Protein C-θ Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow

This information is current as Anna Bertram, Hong Zhang, Sibylle von Vietinghoff, of September 26, 2021. Carmen de Pablo, Hermann Haller, Nelli Shushakova and Klaus Ley J Immunol 2012; 188:4043-4051; Prepublished online 7 March 2012;

doi: 10.4049/jimmunol.1101651 Downloaded from http://www.jimmunol.org/content/188/8/4043

References This article cites 51 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/188/8/4043.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Protein Kinase C-u Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow

Anna Bertram,*,† Hong Zhang,† Sibylle von Vietinghoff,*,† Carmen de Pablo,† Hermann Haller,* Nelli Shushakova,* and Klaus Ley†

Protein kinase C (PKC)-u is involved in activation via regulating the avidity of the b2 integrin LFA-1 in the . LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-u in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP+ (wild-type [WT]) and PKC-u - deficient (Prkcq2/2) mice. Following stimulation with CXCL1, both WT and Prkcq2/2 cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq2/2 neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq2/2 neutrophils started 30 s after CXCL1 2 2 2 2 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq / neutrophils was delayed. Prkcq / Downloaded from neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced perito- nitis, and LPS-induced lung injury. We conclude that PKC-u mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-u in outside–in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo. The Journal of Immunology, 2012, 188: 4043– 4051. http://www.jimmunol.org/

