DAPK2 positively regulates motility of neutrophils and

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Nov 1, 2013 - E-mail: hus@pki.unibe.ch. Abbreviations: DAPK ...... Sasaki, T., Irie-Sasaki, J., Jones, R. G., Oliveira-dos-Santos, A. J., Stanford,. W. L., Bolon, B.
Epub ahead of print November 1, 2013 - doi:10.1189/jlb.0813462

Article

DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants Barbara Geering,*,1 Christina Stoeckle,* Saša Rožman,* Kevin Oberson,* Charaf Benarafa,† and Hans-Uwe Simon*,2 *Institute of Pharmacology and †Theodor Kocher Institute, University of Bern, Switzerland RECEIVED AUGUST 26, 2013; REVISED OCTOBER 3, 2013; ACCEPTED OCTOBER 6, 2013. DOI: 10.1189/jlb.0813462

ABSTRACT The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca2⫹/CaM-sensitive serine/ threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies. J. Leukoc. Biol. 95: 000 – 000; 2014.

Introduction Granulocyte chemotaxis is one of the critical components of the innate immune response to infection or injury, as for an effective response, cells need to be recruited to the site of inflammation. Migration toward a chemoattractant requires polarization in the direction of the chemoattractant source. Both polymerization of F-actin at the leading edge and assembly of myosin II in actomyosin filaments at the trailing edge provide the pushing and pulling forces that mediate cell motility. For

Abbreviations: DAPK, death-associated protein kinase, DAPKi⫽death-associated protein kinase inhibitor, LSM⫽laser-scanning microscopy, LTB4⫽leukotriene B4, MLC⫽myosin light chain, PAF⫽platelet-activating factor, PI(3,4,5)-P3⫽phosphatidylinositol (3,4,5)-trisphosphate, PTEN⫽phosphatase and tensin homolog, qPCR⫽quantitative PCR The online version of this paper, found at www.jleukbio.org, includes supplemental information.

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GPCR-driven chemotaxis in neutrophils, a Gi-dependent activation of PI3K at the leading edge results in a PI(3,4,5)-trisphosphate gradient [1]. This, in turn, mediates the local recruitment of the PI(3,4,5)-trisphosphate-dependent guanine exchange factors for the small GTPase Rac [2]. Active Rac then drives polymerization of F-actin [3]. The initiation of signaling at the trailing edge seems to be driven by the very same chemoattractant-receptor complex that mediates signaling at the leading edge, except that the receptor complex there is coupled to G12/13 [4]. Free G12/13 activates the small GTPase RhoA, which promotes actomyosin assembly through activation of the Rho-associated protein kinase and MLC kinase [5], which in turn phosphorylate MLC at Ser 19 and thereby, activate myosin II to promote contraction of the trailing edge [6, 7]. To be recruited from the blood circulation and move toward the site of inflammation, neutrophils must recognize and prioritize chemoattractant stimuli. So-called end-target chemoattractants produced in the vicinity of infection or inflammation (e.g., bacterial-formylated peptides or complement factors) are dominant over intermediary endothelialand immune-derived molecules (e.g., interleukins or leukotrienes) [8, 9]. This dominance of end-target chemoattractants seems to be a result of separate signaling pathways activated upon chemoattractant stimulation [10]. The first pathway stimulated by end-target molecules requires the activation of the MAPK p38; the second pathway stimulated by intermediary molecules depends on PI3K activity [10]. Provided both stimuli are present, dominance of end-target over intermediary chemoattractants was shown to be mediated by the PI3K antagonist PTEN [11], which is localized at the trailing edge if either chemoattractant is present, resulting in p38 activity when end-target chemoattractants are encountered or in PI3K activity when intermediary chemoattractants stimulate neutrophils. In contrast, PTEN localizes around the entire cell membrane when both chemoattrac1. Current address: Dept. of Biosystems Science and Engineering, ETH Zurich, CH-4058, Basel, Switzerland. 2. Correspondence: Institute of Pharmacology, University of Bern, Friedbuehlstrasse 49, CH-3010 Bern, Switzerland. E-mail: [email protected]

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tants are present. This relocalization of PTEN subsequently results in decreased PI3K activity and hence, prevalence of p38 activity at the leading edge. How this remaining p38 activity controls neutrophil motility is poorly understood. Phosphorylation of MLC at Ser 19 is also mediated by DAPKs [12–15], which are serine/threonine kinases best known for promoting cell death [13, 16 –18]. Yet, DAPKs are also emerging as important players in death-independent cellular functions. Indeed, a prominent feature of increased DAPK activity is the induction of cytoskeletal changes, resulting in cell rounding and membrane blebbing caused by integrin inactivation [19] and actin cytoskeletal reorganization [14, 15, 20]. Recently, the DAPK family member DAPK1 was shown to inhibit random migration by reducing directional persistence and to inhibit directed migration by interfering with cell polarization. The effect on DAPK1 on migration is attributed to its inhibitory role in talin association with integrin ␤1, thereby blocking integrin signaling pathways [21]. Here, we demonstrate that DAPK2 is essential for the migration of mature neutrophils and eosinophils toward intermediary chemoattractants. DAPK2 is the most prominent family member present in granulocytes under homeostatic conditions, and its expression is increased further when granulocytes are activated with proinflammatory cytokines. Following a block in DAPK2 activity using small molecule inhibitors, ex vivo motility of granulocytes in response to intermediary chemoattractants was diminished severely. This failure in migration of DAPK2-inactive granulocytes is likely a result of reduced polarization in response to chemoattractants, as nonpolarized, radial F-actin and pseudopod formation could be observed upon DAPK inhibition. Our data implicate that DAPK2 phosphorylates MLC—a direct target of DAPK2 and essential for establishing cellular polarity—in response to intermediary chemoattractants. As neutrophil migration was also reduced upon DAPK inhibition in a peritonitis model, our data suggest a proinflammatory role for DAPK2 in human and mouse granulocytes.

