombinant protein introduction, as determined by confocal microscopy and flow cytometry (data not shown). Additionally, electroporation alone or in the presence of each of the three Hsp27 proteins did not alter fMLP-stimulated superoxide release (data


2425

FIGURE 6. Hsp27 phosphorylation is required for neutrophil chemotaxis. Wild-type (Hsp27-wt), phosphorylation-resistant (Hsp27-3A), or phosphorylation-mimicking (Hsp27-3D) mutants of Hsp27 were introduced into neutrophils by electroporation (EP). Chemotaxis was determined using transwell chambers as described in Materials and Methods. Hsp27-3A significantly decreased fMLP stimulated migration, while Hsp27-wt and Hsp27-3D had no effect. ⴱ, p ⬍ 0.001 when compared with migration under basal conditions; §, p ⬍ 0.001 when compared with fMLP-stimulated migration in control cells. The data are represented as mean ⫾ SEM of four independent experiments.

not shown). Neutrophils were electroporated in the presence or absence of recombinant proteins, and fMLP-stimulated chemotaxis and exocytosis were measured. The data in Fig. 6 show the effect of introducing the three Hsp27 proteins on chemotaxis. Electroporation alone did not significantly alter the ability of neutrophils to undergo directed migration. Introduction of

FIGURE 8. Hsp27 Colocalizes with F-actin upon fMLP stimulation. Neutrophils were incubated with 3 ⫻ 10⫺7 M fMLP for various times, then fixed, permeabilized, and stained for Hsp27 using an anti-Hsp27 mAb followed by rhodamine-anti-mouse Ab and for F-actin using fluorescein-labeled phalloidin. Cells were subjected to imaging at room temperature with Zeiss Axiovert 100 M microscope with a Zeiss Plan-Neofluar ⫻100/1.3 oil immersion lens, using LSM510 (version 3.2) software. The excitation wavelength was set at 488/543 and emission at band pass 505–530 and long pass 560 using an argon and HeNe laser. The right panel shows the intensity profile across the image along the selected line. F-actin formed a cortical ring within 15 s of fMLP stimulation, and Hsp27 colocalized with F-actin at all time points examined.

FIGURE 7. Hsp27 phosphorylation is not required for neutrophil exocytosis. Wild-type (Hsp27-wt), phosphorylation resistant (Hsp27-3A) or phosphorylation mimicking (Hsp27-3D) mutants of Hsp27 were introduced into cells by electroporation (EP). Following stimulation with 1 ⫻ 10⫺7 M fMLP for 3 min (A) or 10 ng/ml TNF-␣ for 10 min (B), specific granule exocytosis was measured as mean fluorescence intensity of plasma membrane expression of CD66b by flow cytometry. Introduction of Hsp27wt, Hsp27-3A, or Hsp27-3D did not alter specific granule exocytosis stimulated by either fMLP or TNF-␣. Inhibition of p38 MAPK by 3 ␮M SB203580, blocked TNF-␣ stimulated specific granule exocytosis (B). ⴱ, p ⬍ 0.001 when compared with exocytosis in absence of stimulation. §, p ⬍ 0.001 when compared with TNF-␣-stimulated exocytosis in control cells. The data are presented as mean ⫾ SEM of 16 independent experiments for fMLP stimulation and as mean ⫾ SEM of 9 independent experiments for TNF-␣ stimulation.

Hsp27-3A significantly reduced migration toward fMLP, while Hsp27-wt and Hsp27-3D had no effect on chemotaxis. Thus, the ability to phosphorylate Hsp27 is required for directed migration of human neutrophils. Electroporation, alone, induced secretory vesicle exocytosis, which did not increase further following stimulation of the cells with fMLP (data not shown). Thus, specific granule exocytosis was used to examine the effect of Hsp27 phosphorylation on exocytosis. Introduction of Hsp27-wt or either mutant form of Hsp27 had no effect on basal or fMLP-stimulated CD66b expression (Fig. 7A). The observation that inhibition of p38 MAPK activity did not alter fMLP-stimulated specific granule exocytosis (Fig. 3B) suggested, however, that fMLP-stimulated exocytosis of neutrophil granules is independent of the p38 MAPK. In contrast to fMLPstimulated exocytosis, we showed previously that p38 MAPK regulated TNF-␣-stimulated exocytosis of specific granules (6, 7). Therefore, we examined the effect of introduction of recombinant wild-type Hsp27 and its mutants on TNF-␣-stimulated exocytosis.