nflammation is the body’s natural defense mechanism against stimuli, the b2 integrin macrophage-1 Ag (Mac-1) can be rapidly different forms of injury, such as invading microorganisms or mobilized to the cell surface after exposure to inflammatory I physical trauma. Although inflammation is critical for sur- stimuli (4). Neutrophil arrest is LFA-1–dependent and Mac-1– vival, massive inflammation can also be deleterious, leading to independent, whereas Mac-1 is required for many b2 integrin- systemic inflammatory response syndrome or other disorders. A dependent neutrophil functions such as phagocytosis and respi- critical step in the inflammatory process is the attraction and ac- ratory burst (5, 6). tivation of leukocytes that transmigrate through the vascular en- Recently, several intracellular have been shown to dothelium and then migrate to the site of inflammation. Neutrophil participate in neutrophil adhesion. E-selectin engagement induces by guest on September 26, 2021 granulocytes are the first line of defense against bacteria and fungi. b2 integrin activation via a signaling cascade involving the Src They migrate to the site of injury in acute inflammation, where they family tyrosine kinase FGR (7), spleen tyrosine kinase (SYK) (8), kill and phagocytose the attacking bacteria and where they release and Bruton’s tyrosine kinase (9). Once adherent, the ligand-bound oxygen radicals, bactericidal peptides, and inflammatory cytokines. integrins initiate outside–in signaling events that involve SYK To get to the site of inflammation, neutrophils follow a sequence of (10) and Src kinases (11, 12). Using pseudosubstrate inhibition, well-orchestrated events. This adhesion cascade involves tethering, the atypical isoform protein kinase C (PKC)-z has been implicated rolling and slow rolling, arrest, adhesion strengthening and intra- in outside–in signaling downstream of the b2 integrin Mac-1 (13), vascular crawling, and transmigration through the endothelium, but Mac-1 has no known role in neutrophil adhesion under flow. followed by migration through the tissue (1, 2). The functional role of PKC has not yet been investigated in the Inflammation induces surface expression of selectins on endo- context of neutrophil adhesion to the vascular endothelium (14). thelial cells, increasing the portion of rolling cells. Activation of PKC isoforms are involved in cell differentation, migration, rolling leukocytes by chemokines leads to a conformational change proliferation, and activation (15). In other cell types, PKC isoforms of the b2 integrin LFA-1, enabling it to bind to ICAM-1 and other have been shown to be involved in the activation of different ligands (3), which leads to neutrophil arrest. Whereas the level of integrins (16–19). PKCs have been grouped into the conventional, LFA-1 expression on neutrophils is unaltered by inflammatory novel, and atypical isoforms. PKCs are known to be activated by G protein-coupled receptors (GPCRs), tyrosine kinase receptors, *Department of Nephrology and Hypertensiology, Hannover Medical School, 30625 and growth factor receptors (15). Using knockout strategies, PKC Hannover, Germany; and †Division of Inflammation Biology, La Jolla Institute for isoforms have been implicated in a multitude of diseases and or- and Immunology, La Jolla, CA 92037 gan dysfunctions, including inflammatory processes (20). In this Received for publication June 6, 2011. Accepted for publication February 7, 2012. study, we investigate the role of PKC-u, a novel PKC isoform (14), This work was supported by Dr. Werner Jackstaedt Foundation (Germany) Grant which is calcium-insensitive, but activated by 1,2-diacylglycerol S134-10.056 (to A.B.). or phorbol esters, in neutrophil adhesion. The main PKC isoforms Address correspondence and reprint requests to Dr. Anna Bertram, Department of Nephrology and Hypertensiology, Hannover Medical School, Carl-Neuberg-Strasse expressed in neutrophils are thought to be PKC-a, PKC-b, PKC-d, 1, 30625 Hannover, Germany. E-mail address: [email protected] and PKC-z (21, 22). Expression of PKC-u and PKC-i/l has also Abbreviations used in this article: GPCR, G protein-coupled receptor; KO, knockout; been demonstrated (23). MPO, myeloperoxidase; PKC, protein kinase C; RGD, Arg-Gly-Asp; SYK, spleen PKC-u has a well-documented role in T cell activation. Upon tyrosine kinase; WT, wild-type. TCR stimulation, proliferation, IL-2 production, and anti- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 apoptotic mechanisms were markedly reduced in PKC-u knock- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101651 4044 PKC-u IN NEUTROPHIL ADHESION out T cells (24–26). Recently, PKC-u has been shown to regulate For the degranulation assay, isolated neutrophils were primed with 5 the affinity of the b integrin LFA-1 downstream of the TCR by mg/ml cytochalasin B (Sigma-Aldrich, Munich, Germany) for 15 min on 2 ice and 15 min at 37˚C, and 2 3 106 cells in 400 ml were incubated at 37˚ phosphorylating the guanine nucleotide exchange factor Rap- C on a 24-well plate coated with 5 mg/cm2 poly-RGD (Sigma-Aldrich, GEF2, an activator of the small GTPase Rap1. This leads to Munich, Germany), with 100 ng/ml PMA (Sigma-Aldrich, Munich, a stable immunological synapse (27). The signaling cascade Germany) as positive control or the vehicle PBS as negative control. Cell- leading to LFA-1 activation after TCR stimulation shares some free supernatants were collected after 15, 30, and 60 min and were an- similarities with the inside–out signaling pathway following the alyzed for myeloperoxidase (MPO) activity as described (30). Briefly, 20 ml supernatant was incubated with 230 ml 0.167 mg/ml o-dianisidine activation of neutrophil GPCRs by chemokines. Binding of the dihydrochloride solution (Sigma-Aldrich, Munich, Germany) with 160 chemokine CXCL1 to its GPCR CXCR2 is followed by a rapid mM hydrogen peroxide (Sigma-Aldrich, Munich, Germany) for 15 min at signaling cascade involving PLC-b, the guanine exchange factor room temperature. The reaction was stopped by adding 50 ml1Msulfuric CALDAG-GEF1, and the small GTPase Rap1 (3, 28). This inside– acid (Sigma-Aldrich, Munich, Germany). MPO activity was assessed via changes in OD at 405 nm on a Multiskan Ascent plate reader (Thermo out signaling results in the activation of LFA-1, its conformational Scientific, Schwerte, Germany). Total cellular content was determined in change to the high-affinity state, and subsequent arrest of the cell lysates in 0.5% Triton X-100 (Sigma-Aldrich, Munich, Germany) neutrophil on the endothelium (2). The role of PKC-u in neu- and 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich, trophils has not been studied. Munich, Germany) in 50 mM potassium phosphate buffer (pH 6.0), and The objective of this study was to elucidate the role of PKC-u MPO degranulation was determined by dividing MPO activity of the supernatants by MPO activity of whole-cell lysates. A serial dilution of in neutrophils in the inflammatory response and particularly in MPO from human neutrophils (Calbiochem, Bad Soden, Germany) events associated with the activation of b2 integrins in neu- served as standard. trophils. Neutrophil spreading on a glass coverslip was evaluated using differential Downloaded from interference contrast and conventional light microscopy on a BX51WI fixed-stage microscope with a 360 water immersion objective (Olympus, Materials and Methods Center Valley, PA). In the beginning of the experiment, 2 3 104 cells were Animals pipetted onto each coverslip. After 2, 3, 4, 5, 10, or 15 min, spreading was stopped by fixation with 2% paraformaldehyde (Electron Microscopy All animal experiments were conducted in accordance with the regulations Sciences, Hatfield, PA), and the percentage of spread cells was evaluated by counting 200 cells. Data were acquired with SlideBook 4.2 (Olympus).

of the local authorities. Mixed bone marrow chimeric mice were generated http://www.jimmunol.org/ as previously described (9). Briefly, bone marrow cells isolated from male The chemotaxis assay was performed as described (10). Transwell LysM-GFP+ (29) and PKC-u gene-deficient Prkcq2/2 mice (24) were inserts of 3-mm-pore polycarbonate chemotaxis chambers (Costar/ 5 mixed 1:1, and unfractioned cells were i.v. injected into lethally irradiated Corning, Corning, NY) were filled with 7.5 3 10 cells/ml RPMI 1640/ 6-wk-old male C57BL/6 mice. LysM-GFP+ mice strongly express GFP in 0.25% BSA. The inserts were placed into RPMI 1640/0.25% BSA con- neutrophils. The GFP-expressing cells have a normal function and are taining 50 ng/ml recombinant human C5a (Sigma-Aldrich, Munich, Ger- regularly used as a fluorescent wild-type (WT) control (29). Experiments many) or into medium alone (control), and the chambers were incubated were performed 6–8 wk after transplantation. Female, 8- to 12-wk-old for 5, 10, 15, 30, 60, or 120 min at 37˚C. Transmigrated neutrophils were PKC-u gene-deficient Prkcq2/2 mice (25) were used for the model of counted using the Vet ABC hematology analyzer (Scil Animal Care, thioglycollate-induced peritonitis. Animals were housed in a barrier fa- Gurnee, IL), and transmigration was quantified as percentage of total cility under specific pathogen-free conditions in the animal facilities of the neutrophils loaded into the insert.