MATERIALS AND METHODS

Reagents Antibodies to DAPK2 were tested rigorously for specificity (Supplemental Fig. 1). Antibodies were purchased from the following sources: DAPK1 (D2178, 1:500; Sigma-Aldrich, St. Louis, MO, USA), DAPK2 (2323PR, MoBiTec, Göttingen, Germany; SP7055P, Acris Antibodies, Herford, Germany; IMG3028, Imgenex, San Diego, CA, USA), DAPK3 (2928, 1:500; Cell Signaling Technology, Danvers, MA, USA), phospho-MLC (3674, 1:500; Cell Signaling Technology), GAPDH (MAB374; 1:10,000; Merck Millipore, Darmstadt, Germany), FAS (CH11; MBL International, Woburn, MA, USA). Cytokines and other stimuli were obtained from the following companies: C5a (HC2101; HyCult Biotech, Netherlands), fMLF (47729; Fluka, Sigma-Aldrich, Switzerland), G-CSF (Lenograstim; Sanofi-Aventis, Singapore), GM-CSF (Leukomax 300; Novartis, Basel, Switzerland), IL-5 (205-IL; R&D Systems, Minneapolis, MN, USA), CXCL8/IL-8 (208-IL; R&D Systems), LTB4 (434625; Calbiochem, EMD Millipore, Billerica, MA, USA), PAF (511075; Calbiochem, EMD Millipore), TNF-␣ (210TA; R&D Systems). Inhibitors to DAPK family members are described in Supplemental Table 1. Cell culture reagents and protein standards were purchased from Invitrogen (Carlsbad, CA, USA); other buffer components, from Sigma-Aldrich (St. Louis, MO, USA).

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Isolation and culture of human myeloid precursor cells Bone marrow samples were mixed with an equal volume of 2% dextran sulfate in 0.9% NaCl to sediment erythrocytes using gravity flow. The bone marrow leukocytes were layered on top of a two-step discontinuous Percoll (170891; Amersham, GE Healthcare, Little Chalfont, UK) density gradient (1.085 g/ml and 1.065 g/ml, respectively). To enrich for immature granulocytes, negative selection—with CD7-PE (555361; Pharmingen, BD Biosciences, San Jose, CA, USA), CD36-PE (555455; Pharmingen), and CD19-PE (345777; BD Biosciences), followed by incubation with anti-PE microbeads (130048801; Miltenyi Biotec, Bergisch Gladbach, Germany)—was performed with MACS columns, according to the manufacturer’s protocol (Miltenyi Biotec). The collected cells were resuspended in RPMI complete medium [RPMI 1640⫹Glutamax containing 10% FCS (endotoxin ⬍1 EU/ ml) and 1% Pen/Strep (15140122; Invitrogen)] and cultured in the presence or absence of 25 ng/ml G-CSF or 10 ng/ml IL-5 in a humidified 37°C incubator with 5% CO2. Cells were collected on glass slides every other day (cytospin) and stained with Diff-Quik solution (Medion Diagnostics AG, Switzerland). Mature granulocytes were distinguished from meta-/myelocytes by morphological features, such as cell size, granularity, and nuclear shape. Written consent was obtained from all donors. The study was approved by the Ethics Committee of Canton Bern.

Isolation of human leukocytes Blood diluted with PBS was layered onto Ficoll (Biocoll; Biochrom AG, Berlin, Germany) and centrifuged at 800 g. Mononuclear cells were collected. PMN cells and erythrocytes were transferred to cold lysis buffer (155 mM Na4Cl, 100 mM KHCO3, 0.1 mM EDTA). Granulocytes were subsequently harvested. The resulting cell population usually contained ⬎95% neutrophils, assessed by staining of cells with Diff-Quik and light microscopy. Isolation of B cells, monocytes, and T cells from the mononuclear cell fraction was performed by MACS (Miltenyi Biotec), according to the manufacturer’s protocol using PE-coupled anti-CD19, FITC-coupled antiCD14 (347493; BD Biosciences), and FITC-coupled anti-CD4 and anti-CD8 antibodies (345768 and 347313; BD Biosciences). NK cells were isolated from the mononuclear cell fraction and eosinophils from the granulocyte fraction using isolation kits (19055 and 19256; Stemcell Technologies, Vancouver, BC, Canada). Cell purities were routinely ⱖ90%; cell purities of eosinophils were ⱖ95%. Written consent was obtained from all healthy donors. The study was approved by the Ethics Committee of Canton Bern.

Culture of Jurkat T cells Jurkat T cells (ATCC; TIB-152) were grown in RPMI complete medium in a humidified 37°C incubator with 5% CO2. Lentiviral production and Jurkat T cell transduction were performed as described previously [22].

Immunoblotting Cell pellets were lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1 mM EGTA, 0.5% Na-deoxycholate), containing protease (protease inhibitor cocktail, P8340; SigmaAldrich, St. Louis, MO, USA) and phosphatase inhibitors (5 mM NaF, 1 mM Na3VO4, 1 ␮M okadaic acid). Proteins were separated by SDS-PAGE and electroblotted onto PVDF membranes (Immobilion-P; EMD Millipore). Membranes were blocked routinely in PBS/0.1% Tween-20/5% nonfat milk and incubated overnight with primary antibodies. The membranes were subsequently incubated with the HRP-coupled secondary antibody (anti-mouse IgG, NA9310, GE Healthcare; anti-rabbit IgG, NA9340, GE Healthcare; anti-goat IgG, P0449, Dako, Denmark) and the filters developed by an ECL technique, according to the manufacturer’s instructions (ECL kit; GE Healthcare).