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Hsp27 REGULATES CHEMOTAXIS AND EXOCYTOSIS

FIGURE 10. Hsp27 phosphorylation does not regulate neutrophil actin polymerization. Neutrophils (4 ⫻ 106/ml) were stimulated with 10⫺7 M fMLP or buffer for 45 s after introducing Hsp27-wt, Hsp27-3A, or Hsp27-3D by electroporation (EP). Fixed and permeabilized cells were stained with 1.5 U of fluorescein-phalloidin and analyzed by flow cytometry. F-actin was determined as the mean fluorescence intensity (MFI). The fMLP-stimulated increase in F-actin content was not affected by electroporation or the introduction of Hsp27-wt, Hsp27-3A, and Hsp27-3D. Data are presented as mean ⫾ SEM for three experiments. ⴱ, p ⬍ 0.01 compared with the basal level of F-actin.

FIGURE 9. Effect of phosphorylation on Hsp27 colocalization with Factin. Neutrophils were stained for phospho-Hsp27 using an anti-phosphoHsp27 Ser82 Ab followed by rhodamine-anti-rabbit Ab and F-actin using fluorescein-labeled phalloidin. Cells were then visualized by confocal microscopy as described for Fig. 8. Images were analyzed using LSM 510 software and the right panel shows the intensity profile across the image along the selected line. Phosphorylated Hsp27 colocalized with F-actin at all time points examined. Beginning at 30 s after fMLP stimulation, however, phosphorylated Hsp27 was also detected in the cytoplasm independent of F-actin localization.

Stimulation with TNF-␣ resulted in a significant increase in CD66b expression in control cells, which was inhibited by SB203580 (Fig. 7B). Introduction of Hsp27-wt, Hsp27-3A, or Hsp27-3D had no effect on TNF-␣-stimulated CD66b expression. These results suggest that Hsp27 phosphorylation does not mediate p38 MAPK-dependent regulation of specific granule exocytosis. Stimulation with fMLP gives rise to a population of phosphorylated Hsp27 that does not colocalize with F-actin To determine whether stimulation with fMLP alters Hsp27 colocalization with actin, neutrophils were incubated with 1 ⫻ 10⫺7 M fMLP for varying lengths of time, stained with anti-Hsp27 Ab, followed by rhodamine-anti-mouse Ab and fluorescein-phalloidin, and imaged by confocal microscopy. The images in Fig. 8 show diffuse cytoplasmic staining of both F-actin and Hsp27 in unstimulated cells. Increased cortical F-actin was observed as early as 15 s after fMLP stimulation and this increased staining remained through the 90 s of observation. Hsp27 colocalized with F-actin at all time points examined. To determine whether phosphorylation altered the interaction between Hsp27 and F-actin, neutrophils were simultaneously stained with Ab against Hsp27 phosphorylated at Ser82 followed by rhodamine-anti-rabbit Ab and fluorescein-phalloidin. Phosphorylated Hsp27 was present in unstimulated cells and colocalized with F-actin (Fig. 9). After 15 s of fMLP stimulation, there was an increase in cortical F-actin and an