La Jolla Institute for Allergy and Immunology (La Jolla, CA) and Hann- by guest on September 26, 2021 over Medical School (Hannover, Germany). Autoperfused flow chambers Neutrophil adhesion, superoxide release, degranulation, Autoperfused flow chamber experiments were performed as described 3 spreading, and chemotaxis in vitro previously (7). Briefly, rectangular glass capillaries (20 mm 200 mm 3 30 mm) were coated with 30 mg/ml E-selectin (R&D Systems, Neutrophils were isolated from bone marrow collected from both femurs Minneapolis, MN) with or without 15 mg/ml ICAM-1 (15 mg/ml; R&D and tibiae of Prkcq2/2 and C57BL/6 WT mice using a 62% Percoll Systems) for 2 h and blocked with 10% casein (Pierce Chemicals, (Sigma-Aldrich, Munich, Germany) gradient after RBC lysis with am- Rockford, IL) for 1 h. Bone marrow chimeric mice were anesthetized by monium chloride solution (Stemcell Technologies, Vancouver, BC, Can- i.p. injection of ketamine hydrochloride (125 mg/kg; Sanofi, Bridge- ada). The neutrophils were resuspended in calcium- and magnesium-free water, NJ), xylazine (12.5 mg/kg; Phoenix, Auckland, New Zealand), PBS (PAA, Pasching, Austria) and counted in a hemocytometer after and atropine sulfate (0.025 mg/kg; Fujisawa USA, Deerfield, IL) and staining with Tuerk’s reagent (Merck, Darmstadt, Germany). were placed on a heating pad maintained at 37˚C. The left carotid artery Adhesion assays were performed in 96-well plates (BD Biosciences, was cannulated with heparinized PE-10 tubing and connected to the San Jose, CA) coated with 20% FCS with or without poly–Arg-Gly-Asp flow chamber. Microscopy was conducted using a Zeiss Axioscope (poly-RGD, 20 mg/ml; Sigma-Aldrich, St. Louis, MO), a synthetic (Carl Zeiss, Thornwood, NY) with a SW40/0.75 salt immersion ob- integrin ligand. Neutrophils were labeled with calcein AM (Molecular jective, and images were recorded with a CCD camera (VE-1000CD; Probes, Eugene, OR), and 5 3 105 cells per well were incubated for 15 Dage-MTI, Michigan City, IN) connected to a VHS recorder. After 6 min at 37˚C with fMLP (1 mM; Sigma-Aldrich, St. Louis, MO) or the min blood perfusion with a shear rate of 6–8 dynes/cm2, one repre- vehicle HEPES buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2,2mM sentative field of view was recorded for 1 min. Rolling velocity of the CaCl2, 10 mM HEPES [pH 7.2]), and neutrophil adhesion was assessed cells was calculated using a custom tracker written in Matlab 2009b after stimulation with fMLP, poly-RGD, or without any stimulus. The (MathWorks, Natick, MA). total number of adherent cells was quantified with a SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA). The plates Intravital microscopy of the mouse cremaster were washed every 30 s with sterile PBS to remove nonadherent cells, and the remaining fluorescence was measured after every wash step. Cell The tracheae of bone marrow chimeric anaesthesized mice were intu- adhesion was determined by dividing remaining fluorescence by initial bated and the left carotid arteries were cannulated with heparinized PE- fluorescence. 10 tubing for intra-arterial injections. The cremaster was prepared for Superoxide release from isolated neutrophils (5 3 105 cells/well) was intravital microscopy as previously described (31). In brief, the skin of measured by a cytochrome C reduction assay using the same stimuli the scrotum was opened transversely, and the whole cremaster pouch (poly-RGD, fMLP, HEPES buffer as control) as in the adhesion assay. was exteriorized and pinned to a translucent microscope stage. The Cytochrome C is reduced by reaction with superoxide, resulting in the cremaster was incised longitudinally, spread over the stage, and generation of ferrocytochrome C with a detectable absorbance at 550 superfused with 37˚C bicarbonate buffered saline (132 mM NaCl, 18 nm. It was determined with a SpectraMax M5 fluorescence plate reader mM NaHCO3,4.7mMKCl,2mMCaCl2 3 2H2O, 1.2 mM MgCl2;all every 2 min starting immediately after application of the stimuli. For Sigma-Aldrich, St. Louis, MO) equilibrated with 5% CO2 in N2 during statistical analysis, all values were normalized to the maximum fluo- the whole procedure. Microscopy was conducted using a Zeiss Axio- rescence obtained in WT neutrophils after fMLP application, which was scope (Carl Zeiss) with a SW40/0.75 salt immersion objective, and set at 100%. images were recorded with a CCD camera (VE-1000CD; Dage-MTI) The Journal of Immunology 4045 connected to a VHS recorder. Postcapillary venules with a diameter Results between20and50mm were recorded, and firmly adherent cells (defined Differential impact of PKC-u gene deficiency on neutrophil as no movement for at least 30 s) were counted before and after intra- arterial injection of 600 ng CXCL1 (PeproTech, Rocky Hill, NJ). The functions in vitro 3 3 number of adherent cells was divided by surface area (S = p d l, Adhesion of isolated bone marrow neutrophils from WT and where d is diameter and l is length of the vessel) of the vessel wall 2/2 within one field of view. Detachment of adherent cells and new rolling Prkcq mice was induced by fMLP stimulation in vitro. fMLP cells were recorded within 3 min after CXCL1 injection. Afterwards, binds to the GPCR FPR-1 and leads to rapid activation of LFA-1 2 2 the prepared cremaster was rested for 1 h, before velocity and flux of through inside–out signaling (35). Both WT and Prkcq / neu- rolling neutrophils were measured. Wall shear rate was estimated using trophils similarly adhered in the presence of fMLP with an ∼50% 3 the formula gw =24.5 vcenter/d, where blood centerline velocity decline after the first wash step and a nearly complete detachment (vcenter) was measured with a dual-slot photodiode sensor with a digital cross-correlation program (CircuSoft Instrumentation, Hockessin, DE). of cells after seven to eight wash steps (Fig. 1A). For the visualization of neutrophil transmigration into the interstitium, Neutrophil adhesion can also be induced on a poly-RGD surface. postcapillary venules with a diameter between 20 and 40 mmwere RGD binds integrin avb3, which then activates b2 integrins (36, recorded using reflected light oblique transillumination (32) with a 37). Adhesion to the RGD surface was markedly reduced for SW20/0.5 salt immersion objective. Transmigrated cells were counted 2/2 2/2 before and 40 min after superfusion with 10 mMfMLPinanarea Prkcq neutrophils (Fig. 1B). About 60% of Prkcq neu- extending 25 mm to each side of the vessel and over a length of 50 mm trophils already detached after the first wash step, whereas WT (area = 50 3 50 mm2) and normalized to cell number per 10000 mm2 neutrophils showed sustained adhesion. This finding suggests and to the WT-to-knockout (KO) proportion of blood neutrophils for defective integrin outside–in signaling in Prkcq2/2 neutrophils. each animal. We next assessed neutrophil respiratory burst by assessment of Downloaded from superoxide release. Both WT and Prkcq2/2 neutrophils showed Thioglycollate-induced peritonitis a strong release of superoxide after stimulation with fMLP (Fig. 2/2 Prkcq , WT, and bone marrow mixed chimeric mice were i.p. injected 1C). The respiratory burst can also be induced by poly-RGD via with 4% thioglycollate (1 ml/25 g body weight) to induce sterile peri- integrin outside–in signaling (38). There was no difference in the tonitis or with 1 ml sterile saline (control). Mice were sacrificed 4 h after 2/2 thioglycollate injection, and peritoneal lavage was performed using 10 amount of released superoxide anions in Prkcq neutrophils ml PBS (2 3 5 ml). For quantification of neutrophil accumulation in the in comparison with WT neutrophils after binding to poly-RGD