Real-time qPCR RNA was extracted using the SV Total RNA Isolation system (Z3100; Promega, Madison, WI, USA). RT of RNA was performed in duplicate using

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Geering et al. DAPK2 mediates granulocyte migration Superscript III RT, according to the manufacturer’s protocol (Invitrogen). Two control experiments were performed before qPCR. Firstly, a cDNA sample was used to test the newly designed primers for PCR amplification (correct length and amplification of a single DNA sequence only) of DAPK1, DAPK2, DAPK3, and GAPDH cDNA. Primer sequences were as follows: DAPK1 (NM_004938) GCA CCT TCT GGG CTC ATT AT and TTT GAA TAT TCC CAC AGC CA; DAPK2 (NM_014326) GGA AGC TTT CCT TCA GCA TC and AGT TCC TCA GGT CCT CAT CC; DAPK3 (NM_001348) ACA TGT GGA GCA TCG GTG T and AGT TCA CGG CTG AGA TGT TG; GAPDH (NM_002046) TTC CAA TAT GAT TCC ACC CA and GAT CTC GCT CCT GGA AGA TG. Secondly, a GAPDH control qPCR was performed to verify that similar amounts of starting material were present in each sample. Two qPCRs were then performed on consecutive days with samples of each RT and each primer pair. qPCR reactions contained 3.5 ␮l cDNA, 200 nM forward and reverse primer, and 12.5 ␮l SYBR Green Master Mix in 25 ␮l total volume. Samples were heated to 95°C for 15 s, 57°C for 20 s, and 72°C for 20 s (40 cycles). Comparative threshold values were evaluated using iQ5 Optical Systems Software (Version 2.0).

Nonradioactive in vitro kinase assay The DAPK Z-’LYTE in vitro kinase assay was adapted from protocols provided by Invitrogen (PV3793). rDAPK1-GST (10 –1000 nM; PV3969; Invitrogen), rDAPK2-His (PV3686; Invitrogen), or rDAPK3-GST (PV3686; Invitrogen) was added to kinase buffer B (50 mM HEPES, 20 mM MgCl2, 0.1 mg/ml BSA, 0.5 mM CaCl2) in the presence or absence of 500 –1000 nM CaM (P2277; Sigma-Aldrich, St. Louis, MO, USA). Peptide substrate was added to a final concentration of 2 ␮M; ATP was added at 10 –100 ␮M. Phosphorylated peptide substrate was used as the positive control. The reaction was incubated at room temperature overnight. The emission signal was analyzed by a SpectraMax M2e (Molecular Devices, Sunnyvale, CA, USA). Calculations were performed according to the manufacturer’s protocol (Invitrogen).

Radioactive in vitro kinase assay Active, GST-tagged DAPK2 (0.16 ␮M; 14 – 657; EMD Millipore) in kinase reaction buffer (50 mM HEPES, pH 7.5, 50 ␮M ATP, 20 mM MgCl2, 0.1 mg/ml BSA) was incubated with 1.2 ␮M MLC (M1636; Sigma-Aldrich, St. Louis, MO, USA). Kinase reactions in the presence of 1.2 ␮M CaM (P1431; Sigma-Aldrich, St. Louis, MO, USA) were supplemented with 0.5 mM CaCl2. The mix was incubated for 30 min at 30°C in buffer containing 15 ␮Ci ␥-32ATP. SDS sample buffer was added to terminate the reaction. After boiling, proteins were separated on a 12% polyacrylamide gel by SDSPAGE. The gel was fixed and exposed to a phosphor screen. Once radioactivity was decayed, the gel was stained with colloidal Coomassie blue (GelCode Blue Stain reagent; Pierce, Thermo Fisher Scientific, Rockford, IL, USA) to check for equal protein loading.

Determination of viability Viability was assessed by uptake of 25 ␮M ethidium bromide and flow cytometric analysis (FACSCalibur; BD Biosciences), as described previously [23]. Data analysis was done with FlowJo software.

Transwell assay Cells ⫾ DAPKi were put in the upper chamber of a Transwell system [5 ␮m pores (3421; Costar, Corning, NY, USA)], whereas chemotactic stimuli were added to the lower chamber. The cells were left to migrate between 90 and 180 min. Cells collected from the lower chamber were analyzed by flow cytometry (FACSCalibur) in the presence of counting beads (CountBright Absolute Counting Beads; Invitrogen). Data analysis was done with FlowJo software.

Phagocytosis assay Fluoresbrite beads (1 ␮m; 18660-5; Polysciences, Warrington, PA, USA) were opsonized with autologous serum in HBSS containing Mg2⫹ and Ca2⫹

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for 60 min at 37°C. The beads were washed subsequently with HBSS containing Mg2⫹ and Ca2⫹ and 5% autologous serum and incubated for 30 min at 37°C with freshly isolated human neutrophils at a 1:10 cell:bead ratio. A control sample was kept for the same time at 4°C to control for adhesion versus phagocytosis. The cells were washed with HBSS without Mg2⫹ and Ca2⫹ containing 0.02% EDTA, harvested, and analyzed by flow cytometry. Data analysis was done with FlowJo software.

Adoptive transfer and mouse peritonitis model The in vivo efficacy of DAPKi-13 was analyzed using a mouse model of sterile peritonitis. Neutrophils were isolated from the bone marrow of C57BL/6 (CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) male mice by negative selection, according to the manufacturer’s protocol (19762; Stemcell Technologies). Cell purity was ⬎90%. The cells were incubated subsequently in RPMI 0.5% BSA with 20 ␮M DAPKi-13 or control vehicle for 40 min, washed, and resuspended to 10 ⫻ 106/ml in warm PBS. Equal amounts of CD45.1- and CD45.2-positive granulocytes were mixed and injected (100 ␮l/mouse) in the retro-orbital venous plexus of anesthetized GFP⫹ transgenic [B6-Tg(CAG-EGFP)1Osb] male mice. Then, 1 ml 3% thioglycollate broth was injected i.p. into each mouse. After 3 h, blood samples were collected and the mice killed. Peritoneal lavage was performed, and cells were analyzed by flow cytometry (FACSCalibur) after staining with anti-Ly-6G (clone 1A8) and anti-CD45.1 (clone A20) antibodies (BioLegend, San Diego, CA, USA). All animal studies were approved by the Cantonal Veterinary Office of Bern and conducted in accordance with the Swiss federal legislation on animal welfare.

Immunocytochemistry Freshly isolated neutrophils were incubated for 1 h in the presence or absence of DAPKi and transferred onto glass coverslides prior to stimulation with 20 ng/ml IL-8. Nonadherent cells were removed carefully, and the cells were fixed in 4% PFA. Subsequently, slides were washed in PBS and cells permeabilized with 10% saponin-PBS solution. Cells were then put for 10 min at ⫺20°C in acetone, washed in PBS, and incubated for 30 min with phalloidin coupled to Alexa Fluor 488 (A12379; 1:40 in 1% BSA-PBS; Invitrogen). PI was added at 1:7000 to stain nuclei; slides were washed in PBS and mounted with Mowiol mounting medium (475904; Calbiochem, EMD Millipore) onto glass plates. For analysis, a confocal LSM (LSM 510; Carl Zeiss, Thornwood, NY, USA), equipped with argon and helium-neon lasers and a 63⫻/1.4 objective lens, was used. Images were acquired using LSM confocal software.