increased staining for phosphorylated Hsp27 that colocalized with F-actin (Fig. 9). At 30 s and later, however, phosphorylated Hsp27 demonstrated two populations, one that colocalized with cortical F-actin and a second cytoplasmic population that was independent of F-actin (Fig. 9). We showed previously that Akt phosphorylates Hsp27 on Ser82 (39). Thus, these studies cannot distinguish between Hsp27 phosphorylated by MAPKAPK2 or Akt. We have shown, however, that Akt activation by fMLP in human neutrophils is dependent on MAPKAPK2 phosphorylation of Ser473 on Akt (47), indicating that phosphorylation of Ser82 on Hsp27 is dependent on activation of the p38 MAPK pathway. Hsp27 phosphorylation does not regulate fMLP-stimulated actin polymerization in neutrophils It was suggested previously that phosphorylation of Hsp27 resulted in its dissociation from the barbed end of actin filaments (25). To determine whether phosphorylation of Hsp27 regulated fMLP-stimulated actin polymerization, F-actin content was determined as fluorescein-phalloidin binding by flow cytometry in unstimulated cells and cells incubated with 1 ⫻ 10⫺7 M fMLP for 45 s. Neither electroporation, alone, nor introduction of Hsp27-wt, Hsp27-3A, or Hsp27-3D into neutrophils, altered basal or fMLPstimulated actin polymerization (Fig. 10). These data suggest that MAPKAPK2 phosphorylation of Hsp27 does not regulate actin polymerization in human neutrophils, and the ability of phosphorylated Hsp27 to regulate chemotaxis is independent of actin reorganization.

Discussion The present study identified two different mechanisms by which Hsp27 regulates neutrophil chemotaxis and exocytosis. First, regulation of actin reorganization by Hsp27 in a phosphorylationindependent manner was necessary for fMLP-stimulated chemotaxis and exocytosis. Second, Hsp27 regulated chemotaxis, but not exocytosis, through a phosphorylation-dependent process that did not involve actin reorganization. Introducing an anti-Hsp27 Ab to sequester Hsp27 significantly reduced migration of unstimulated and fMLP-stimulated neutrophils. Sequestration of Hsp27 also enhanced actin polymerization, as evidenced by increased cortical F-actin in ⬎90% of unstimulated cells. This effect of Hsp27 sequestration was consistent with the proposed function of Hsp27 as an actin-capping protein (24, 25). We postulate that this loss of cell motility was due to the loss

The Journal of Immunology of dynamic actin polymerization-depolymerization necessary for protrusion of the leading edge and retraction of the uropod. The effect of Hsp27 sequestration on neutrophil granule exocytosis was more complex. Introduction of anti-Hsp27 Ab significantly inhibited basal and fMLP-stimulated exocytosis of secretory vesicles, as measured by plasma membrane CD35 expression. In contrast, sequestration of Hsp27 induced exocytosis of specific and gelatinase granules, although it inhibited further exocytosis upon fMLP-stimulation. Disrupting actin polymerization with latrunculin A restored normal basal and fMLP-stimulated exocytosis of specific and gelatinase granules. This observation suggests that alterations in basal and stimulated exocytosis induced by Hsp27 sequestration are mediated by changes in actin reorganization. One possible explanation for the gelatinase and specific granule exocytosis induced by Hsp27 sequestration could be the presence of ready release populations of these granules close to the plasma membrane, similar to that described for vesicles in adrenal chromaffin cells (21, 48, 49). Fusion of this ready release population with the plasma membrane might be induced by cortical actin polymerization (50 –53). The inability of Hsp27 sequestration to induce secretory vesicle exocytosis could be explained by the lack of a ready release population of these granules. Previous studies showed that the cortical actin network provides a barrier to stimulated exocytosis in a variety of cell types (15, 20, 21, 41– 43, 54 –56). The failure of fMLP to stimulate exocytosis in the presence of Hsp27 sequestration might be the result of the associated actin polymerization increasing the cortical actin barrier and impeding exocytosis of granules from a core population. Although separate populations of ready release and core granules have not been described in neutrophils, electron microscopy has shown the presence of some granules at the plasma membrane of unstimulated neutrophils (57, 58). Hsp27 was previously identified as a substrate of MAPKAPK2, a serine threonine kinase activated by p38 MAPK, in human neutrophils (22, 23, 59). The requirement for Hsp27 phosphorylation for normal neutrophil chemotaxis is consistent with a number of previous reports. As confirmed by the data in Fig. 2, inhibition of p38 MAPK activity impairs neutrophil chemotaxis both in vitro and in vivo (2, 3, 7, 9, 10, 60). Studies using inhibitory peptides and MAPKAPK2-deficient neutrophils have provided evidence that MAPKAPK2 mediated p38 MAPK-dependent neutrophil chemotaxis (7, 61, 62). That Hsp27 phosphorylation might mediate p38 MAPK-dependent neutrophil chemotaxis was suggested by studies showing that migration of smooth muscle cells was inhibited by overexpression of a dominant-negative mutant of p38 MAPK and a nonphosphorylatable mutant form of Hsp27 (32, 34). We introduced recombinant wild-type, a phosphorylation resistant mutant, or a phosphorylation mimicking mutant Hsp27 into neutrophils by electroporation to determine whether Hsp27 phosphorylation mediated p38 MAPK-dependent chemotaxis. Only the phosphorylation resistant mutant of Hsp27 significantly reduced migration toward fMLP. Taken together, these data suggest that Hsp27 phosphorylation by the p38 MAPK pathway regulates chemotaxis. In contrast to the effect on exocytosis, introduction of a mutant of Hsp27 resistant to MAPKAPK2 phosphorylation did not inhibit fMLP-stimulated actin polymerization in human neutrophils. Further, while immunostaining of endogenous Hsp27 showed that it colocalized with F-actin under both unstimulated and stimulated conditions, phosphorylated Hsp27 existed in two populations, one that colocalized with F-actin and another cytoplasmic population that was independent of F-actin. These data suggest that Hsp27 regulates actin reorganization independently of phosphorylation, despite the observation that phosphorylation induced dissociation