2 2 http://www.jimmunol.org/ peritoneal lavage of Prkcq / and WT mice, differential cell counts (Fig. 1D). 3 were performed on cytospins (55 g for 10 min) stained with H&E. For Upon activation, degranulation of preformed vesicles containing bone marrow mixed chimeric mice, the cell counts of GFP-positive WT and GFP-negative Prkcq2/2 neutrophils were determined by FACS such as proteases is an integral part of neutrophil immune analysis. function. Upon activation by the phorbol ester PMA, which strongly activates neutrophils independently of integrin binding LPS-induced lung injury (39), MPO, serving as a marker of primary, azurophilic granula, 2/2 LPS-induced lung injury experiments were performed as described was similarly released from WT and Prkcq neutrophils (Fig. elsewhere (33). Briefly, LPS from Salmonella enteritidis (Sigma- 1E). Integrin-dependent degranulation was induced by neutrophil 2/2

Aldrich, St. Louis, MO) was dissolved in 0.9% saline (500 mg/ml) adhesion to poly-RGD. After 15, 30, and 60 min, WT and Prkcq by guest on September 26, 2021 and aerosolized with the MicroAir nebulizer (Omron Healthcare, Ver- neutrophils showed no statistically significant difference in the non Hills, IL). Bone marrow mixed chimeric mice were exposed to the amount of MPO release (Fig. 1E, 1F). aerosolized LPS for 30 min in a 20 3 9 cm plastic chamber with a constant airflow of 15 ml/min. At 24 h after LPS exposure, the mice Neutrophils can adhere to and spread on glass, most likely were anesthetized, the tracheae were cannulated with PE-50 tubing, and by secretion of adhesive molecules (40). On a glass surface, 1 ml PBS was infused intratracheally and withdrawn. This step was WT neutrophils showed the expected time-dependent spreading. repeated five times with 1 ml PBS each time. Bronchoalveolar lavage Prkcq2/2 neutrophils failed to spread at 2 min, displayed reduced fluid was centrifuged at 300 3 g for 5 min, and the pellet was resus- pended in PBS/1% BSA. Cells were counted with a hemocytometer and spreading at 3 min, and only at 4 min and at later time points was further analyzed using FACS. spreading equal to WT neutrophils (Fig. 2A, 2B). Thus, absence of PKC-u in Prkcq2/2 neutrophils induced delayed spreading in this FACS analysis assay. 2/2 2/2 Prkcq Cell counts of GFP-positive WT and GFP-negative Prkcq neutrophils We next compared the in vitro capacity of WT and were determined by FACS analysis on an LSR-II (BD Biosciences, San neutrophils to migrate toward the chemoattractant C5a. The mi- 2 2 Jose, CA) using forward and side scatter, PerCP-conjugated rat anti- gratory capacity of Prkcq / neutrophils was not reduced com- mouse CD45, and allophycocyanin-conjugated rat anti-mouse Gr-1– pared with WT cells. In the early phase (5 min), there even specific Abs. Data were analyzed using FlowJo software (Tree Star, was a slightly increased migration of Prkcq2/2 neutrophils (WT, Ashland, OR). 12.5 6 0.5%; KO, 15.8 6 0.4%; p = 0.0015). This difference was For the analysis of b2 integrin surface expression, isolated bone marrow neutrophils from WT and Prkcq2/2 mice were suspended in serum-free not evident at later time points. At 30 min, the number of migrated RPMI 1640 medium and incubated with 100 ng/ml recombinant murine neutrophils reached maximum values for both WT and Prkcq2/2 TNF-a, 50 ng/ml recombinant human C5a, or medium for 30 min at 37˚C neutrophils (Fig. 2C), with no further increase found at later time (4, 34). Then, cells were washed and stained with PE-conjugated rat anti-mouse CD11a (LFA-1), allophycocyanin-conjugated rat anti-mouse points (data not shown). CD11b (Mac-1), and FITC-conjugated rat anti-mouse Gr-1 (neutrophils) In summary, these data suggest a role of PKC-u in neutrophil mAbs (BD Biosciences, Heidelberg, Germany). Mean fluorescent inten- adhesion, but not other integrin-dependent cell functions in vitro. sities of LFA-1 and Mac-1 with or without stimulation were determined using a FACSCanto (BD Biosciences, Rockville, MD). PKC-u is dispensable for rapid chemokine-induced arrest but Statistical analysis is required for sustained neutrophil adhesion and To analyze differences between the mean values of two groups, a two-sided transmigration in vivo unpaired Student t test was used. To determine differences between mean values of more than two groups, ANOVA was used. A p value of ,0.05 Intravital microscopy of murine cremaster postcapillary venules was considered to be statistically significant. Statistical analysis was per- was employed to examine chemokine-induced arrest in vivo. The formed using GraphPad Prism 4 software. average shear rate in the examined vessels was 2043 6 387/s. Intra- 4046 PKC-u IN NEUTROPHIL ADHESION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Adhesion, but not superoxide release or degranulation, of PKC-u gene-deficient neutrophils is defective in vitro. (A) Adhesion of fluo- rescently labeled Prkcq2/2 and WT bone marrow neutrophils was induced by stimulation with 1 mM fMLP or (B)20mg/ml poly-RGD, and fluorescence was measured after every wash step using the fluorescence plate reader SpectraMax M5 at 485-nm excitation and 530-nm emission wavelengths. Cell adhesion was determined by dividing remaining fluorescence by initial fluorescence. (C) Superoxide release from isolated neutrophils was induced by stimulation with 1 mM fMLP or (D)20mg/ml poly-RGD using a cytochrome C reduction assay, and fluorescence was determined with the SpectraMax M5 fluorescence plate reader every 2 min. (A–D) were performed in duplicates from n = 3 mice. Data are presented as means 6 SEM. (E) MPO release was induced by stimulation with 100 ng/ml PMA or on a plate coated with 5 mg/cm2 poly-RGD (E) and (F), and cell-free supernatants were analyzed for MPO activity after 15, 30 (F), and 60 min (E) and (F)(n = 6–12 mice). Data are presented as means 6 SEM. arterial injection of CXCL1 (600 ng) induced immediate arrest of (23.4 6 2.4 mm/s for WT neutrophils; 20.7 6 4.0 mm/s for all rolling neutrophils (41), resulting in a 3.6 6 0.8-fold increase Prkcq2/2 neutrophils; n = 7 mice) were not significantly different. of WT and a 3.6 6 1.0-fold increase of Prkcq2/2 firmly adherent Also, no significant difference was seen when neutrophils were neutrophils (n = 6 mice), with firm adhesion being defined as “no observed in autoperfused flow chambers on E-selectin with or movement for 30 s.” Whereas most adherent WT neutrophils without ICAM-1 (data not shown). remained adherent for at least 180 s after CXCL1 injection, 50% After firm adhesion, neutrophils transmigrate through the en- of Prkcq2/2 neutrophils were already detached after 105 s (Fig. dothelial wall. Superfusion with fMLP induces a robust transmi- 3A). Chemokine-induced neutrophil arrest stops rolling, because gration of neutrophils in the mouse cremaster model (42), which essentially all rolling cells become adherent (41). Rolling of all was visualized by reflected light oblique transillumination (32). In WT neutrophils stopped for 90 s, but rolling of Prkcq2/2 neu- an area of 10,000 mm2, the number of interstitial neutrophils rose trophils started as early as 30 s after CXCL1 injection (Fig. 3B). from 0 to 20.6 6 0.6 for WT neutrophils and from 0 to 9.9 6 1.3 Rolling flux (44.3 6 4.6% for WT neutrophils; 55.7 6 4.6% for for Prkcq2/2 neutrophils (n = 4 mice; p = 0.0003) after 40 min Prkcq2/2 neutrophils; n = 7 mice) as well as rolling velocity superfusion with fMLP (Fig. 3C–E). The Journal of Immunology 4047 Downloaded from http://www.jimmunol.org/