Adhesion assay Adhesion assays were done on serum-coated glass and serum-coated or fibronectin-coated plastic surfaces. Briefly, neutrophils were resuspended in RPMI complete medium (1% FCS) in the presence or absence of DAPKi. Following stimulation with 20 ng/ml IL-8, nonadherent cells were removed and the cells fixed and stained with Diff-Quik solution. Cells were analyzed on a DM IL light-emitting diode microscope equipped with a 40⫻/0.5 objective lens (Leica Microsystems, Buffalo Grove, IL, USA). Pictures were acquired using a DFC295 digital color camera and Application Suite Core software (Leica Microsystems). Images were processed with Photoshop 5.0 software (Adobe, San Jose, CA, USA) and quantified using ImageJ (http:// rsbweb.nih.gov/ij/).

Live-cell imaging Freshly isolated neutrophils were stained for 20 min with 2 ␮M CellTracker Green (C2925; Invitrogen). Following a wash step, neutrophils were incubated in RPMI complete medium (1% FCS) in the presence or absence of DAPKi. Cells were then transferred into a live-cell imaging device and analyzed in the presence or absence of 20 ng/ml IL-8 using a confocal LSM (LSM 510; Carl Zeiss), equipped with a 63⫻/1.4 objective lens. Images were acquired every 8 s for up to 15 min using LSM confocal software.

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RESULTS

Identification and characterization of DAPK2 small molecule inhibitors in vitro and in cellulo Recent reports revealed that small-molecule inhibitors with a 4-(3-pyridinylmethylene)-5(4H)-oxazolone core structure inhibit rDAPK1 and rDAPK3 in vitro [24, 25]. Furthermore, Okamoto et al. [24, 25] analyzed the selectivity of DAPKi toward a panel of Ser/Thr/Tyr kinases and showed that some of the compounds tested selectively inhibit DAPK1 and DAPK3. We therefore used these same inhibitors (DAPKi-1, DAPKi-8, DAPKi-9, DAPKi-10, DAPKi-11, DAPKi-13; Supplemental Table 1) to analyze their efficacy to block DAPK2. An in vitro kinase assay was set up with rDAPKs, rCaM, ATP, and a fluorescent substrate peptide derived from the endogenous DAPK substrate MLC (Fig. 2A and B and Supplemental Fig. 2). DAPKi concentrations ranging from 1 to 100 ␮M were used in the in vitro kinase assay with rDAPK1, rDAPK2, or rDAPK3 (Fig. 2C). Whereas inhibitor concentrations below 10 ␮M did not significantly inhibit the phosphorylation of the MLC peptide by Volume 95, February 2014

mature neutrophils

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Following evaluation of commercially available anti-DAPK2 antibodies (Supplemental Fig. 1), we investigated DAPK2 expression during ex vivo differentiation of granulocytes (Fig. 1). Myeloid precursors were isolated from human bone marrow and subjected to G-CSF or IL-5 treatment to differentiate the cells toward neutrophilic and eosinophilic granulocytes (Fig. 1A). Whereas DAPK2 protein expression was very low or undetectable in myeloid precursors, it increased during ex vivo differentiation, resulting in high DAPK2 levels in more mature neutrophils (Day 6) and eosinophils (Day 8; Fig. 1B). We then asked whether other leukocyte cell types expressed DAPK2 and in which cells the other DAPK family members— DAPK1 and DAPK3—were expressed. DAPK2 protein levels for T, B, and NK cells, monocytes, and granulocytes were compared (Fig. 1C). Although DAPK2 expression was highest in eosinophils, all cells tested expressed DAPK2. Strikingly, DAPK1 and DAPK3 expression was very low or undetectable in granulocytes compared with lymphocytes, indicating that DAPK2 is the main DAPK family member in neutrophils and eosinophils. The protein expression data were corroborated using real-time qPCR analyses (Fig. 1D), which showed that DAPK2 mRNA expression is also detected predominantly in neutrophils and eosinophils, whereas DAPK1 and DAPK3 mRNA expression is low in these cells.

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Figure 1. DAPK2 is the prevailing DAPK family member in granulocytes. (A) Ex vivo differentiation of myeloid precursor cells derived from human bone marrow resulted in formation of neutrophil- and eosinophil-like cells. (Upper) Immature granulocytes derived from human bone marrow were exposed ex vivo to G-CSF (25 ng/ml) or IL-5 (10 ng/ml) and harvested by centrifugation onto glass slides on the indicated days. (Lower) Cells were analyzed for morphological features of meta-/myelocytes or mature neutrophils/ eosinophils by light microscopy. Values are means ⫾ sd (n⫽4; *P⬍0.05). (B) DAPK2 protein expression was up-regulated during granulocyte differentiation. Bone marrow-derived immature granulocytes were stimulated with cytokines as above and cell lysates analyzed by immunoblotting for DAPK2 and GAPDH expression. Representative experiments are shown (n⫽4). (C) DAPK1 and DAPK3 protein expression was absent in granulocytes. Different cell types were enriched by antibody-mediated magnetic sorting from human blood. DAPK1, DAPK2, DAPK3, and GAPDH protein expression was analyzed by immunoblotting. Representative experiments are shown (n⫽3). (D) DAPK2 mRNA expression was highly enriched in granulocytes compared with PBMCs. GAPDH was used as a control. Values are means ⫾ sd (n⫽3).