2427 of a population of Hsp27 from the actin cytoskeleton. We interpret these results to indicate that Hsp27 regulates chemotaxis by two independent mechanisms. First, Hsp27 acts to inhibit the basal rate of actin polymerization, allowing appropriate reorganization to take place following receptor-mediated stimulation. This activity is independent of Hsp27 phosphorylation. Second, Hsp27 phosphorylation regulates chemotaxis by an unidentified mechanism that is independent of actin reorganization. We reported previously that MAPKAPK2 regulated neutrophil granule exocytosis (6, 7). FMLP-stimulated exocytosis of specific granules was inhibited by Hsp27 sequestration, but not by introduction of a phosphorylation resistant mutant of Hsp27. These results suggest that phosphorylation of Hsp27 by the p38 MAPK pathway is not required for exocytosis. However, pretreatment of neutrophils with a pharmacological inhibitor of p38 MAPK also did not inhibit fMLP-stimulated exocytosis. Thus, failure to define a role for Hsp27 phosphorylation in neutrophil exocytosis may be due to the fact that fMLP-stimulated neutrophil exocytosis is independent of p38 MAPK. As we reported previously, and confirmed by the data in Fig. 7B, TNF-␣-stimulated neutrophil exocytosis is regulated by p38 MAPK (6). However, introduction of a phosphorylation-resistant mutant of Hsp27 had no effect on TNF␣-stimulated exocytosis, supporting the conclusion that Hsp27 is not a target of phosphorylation by the p38 MAPK-signaling pathway that regulates neutrophil exocytosis. In conclusion, our data suggest that Hsp27 has a dual role in neutrophil functional responses. First, the ability of nonphosphorylated Hsp27 to act as an actin-capping protein inhibits uncontrolled actin reorganization and Hsp27 regulation of actin reorganization is required for normal neutrophil chemotaxis and exocytosis. Second, phosphorylated Hsp27 mediates p38 MAPK-dependent regulation of chemotaxis by an actin-independent mechanism, the molecular details of which remain to be determined. Finally, the targets of the p38 MAPK pathway that control exocytosis remain to be identified.

Disclosures The authors have no financial conflict of interest.

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