FIGURE 2. PKC-u gene-deficient neutrophils show a time-dependent defect in spreading in vitro, but normal chemotaxis to rhC5a over time. (A) Prkcq2/2 and WT neutrophils were pipetted onto glass coverslips, and spreading after 2, 3, and 4 min was evaluated using differential interference contrast microscopy on a BX51WI fixed stage microscope with a 360 water immersion objective. The percentage of spreading/total cells was evaluated by counting 200 cells (n = 3 mice). Data are presented as means 6 SEM. Significance was determined by a Student t test. *p , 0.05, ***p , 0.001. (B) One representive field of view from the spreading assay for WT and Prkcq2/2 neutrophils after 3 min using differential interference contrast microscopy. (C) Chemotaxis of WT and Prkcq2/2 neutrophils to 50 ng/ml rhC5a was analyzed using 3-mm-pore polycarbonate chemotaxis chambers. Transmigrated neutrophils were counted after 5, 10, 15, and 30 min, and transmigration was quantified as percentage of total neutrophils loaded into the insert (n = 5 mice). Data are presented as means 6 ,

SEM. Significance was determined by a Student t test. **p 0.01. by guest on September 26, 2021

These data suggest a role for PKC-u in neutrophil firm adhesion, In Prkcq2/2 mice, PKC-u is absent in all cells, including en- resulting in a significantly decreased number of extravasating cells dothelial cells. To investigate the specific role of PKC-u in neu- in the absence of PKC-u. trophils, we constructed bone marrow chimeric mice from LysM- GFP+ and Prkcq2/2 mice, resulting in equal numbers of WT and Migration of PKC-u gene-deficient neutrophils is diminished in Prkcq2/2 neutrophils in blood. After thioglycollate injection, both thioglycollate-induced peritonitis and LPS-induced lung these mice showed a strong migration of cells into the peritoneal injury cavity. The number of GFP-positive WT neutrophils was signifi- To investigate the potential role of PKC-u in the inflammatory cantly higher than the number of GFP-negative Prkcq2/2 neu- response in vivo, we induced chemical peritonitis with thiogly- trophils, resulting in preferential recruitment of WT neutrophils 2/2 collate in WT and Prkcq mice. Similar numbers of leukocytes (59.2 6 3.0%, n = 6 mice; p , 0.01) (Fig. 4C). 2/2 were observed in peritoneal lavage obtained from WT and Prkcq Inhaled LPS induces a robust recruitment of neutrophils (33). 6 mice immediately after thioglycollate injection (1.49 6 0.26 3 10 In this model of LPS-induced lung injury, the numbers of GFP- 6 2/2 cells and 1.99 6 0.31 3 10 cells for WT and Prkcq mice, positive WT neutrophils was twice as high as the number of GFP- respectively). Differential cell counts revealed that the peritoneal negative Prkcq2/2 neutrophils, accounting for 66.6 6 1.3% of all lavage obtained at this time (0 h) predominantly contained resident neutrophils in the bronchoalveolar lavage at 24 h (n = 5 mice; p , 2/2 peritoneal macrophages for WT and Prkcq mice (∼95%). At 0.0001) (Fig. 4D). 4 h after thioglycollate injection, the peritoneal lavage showed a strong leukocyte accumulation in WT and Prkcq2/2 mice (Fig. Mobilization of Mac-1 to the cell surface is not impaired in u 4A). However, this effect was significantly less pronounced in activated PKC- gene-deficient neutrophils Prkcq2/2 mice (6.35 6 0.63 3 106 and 3.91 6 0.50 3 106 cells for LFA-1 expression was similar for WT and Prkcq2/2 neutrophils WT and Prkcq2/2 mice, respectively). Differential cell counts before and after stimulation with TNF-a or C5a (data not shown). revealed a dramatic increase in peritoneal neutrophil influx oc- Neutrophil activation by chemoattractants leads to increased sur- curring 4 h after thioglycollate injection, reaching 72% of perito- face expression of the b2 integrin Mac-1 (4). Mac-1 expression neal leukocytes obtained from WT mice (4.57 6 0.64 3 106 cells), was significantly higher on Prkcq2/2 compared with WT neu- but only 42% of peritoneal leukocytes obtained from Prkcq2/2 trophils already under resting conditions. After stimulation with mice (1.89 6 0.35 3 106 cells) (Fig. 4B). Neutrophil cell counts in TNF-a or C5a, a similar degree of Mac-1 mobilization to the cell the peripheral blood before and 4 h after thioglycollate injection surface could be demonstrated for WT and Prkcq2/2 neutrophils were similar for WT and Prkcq2/2 mice (data not shown). (Fig. 5). 4048 PKC-u IN NEUTROPHIL ADHESION Downloaded from