any DAPK family member (data not shown), DAPKi-8, DAPKi-9, DAPKi-10, DAPKi-11, and DAPKi-13, used at 10 ␮M, blocked DAPK1 and DAPK2 activity up to 40%. Addition of the 100-␮M inhibitor blocked activity of all three recombinant proteins by ⬎50%. An exception was DAPKi-10, which failed to block DAPK3. To evaluate whether DAPKi would also block DAPK activity in cellulo, we analyzed the phosphorylation status of the known DAPK substrate MLC. Previous studies using DAPK2 as a readout for enzymatic activity following protein transduction showed that overexpression of WT DAPK2 in Jurkat T cells resulted in increased phosphorylation of MLC [22], whereas overexpression of GFP (control) or inactive DAPK2 only led to

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Geering et al. DAPK2 mediates granulocyte migration

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Figure 2. Evaluation of DAPK small molecule inhibitors. (A) Nonradioactive in vitro kinase assay to study DAPK2 activity. The phosphorylation of i-1 i-8 i-9 i-10 i-11 i-13 2 ␮M of a synthetic substrate (peptide sequence based on the phosphorylation site in MLC) was assessed spectroscopically using increasing conµM centrations of rDAPK2 and of ATP in the presence of 500 nM rCaM. ValpMLC ues are means ⫾ sd (nⱖ3). (B) DAPK2 activity was threefold increased in the presence of CaM. In vitro DAPK2 activity of 100 nM rDAPK2 toGAPDH ward 2 ␮M synthetic substrate in the presence of 10 ␮M ATP was asJurkat T cells WT DAPK2-overexpressing Jurkat T cells sessed in the presence or absence of 600 nM rCaM. Values are means ⫾ sd (nⱖ3; *P⬍0.01). (C) DAPKi block DAPK family members nonspecifically. Increasing concentrations (10 and 100 ␮M) of DAPKi (i-1, i-8, i-9, i-10, i-11, i-13) were added to rDAPK family members (10 nM DAPK1, 100 nM DAPK2, 30 nM DAPK3) in the presence of 2 ␮M synthetic substrate, 10 ␮M ATP, and for DAPK1 and DAPK2 samples, 600 nM rCaM. Values are means ⫾ sd (nⱖ3). (D) WT DAPK2-overexpressing Jurkat T cells failed to phosphorylate MLC (pMLC) after treatment with DAPKi. (Left) Overexpression of WT but not inactive DAPK2 results in an increase in the phosphorylation of MLC. (Right) WT DAPK2-overexpressing Jurkat cells were treated with indicated concentrations of DAPKi. Cell samples were collected 4 h following addition of DAPKi and analyzed by immunoblotting for pMLC and GAPDH. A representative experiment is shown (n⫽5).

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low levels of phospho-MLC (Fig. 2D). We therefore subjected DAPK2-overexpressing Jurkat T cells to increasing concentrations of DAPKi. Most inhibitors blocked phosphorylation of MLC at 200 ␮M, with the exception of DAPKi-10 (Fig. 2D). These data show that phosphorylation of MLC can be blocked by DAPKi intracellularly. As DAPKi-9 and DAPKi-13 were most efficient in blocking DAPK2 in in vitro and in cellulo assays and as DAPKi-9 and DAPKi-13 exhibit the most similar structure among the inhibitors, we performed subsequent experiments with DAPKi-9 or DAPKi-13 interchangeably.

Viability is not affected by DAPK2 inhibition in granulocytes Granulocytes undergo spontaneous apoptosis in the absence of prosurvival stimuli ex vivo [26]. As DAPK family members have been shown to promote cell death in a variety of cell types [13, 16 –18], we tested whether inhibition of DAPK by DAPKi-9 or DAPKi-13 in granulocytes would result in increased viability compared with control cells. Incubation of neutrophils or eosinophils with low (ⱕ10 ␮M) or high (ⱖ50 ␮M) concentrations of DAPKi-9 or DAPKi-13 failed to cause increased viability (Fig. 3A). In contrast, a slight decrease in viability could be detected in cells treated with ⬎50 ␮M DAPKi-13, suggesting a toxic effect of this compound at higher concentrations. In addition to spontaneous apoptosis, neutrophils and eosinophils undergo apoptosis in response to FAS ligation [27]. Yet, DAPKi-9 or DAPKi-13 also did not rescue anti-FAS antibodystimulated neutrophil or eosinophil cell death (Fig. 3B). Thus,

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inhibition of DAPK2 did not decrease viability of granulocytes undergoing spontaneous or FASR-induced cell death.

DAPK2 inhibition blocks motility of granulocytes ex vivo DAPK1 has been shown to regulate random and directed migration [21]. Because treatment of neutrophils and eosinophils with proinflammatory cytokines, which can stimulate granulocyte migration, resulted in the up-regulation of DAPK2 protein expression within a few hours (Fig. 4A), we hypothesized that DAPK2 may also be important for granulocyte motility. Therefore, we assessed migration of neutrophils and eosinophils ex vivo using Transwell assays. Approximately 80% of granulocytes migrated toward IL-8 or eotaxin within 1 and 3 h, respectively, as compared with an input control under nonblocking conditions (data not shown). In the presence of 10 ␮M DAPKi-9 or DAPKi-13, motility of granulocytes in response to the respective chemokine was inhibited (Fig. 4B and C), and at 50 ␮M, migration was virtually abolished, suggesting that granulocyte migration can be blocked by DAPKi in a concentration-dependent manner. As granulocytes express very little or no DAPK1 and DAPK3 (Fig. 1), we assumed that any effect of DAPKi on granulocyte migration could be attributed to an inhibition of DAPK2. We conclude that blocking DAPK2 dramatically diminishes granulocyte motility. To understand whether DAPK2 may be important for motility in response to diverse chemoattractants, we analyzed migration of neutrophils in response to the intermediary chemoatVolume 95, February 2014

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Figure 3. Inhibition of DAPK2 does not enhance granulocyte viability. (A) Spontaneous decrease in neutrophil (left) and eosinophil (right) viability is not affected by DAPKi. Granulocytes were isolated from human blood and incubated with increasing concentrations of DMSO or DAPKi-9 and DAPKi-13. Viability was assessed at 24 h of ex vivo culture by analysis of ethidium bromide-negative (i.e., viable) cells by flow cytometry. Decreased viability— only observed after treatment of cells with high concentrations of DAPKi-13—is likely a result of cytotoxic effects of DAPKi-13 under these conditions (nⱖ3). (B) FAS-induced decrease in neutrophil (left) and eosinophil (right) viability is not affected by DAPKi. Neutrophils were isolated from human blood and incubated for 60 min with increasing concentrations of DMSO, DAPKi-9, or DAPKi-13 prior to stimulation of the cells with anti-FAS antibody (CH11; 1 ␮g/ml; nⱖ2).

tractants LTB4 or PAF and toward the end-target chemoattractant fMLF. Whereas motility of neutrophils upon LTB4 and PAF stimulation was reduced in the presence of DAPKi-9 or DAPKi-13, fMLF-induced migration could not be blocked (Fig. 4D). Similarly, motility of eosinophils in response to the intermediary chemoattractants LTB4 or PAF was blocked by DAPKi, whereas migration to the end-target chemoattractant C5a was unaffected (Fig. 4E). These data suggest that DAPK2 is essential for migration toward intermediary chemoattractants, whereas granulocyte chemotaxis toward end-target chemoattractants is DAPK2-independent. To exclude possible nonspecific effects of the DAPKi, e.g., as a result of subtoxicity, we tested neutrophil phagocytosis in the absence or presence of DAPKi. Phagocytosis of serum-opsonized fluorescent beads by primary human neutrophils was not affected by treatment of the cells with 20 ␮M DAPKi-9 or DAPKi-13, independent of IL-8 stimulation (Fig. 4F), suggesting that the observed effects of these DAPKi on chemotaxis were specific.

Polarization but not adhesion is diminished upon DAPK2 inhibition To understand the cellular mechanisms underlying inhibition of motility upon DAPK2 inactivation, we investigated neutrophil adhesion, spreading, polarization, and chemotaxis. In a first step, adhesion of neutrophils in the presence or absence of DAPKi-13 to serum- or fibronectin-coated surfaces was analyzed. As shown in Fig. 5A, adhesion of neutrophils to fibronectin was increased upon stimulation with IL-8, independent of DAPK2 activity. Similar results were obtained when adhesion to serum-coated surfaces was analyzed (data not shown). As we observed strikingly different morphologies in these experiments in neutrophils treated with DAPKi-13 compared with control cells, we investigated shape changes by flow cytometry. Hence, neutrophils and eosinophils increased in cell size upon stimulation with intermediary (IL-8 and eotaxin) and end-target (fMLF) chemoattractants (Fig. 5B). Whereas treatment of neutrophils with DAPKi-13 prior to stimulation with intermediary chemoattractants abolished the observed 6 Journal of Leukocyte Biology

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shape change, this could not be observed upon stimulation with the end-target chemoattractant fMLF. A similar observation was made when neutrophil morphology was analyzed by immunocytochemistry, showing that neutrophils treated with DAPKi-13 prior to IL-8 stimulation neither increased in size (Fig. 5C, middle) nor polarized (Fig. 5C, right). Surprisingly, staining the cells with phalloidin revealed increased F-actin staining in DAPKi-13-treated cells (Fig. 5C, left). However, such F-actin staining was detected uniformly along the entire neutrophil membrane and not in punctuated stretches of leading and trailing edges, as seen in neutrophils stimulated with IL-8 only. Furthermore, radial pseudopodia could be detected upon DAPKi-13 treatment (Fig. 5C, left). Neutrophil motility in response to IL-8 was also assessed by live-cell imaging in the presence or absence of DAPKi-13. Whereas the vast majority of unstimulated neutrophils floated in glass chambers (Fig. 5D, right), 80% of neutrophils crawled on or adhered to the glass shortly after IL-8 stimulation (Fig. 5D, right and left). If the cells were pretreated with DAPKi-13, adhesion of neutrophils to glass slides was unchanged, but ⬍10% of neutrophils showed crawling movements compared with 40% in the vehicle-treated control. Hence, data presented in Fig. 5 suggest that granulocyte motility toward intermediary chemoattractants is reduced upon DAPK inhibition, as the cells cannot polarize, likely as a result of a reduced ability to localize F-actin to the leading and trailing edges.

Phosphorylation of MLC is mediated by DAPK2 in response to intermediary chemoattractants Given the strikingly similar phenotype of DAPK2-inactive and MLC kinase-inactive neutrophils [28], combined with the knowledge that MLC is a DAPK2 substrate [12–15], we assessed whether DAPK2 may mediate granulocyte migration through regulation of MLC. In a first step, we confirmed that DAPK2 indeed phosphorylates MLC directly, by performing a radioactive in vitro kinase assay with recombinant protein (Fig. 6A). Subsequently, we assessed whether MLC is phosphorylated by DAPK2 upon IL-8 stimulation. Indeed, following stimulation of

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Geering et al. DAPK2 mediates granulocyte migration

Figure 4. Inhibition of DAPK2 blocks neutrophil and eosinophil chemotaxis. (A, left) DAPK2 protein expression is up-regulated under inflammatory conditions. Neutrophils were stimulated with or without 10 ng/ml GM-CSF for the indicated times and analyzed by immunoblotting for DAPK2 and GAPDH expression. A representative experiment is shown (n⫽3). (Middle) Neutrophils were stimulated with GM-CSF (10 ng/ml), G-CSF (25 ng/ml), TNF-␣ (10 and 100 ng/ml), and IL-8 (10 ng/ ml) for 3 h and analyzed by immunoblotting for DAPK2 and GAPDH expression. A representative experiment is shown (n⫽3). (Right) Eosinophils were stimulated with 10 ng/ml IL-5 for 3 h and analyzed by immunoblotting for DAPK2 and GAPDH expression. A representative experiment is shown (n⫽6). (B and C) Chemotaxis toward intermediary chemoattractants was blocked by DAPKi. (B) Neutrophils were incubated with increasing concentrations of DAPKi-9 or DAPKi-13 for 60 min. The cells were subsequently subjected to 10 ng/ml IL-8 in a Transwell assay. Values are means ⫾ sd (n⫽7; *P⬍0.05). (C) Eosinophils were allowed to migrate toward eotaxin (100 ng/ml) for 150 min. Values are means ⫾ sd (n⫽5; *P⬍0.05). (D and E) Chemotaxis toward intermediary chemoattractants was DAPK2-dependent; toward end-target chemoattractants, DAPK2-independent. Neutrophils (D) and eosinophils (E) were pretreated with 20 ␮M DAPKi-9 or DAPKi-13 and subjected to chemokine gradients in Transwells. The following chemoattractant concentrations were used: IL-8 (10 ng/ml), LTB4 (1 ␮M), PAF (1 ␮M), fMLF (10 nM), eotaxin (100 ng/ml), C5a (1 nM). Values are means ⫾ sd (n⫽4; *P⬍0.05). (F) Phagocytosis is not affected by DAPKi. Neutrophils were isolated from human blood, pretreated with 20 ␮M DAPKi-9 and DAPKi-13, and incubated with control or serum-opsonized fluorescent polystyrene beads in the presence or absence of 10 ng/ml IL-8 for 30 min. Phagocytosis of fluorescent beads was assessed by flow cytometry (n⫽3).