FIGURE 3. PKC-u is required for sustained adhesion and transmigration in the inflamed cremaster in vivo. (A) The number of adherent neutrophils after intra-arterial injection of 600 ng CXCL1 at t = 0 was assessed every 15 s, beginning 30 s after injection (when no rolling cells could be detected) and ending http://www.jimmunol.org/ 3 min after injection in cremaster postcapillary venules of bone marrow mixed chimeric mice 6 wk after transplantation. The number of adherent cells at each time point is expressed as a percentage of maximum adherent cells after CXCL1 stimulation. (B) The cumulative number of neutrophils rolling through one field of view from the same experiments, starting at 30 s after CXCL1 injection, when all rolling had stopped after CXCL1 stimulation. Cell numbers were normalized to the WT-to-KO proportion of blood neutrophils for each animal (48.8: 52.1 6 13.0%) (n = 7 mice). Data are presented as means 6 SEM. (C) The number of transmigrated neutrophils was assessed after 40 min fMLP superfusion (10 mM) of the cremaster muscle in bone marrow mixed chimeric mice (n = 4 mice). Data are presented as means 6 SEM. Significance was determined by a Student t test. ***p , 0.001. (D and E) One rep- resentative field of view from the transmigration experiment before (D) and after 40 min (E) fMLP superfusion. LysM-GFP+ WT neutrophils are displayed in green; Prkcq2/2 neutrophils are not colored. by guest on September 26, 2021 Discussion comparison with WT neutrophils. This suggested a defect in With this work, to our knowledge we describe the first func- integrin-dependent signaling, but this test did not account for tional role for the novel PKC isoform PKC-u in neutrophils. We shear forces from the blood flow. Our in vivo investigations show demonstrate that PKC-u mediates one of the steps of neutrophil that neutrophil arrest is intact in the absence of PKC-u, but ad- adhesion, that is, neutrophil adhesion strengthening. Adhesion hesion cannot be sustained, resulting in premature release of strengthening follows arrest and depends on integrin outside–in arrested neutrophils back into the circulation. We reasoned that signaling (43, 44). Consistent with adhesion strengthening as this defect would translate into a neutrophil recruitment defect in a critical step in the inflammatory response, Prkcq2/2 neutrophils models of inflammation, analogous to what was seen in mice with showed significantly reduced recruitment in different models of defective integrin outside–in signaling. Deficiency in signaling acute inflammation. molecules downstream from integrins was shown to lead to de- PKC-u has been described as the major PKC isoform of T cells. fective neutrophil resistance to shear forces from the blood flow, Its presence in neutrophils was demonstrated in 2002 (23), but its resulting in detachment from the endothelial surface and impaired function was not investigated. In T cells, PKC-u has been shown recruitment (10, 11, 45, 47). Indeed, we found significant defects 2/2 to mediate the activation of LFA-1 after TCR stimulation (27). in Prkcq neutrophil recruitment in two models of sterile in- This signaling cascade shares some similarities with the inside– flammation. Furthermore, neutrophil transmigration through the out signaling pathway following the activation of neutrophil endothelial wall was significantly impaired in the cremaster GPCRs by chemokines. Initially, we thought that PKC-u might model. activate CALDAG-GEF1 in neutrophils in a similar way as it Although initial arrest is LFA-1–dependent, the main b2 integrin activates RapGEF2 in T cells. If that were true, Prkcq2/2 neu- in the context of neutrophil adhesion strengthening seems to be trophils would not be able to activate LFA-1 in response to che- Mac-1 (5, 6). Thus, we hypothesized that the expression of Mac-1 2/2 mokine and therefore would not be able to arrest. However, the might be reduced in Prkcq neutrophils. Using FACS analy- data we obtained in vitro and in vivo showed that our initial hy- sis, we showed normal LFA-1 and doubled Mac-1 expression in 2/2 pothesis was incorrect. Prkcq neutrophils. The apparent dysregulation of Mac-1 ex- Therefore, we investigated the possibility that integrin outside– pression in these mice deserves further investigation, but certainly in signaling may require PKC-u. This signaling involves SYK does not explain the adhesion defect. and Src kinases, the adaptor proteins DAP12 and FcRg, PI3K, Some previous work had addressed the role of other PKC iso- the guanine exchange factors VAV1 and VAV3, and the small forms in neutrophil adhesion. Chou et al. (48) provided evidence GTPases Cdc42, RhoA, or Rac1 (11, 38, 45–47). On a surface for a role of PKC-d in neutrophil adhesion. Similar to PKC-u, coated with poly-RGD, a synthetic ligand for the aVb3 integrin, PKC-d belongs to the PKC subfamily of novel PKC isoforms. the adhesion of Prkcq2/2 neutrophils was strongly reduced in Upon stimulation with fMLP or IL-8, adhesion of PKC-d gene- The Journal of Immunology 4049 Downloaded from