neutrophils with IL-8, phosphorylation of MLC was increased, which could be blocked by pretreatment of the cells with DAPKi-13 (Fig. 6B). These data thus suggest that inhibition of motility in cells with inactive DAPK2 may be mediated by a decrease in myosin II contractility.

DAPKi block recruitment of neutrophils to the site of inflammation in a mouse peritonitis model We chose a mouse peritonitis model triggered by thioglycollate to test the potency of DAPKi-13 in vivo. To restrict the effect of DAPKi-13 to neutrophils, we performed an adoptive transfer of ex vivo-treated neutrophils into recipient mice. To this end, bone marrow neutrophils purified from mice carrying different CD45 alleles (CD45.1 or CD45.2) were incubated with

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DAPKi-13 or vehicle control. A one-to-one mix of CD45.1 and CD45.2 cells was subsequently injected i.v. into GFP⫹ recipient mice (Fig. 7A). GFP⫹ recipient mice were used to allow exclusion of recipient cells during analysis of adoptively transferred cells. CD45.1 and CD45.2 granulocytes could be distinguished easily by flow cytometric analysis (Fig. 7B), and the one-to-one ratio of CD45.1 versus CD45.2 neutrophils was detected in the preinjection mix (Fig. 7C, left). In agreement with in vitro migration data, DAPKi-13 blocked recruitment of granulocytes to the inflamed peritoneum, resulting in increased DAPKi-13treated neutrophils in the blood (Fig. 7C, middle) and decreased DAPKi-13-treated neutrophils in the peritoneum (Fig. 7C, right). Thus, DAPKi-13 inhibits granulocyte migration in vivo. Volume 95, February 2014

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Figure 5. Inhibition of DAPK2 results in reduced spreading and polarization but not in reduced adhesion. (A) Adhesion of neutrophils to fibronectin-coated surfaces was not affected by DAPK2 inhibition. Neutrophils were plated on fibronectin-coated plastic wells and stimulated with 10 ng/ml IL-8 in the absence or presence of 20 ␮M DAPKi-13. (Left) Representative phase-contrast images. (Right) Relative quantitation of adhering cells. Values are means ⫾ sd (n⫽3). (B) DAPKi blocked shape change upon stimulation of neutrophils and eosinophils with intermediary chemoattractants. Granulocytes were treated with or without DAPKi-13 for 60 min and stimulated with 10 ng/ml IL-8 or 100 nM fMLF (neutrophils) or 100 ng/ml eotaxin (eosinophils) for 15 and 45 min, respectively, and cells analyzed by flow cytometry. Representative flow cytometry histogram (n⫽3). (C) Neutrophil spreading and polarization are impaired following DAPKi treatment. Neutrophils pretreated with DAPKi-13 were plated on glass slides and stimulated with IL-8 for 15 min. F-actin was analyzed following phalloidin staining of neutrophils by fluorescence microscopy. (Left) Images of a representative experiment are shown (n⫽3). (Middle) Cell area was measured using ImageJ. Values are means ⫾ sd (*P⬍0.0001; n⫽50 cells). (Right) Quantitation of cells showing a round or polarized phenotype (n⫽50 cells) (D) Reduced numbers of crawling cells following DAPKi treatment. (Left) Live-cell imaging of neutrophils was recorded. Neutrophils were pretreated with control vehicle or DAPKi-13, followed by stimulation with IL-8. Images were taken before and up to 15 min post-addition of IL-8. A representative sample of this experiment is shown. Cells that were observed adhering to the surface are encircled; cells observed crawling are marked with an arrow. (Right) Quantification of the experiments. Values are means ⫾ sd (*P⫽0.007; **P⫽0.0001; n⫽4).

DISCUSSION It is well documented that DAPK2 mediates apoptosis and autophagy in adherent cells [13, 16 –18]. DAPK2 was moreover shown to mediate granulocyte differentiation [29]. Hence, DAPK2 expression was low in hematopoietic CD34⫹ precursor cells and the human promyelocytic NB4 leukemia cell line, whereas in vitro differentiation toward neutrophils resulted in elevated DAPK2 expression in both models [29]. In addition, neutrophil maturation was enhanced by ectopic expression of WT DAPK2, and depletion of DAPK2 by small interfering RNA slowed neutrophil maturation [29]. Whereas we confirm the increase in DAPK2 mRNA and protein levels during granulopoiesis, in this study, we uncover a novel biological function of 8 Journal of Leukocyte Biology

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DAPK2 in regulating granulocyte motility. Inhibition of DAPK2 by small molecule inhibitors blocks motility of granulocytes in response to intermediary chemoattractants (e.g., IL-8, LTB4, PAF) but not to end-target molecules (e.g., fMLF, C5a). A hierarchy of chemoattractive molecules has been reported, suggesting that end-target molecules are dominant over intermediary molecules [8, 9]. This prioritization process was shown to be a result of separate signaling pathways activated upon chemoattractant stimulation [10]. Exposure of granulocytes to intermediary chemoattractants depends on PI3K activity, whereas end-target chemoattractants use the MAPK p38 signaling pathway [10]. As DAPK2 mediates motility in response to intermediary chemoattractants, our data therefore

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suggest that DAPK2 may be activated downstream of PI3Ks, and indeed, the PI3K pathway can influence DAPK2 activity, as pharmacological inhibition of the PI3K, but not p38, pathway negatively impacted on DAPK2 protein expression (data not shown). We are currently investigating the signal transduction pathways leading to DAPK2 activity in more detail. Our findings imply that DAPK2 mediates granulocyte migration by controlling cellular spreading and polarization. Hence, inhibition of DAPK2 by small molecule inhibitors results in nonelongated cells that show increased radial pseudopod formation. This phenotype is reminiscent of myosin II null mutants in Dictyostelium amoeba [30, 31] and granulocytes treated with pharmacological inhibitors against Rho-associated protein kinase or MLC kinase [28, 32]. In this study, we could indeed confirm that MLC is phosphorylated by rDAPK2 in vitro [12, 33] and by overexpressed DAPK2 ex vivo [22]. We furthermore show that activation of neutrophils by intermediary chemoattractants results in increased DAPK2 activity, as measured by increased MLC phosphorylation, whereas blocking DAPK2 activity in granulocytes by DAPKi impairs phosphorylation of MLC upon stimulation with intermediary chemoattractants. Hence, these data suggest that DAPK2 regulates neutrophil motility by phosphorylation of MLC. However, our data do not exclude the involvement of other yet-to-be-identified DAPK2 substrates. Indeed, stimulation of eosinophils with eotaxin did not result in increased phosphorylation of MLC (data not shown), although chemotaxis of eosinophils was blocked to a similar extent. Whereas ⬃30 interaction partners and a handful of substrates of DAPK1 have been identified, only MLC has been confirmed as a substrate of DAPK2 [12–15]. As the 160-kDa DAPK1 possesses many more protein interaction domains than 42-kDa DAPK2, it would be unwary to conclude that DAPK1 substrates are also DAPK2 substrates. Hence, additional work will be required to identify DAPK2 substrates.

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Figure 7. DAPKi-13 blocks in vivo migration of neutrophils. (A) Illustration of strategy used for adoptive transfer experiments coupled to the mouse peritonitis model. Neutrophils were isolated from bone marrow of CD45.1- and CD45.2-positive C57BL/6 mice and treated ex vivo with vehicle control or 20 ␮M DAPKi-13. Donor cells were injected i.v. into GFP⫹ recipient mice, which were then injected i.p. with thioglycollate (TG). Three hours after injection, mice were killed to collect blood and peritoneal lavage cells. (B) Specificity of anti-CD45.1 antibody. Neutrophils isolated from CD45.1 and CD45.2 mice were stained separately or in a 1:1 mixture with anti-CD45.1 antibody and analyzed by flow cytometry. FL2, Fluorescence 2. (C) Reduced migration of neutrophils treated with DAPKi-13. Transferred neutrophils (GFPneg, Ly6G⫹) were analyzed by flow cytometry, and CD45.1 was used to distinguish DAPKi13-treated from untreated neutrophils. (Left) Equal amounts of CD45.1 and CD45.2 neutrophils were present in the preinjection mix. (Middle) Neutrophils treated with DAPKi-13 are increased in the circulation compared with control neutrophils (*P⫽0.02). (Right) DAPKi-13 reduced migration of neutrophils to inflamed peritoneum (P⫽0.18). Data were from two independent experiments (n⫽6 mice) and analyzed by Mann-Whitney U-test.

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be beneficial during the course of chronic inflammation. Small molecule inhibitors that target granulocytic chemotaxis are of great interest in drug development for inflammatory diseases, including chronic obstructive pulmonary disease, gout, or rheumatoid arthritis. These include LTB4 [34], IL-8 [35], and MCP [36] antagonists, inhibitors of adhesion molecules [37], and inhibitors against intracellular molecules involved in the molecular mechanisms of chemotaxis, such as PI3K inhibitors [38]. We tested and validated small molecule inhibitors against DAPK family members in vitro, ex vivo, and in vivo. These were shown to act selectively on DAPK family members when compared with a panel of kinases [25]. Yet, our data suggest that there is little specificity toward different DAPK family members. Thus, all inhibitors block DAPK activity with equal efficacy, a finding that makes it unlikely that these compounds could be used in the clinic to inhibit DAPK2/ granulocyte migration specifically. However, specific DAPK2i can surely be developed in the future and may prove to be valuable tools for treatment of diseases with excessive granulocyte infiltrations causing immunopathology. In summary, we provide evidence that DAPK2 is the main DAPK family member in granulocytes and positively regulates granulocytic motility by controlling cell spreading and polarization. Based on these observations, a proinflammatory phenotype can be attributed to DAPK2, a conclusion substantiated by the increased DAPK2 protein expression levels and activity in granulocytes stimulated with proinflammatory cytokines. Thus, DAPK2 may be a valid target for anti-inflammatory treatment.

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AUTHORSHIP B.G., C.S., C.B., and H-U.S. designed research. B.G., C.S., S.R., K.O., and C.B. performed experiments and analyzed the results. B.G. and C.S. created the figures. B.G., C.S., and H-U.S. wrote the paper.

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ACKNOWLEDGMENTS

17.

This work was supported by the Swiss National Science Foundation (Grant No. 310030_146181 to H-U.S. and Grant No. 310030_127464 to C.B.). B.G. was supported by the Roche Research Foundation (Switzerland) and L’Oréal for Women in Science (Switzerland) and C.S., by Deutsche Forschungsgemeinschaft (Grant No. STO 906/1-1). We thank Dr. Mario Tschan (Institute of Pathology, University of Bern) for helpful discussions. Mice were kindly provided by Drs. Christoph Müller (Institute of Pathology, University of Bern), Adrian Ochsenbein (Department of Clinical Research, University of Bern), and Jens Stein (Theodor Kocher Institute, University of Bern). Human bone marrow samples were kindly supplied by Dr. Benjamin Gantenbein (ARTORG Center, University of Bern).

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DISCLOSURES

The authors declare no conflicts of interest.

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KEY WORDS: death-associated protein kinase 䡠 granulocyte 䡠 migration 䡠 inflammation 䡠 inhibitor

Volume 95, February 2014

Journal of Leukocyte Biology 11