FIGURE 4. Neutrophil recruitment in a model of thioglycollate-induced neutrophil peritonitis and LPS-induced lung injury is attenuated for PKC-u gene-deficient neutrophils. (A) Total leukocyte count and (B) differential cell count for neutrophils in the peritoneal lavage 4 h after injection of 4% 2/2 thioglycollate into the peritoneum of Prkcq and WT mice. Total numbers of leukocytes and neutrophils in the peritoneal cavity were assessed using http://www.jimmunol.org/ cytospins stained with H&E. Data are means 6 SEM (n = 6–10 mice). (C) The percentage of Prkcq2/2 versus LysM-GFP+ neutrophils in the peritoneal lavage normalized to the respective cell numbers in the peripheral blood 4 h after injection of 4% thioglycollate into the peritoneum of bone marrow chimeric Prkcq2/2/LysM-GFP+ C57BL/6 mice was evaluated using flow cytometry (n = 6 mice). Data are expressed as means 6 SEM. (D) The percentage of Prkcq2/2 versus LysM-GFP+ neutrophils in the bronchoalveolar lavage normalized to the respective cell numbers in the peripheral blood 24 h after LPS inhalation of bone marrow chimeric Prkcq2/2/LysM-GFP+ C57BL/6 mice was evaluated using flow cytometry (n = 5 mice). Data are expressed as means 6 SEM. Significance was determined by a Student t test. **p , 0.01, ***p , 0.001.

deficient neutrophils was reduced ∼50% compared with WT neu- Evidence for a potential involvement of PKC isoforms in by guest on September 26, 2021 trophils in a static in vitro assay. These findings are compara- integrin outside–in signaling also comes from studies with ble to our results for in vitro adhesion on poly-RGD. Similar to . Spreading of platelets on fibrinogen is mediated by poly-RGD, IL-8 induces adhesion in an integrin-dependent way outside–in signaling downstream of aIIbb3 integrin and can be (49). In contrast to PKC-u gene deficiency, absence of PKC-d also prevented by knockdown of PKC-b (51) or PKC-u (17). PKC-u leads to reduced fMLP-induced adhesion. However, fMLP- has been shown to be constitutively associated with aIIbb3 induced neutrophil adhesion is independent of b2 integrin sig- integrin and associated with SYK upon binding of the to naling (50). fibrinogen (17). The neutrophil phenotype of Prkcq2/2 mice we demonstrate in this work resembles that of neutrophils deficient in SYK regarding the impaired sustained adhesion, but intact migration toward chemoattractants. However, SYK-deficient neutrophils also show impaired integrin-dependent respiratory burst and degranulation (10), but both integrin-dependent neu- trophil functions were not disturbed in Prkcq2/2 neutrophils. SYK activates VAV1 and VAV3, which activate different small GTPases and thereby mediate the different integrin-dependent neutrophil functions, such as adhesion strengthening, phagocy- tosis, and the respiratory burst (45). Our findings suggest that PKC-u is a member of the b2 integrin outside–in signaling pathway, where it is potentially located downstream of the VAV GEFs. In summary, to our knowledge, this work is the first to dem- onstrate a functional role of PKC-u in neutrophils. We show that FIGURE 5. PKC-u gene-deficient neutrophils show a higher Mac-1 PKC-u is required for timely neutrophil spreading and sustained expression under resting conditions and after treatment with inflammatory adhesion, suggesting that PKC-u isanewlyrecognizedcom- stimuli. Surface expression levels of Mac-1 of Prkcq2/2 and WT bone ponent of the integrin outside–in signaling cascade. Defective marrow isolated neutrophils were assessed using flow cytometry. Cells a neutrophil recruitment to the airways and peritoneal cavity in were incubated with 100 ng/ml TNF- , 50 ng/ml C5a, or medium for 2/2 30 min at 37˚C and then stained with allophycocyanin-conjugated rat Prkcq mice shows that this mechanism is relevant for a nor- anti-mouse CD11b mAb. Data are presented as means 6 SEM (n = 6–12 mal response to inflammatory stimuli. Targeting PKC-u by mice for each group). Significance was determined by ANOVA. *p , 0.05, pharmacological means may therefore be expected to result in **p , 0.01. defects in host defense, or it could be beneficial in diseases 4050 PKC-u IN NEUTROPHIL ADHESION where overabundant neutrophil recruitment contributes to the 23. Balasubramanian, N., S. H. Advani, and S. M. Zingde. 2002. Protein kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neu- disease process. trophils and changes during myeloid maturation in chronic myeloid leukemia. Leuk. Res. 26: 67–81. 24. Sun, Z., C. W. Arendt, W. Ellmeier, E. M. Schaeffer, M. J. Sunshine, L. Gandhi, Acknowledgments J. Annes, D. Petrzilka, A. Kupfer, P. L. Schwartzberg, and D. R. Littman. 2000. We thank Amnon Altman for providing the Prkcq KO mice. PKC-u is required for TCR-induced NF-kB activation in mature but not im- mature T . Nature 404: 402–407. 25. Pfeifhofer, C., K. Kofler, T. Gruber, N. G. Tabrizi, C. Lutz, K. Maly, M. Leitges, and G. Baier. 2003. 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