Neutrophil infiltration during inflammation is regulated ...

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Neutrophil infiltration during inflammation is regulated by PILRa via modulation of integrin activation

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© 2013 Nature America, Inc. All rights reserved.

Jing Wang1,2, Ikuo Shiratori1,5, Junji Uehori1,2,5, Masahito Ikawa3 & Hisashi Arase1,2,4 Acute inflammatory responses are important in host defense, whereas dysregulated inflammation results in life-threatening complications. Here we found that paired immunoglobulin-like type 2 receptor alpha (PILRa), an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), negatively regulated neutrophil infiltration during inflammation. Pilra−/− mice had increased neutrophil recruitment to inflammatory sites and were highly susceptible to endotoxin shock. Pilra−/− neutrophils showed enhanced transmigration ability and increased adhesion to the b2 integrin ligand ICAM-1. PILRa expressed on neutrophils constitutively associated in cis with its ligands, resulting in clustering of PILRa during stimulation with a chemoattractant. Clustering of PILRa enhanced ITIM-mediated signaling, thus modulating b2 integrin inside-out activation. These data demonstrate that neutrophil recruitment in inflammatory responses is regulated by PILRa via modulation of integrin activation. In response to infection or injury, the host immune system initiates swift and robust inflammatory responses to restrict the spread of harmful agents. This process is characterized mainly by vascular changes with delivery of leukocytes to the sites of infection or injury. However, if the initial host response is dysregulated or overamplified, inflammation results in severe host tissue damage and can eventually lead to life-threatening complications such as multiple organ failure1,2. Neutrophils are considered key players in this process3. Neutrophil recruitment from the blood to the inflamed tissue is initiated by selectin family molecules that facilitate cell rolling followed by chemotactic activation of β2 integrins that mediate firm adhesion and migration across the blood vessel wall4. This process is characterized by a dynamic conformational shift of β2 integrins from low to high binding affinity to their ligand ICAM-1 expressed on endothelial cells. The interaction of activated β2 integrins with ICAM-1 leads to neutrophil arrest and initiates transendothelial migration. An endo genous integrin antagonist, Del-1, has been reported to inhibit αLβ2 integrin–dependent adhesion and thereby suppress recruitment of inflammatory cells5. Therefore, rigorous control of integrin activation is necessary to protect against neutrophil-mediated tissue damage during inflammation. Although several molecules linking chemokine receptors with integrin activation have been reported6,7, molecules that negatively regulate this pathway remain unidentified. The paired immunoglobulin-like type 2 receptor comprises the inhibitory receptor, PILRα, and the activating receptor, PILRβ. PILRα is mainly expressed on macrophages, dendritic cells and granulocytes, whereas PILRβ is mainly expressed on activated natural killer cells8. Both inhibitory PILRα and activating PILRβ are well conserved in most mammals, although human PILRβ seems to be nonfunctional9,10.

PILRα contains two ITIMs in its cytoplasmic domain and recruits the phosphatases SHP-1 and SHP-2 in pervernadate-activated cells11. We and others have reported that PILRα associates with several host ligands such as CD99, PILR-associating neural protein, neuronal differentiation proliferation factor-1 and collectin 12 (refs. 8,12,13). Furthermore, we have found that human PILRα has an important role in membrane fusion during herpes simplex virus 1 infection by associating with envelope glycoprotein B14–16. Sialic acid–containing O-glycosylation of these ligands is required for association with both PILRα and PILRβ13,15,17. However, it has remained unclear how these host ligands regulate PILRα function in vivo. Although studies using Pilrb1−/− mice suggested that activating PILRβ is involved in controlling acute S. aureus–mediated pneumonia18, physiological functions of the inhibitory PILRα in the immune response have not been studied. Here we analyzed the function of PILRα by generating Pilra−/− mice. We found that these mice were highly susceptible to lipopolysaccharide (LPS)-induced endotoxin shock and suffered severe tissue damage accompanied by increased neutrophil accumulation in the tissues. Furthermore, chemoattractant-triggered activation of neutrophil β2 integrin was negatively regulated by PILRα through associating with its ligands in cis. Our findings reveal a self-regulatory mechanism of neutrophil recruitment via regulation of the activation of β2 integrins and show an important role of PILRα in regulating acute inflammatory reactions. RESULTS Pilra−/− mice are highly susceptible to endotoxin shock To investigate the physiological roles of PILRα in immune responses in vivo, we generated Pilra−/− mice (Supplementary Fig. 1a–c).

1Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. 2Laboratory of Immunochemistry, World Premier International (WPI) Immunology Frontier Research Center, Osaka University, Osaka, Japan. 3Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. 4Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8, Honcho Kawaguchi, Saitama, Japan. 5Present addresses: Nippon Electric Company (NEC) Tsukuba Research Laboratories, Miyukigaoka, Tsukuba, Ibaraki, Japan (I.S.) and Kobe Pharma Research Institute, Nippon Boehringer Ingelheim, Kobe, Japan (J.U.). Correspondence should be addressed to H.A. ([email protected]).

Received 6 August; accepted 21 September; published online 11 November 2012; doi:10.1038/ni.2456

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These mice were born at the expected Mendelian ratio, and they developed normally in a specific pathogen–free animal facility. Pilra−/− mice had normal composition of myeloid cells and lympho cyte populations in the spleen, peripheral blood and bone marrow (BM; Supplementary Fig. 2a–c and data not shown). Consistent with a previous report8, analysis of PILRα expression in splenocytes and BM cells demonstrated that PILRα was preferentially expressed on Ly-6C+CD11bhigh cells (granulocytes and neutrophils), but was also expressed on macrophages, monocytes and dendritic cells (Supplementary Fig. 1d). To investigate the role of PILRα in inflammatory responses, we challenged Pilra−/− and wild-type mice with LPS intraperitoneally. Pilra−/− mice had higher mortality rates than wild-type mice did (Fig. 1a). However, there were no differences in circulating cytokine levels in wild-type and Pilra−/− mice (Supplementary Fig. 3a). We also investigated cytokine production by peritoneal macrophages and found that, consistent with the in vivo results, both wild-type and Pilra−/− macrophages produced similar amounts of interleukin 6 (IL-6) and tumor necrosis factor upon stimulation with LPS (Supplementary Fig. 3b). These data suggest that PILRα is not involved in the regulation of cytokine production in response to stimulation of Toll-like receptor (TLR). Endotoxemia in septic shock is associated with multiple organ damage, which sometimes results in death. Therefore, we mea sured serum amounts of alanine aminotransferase, which reflects hepatic injury, as well as blood urea nitrogen, a marker of renal function, and lactate dehydrogenase released from several tissues after administration of LPS. The concentration of these factors was significantly higher in Pilra−/− mice than in wild-type mice (Fig. 1b). In addition, we examined liver sections from Pilra−/− and wild-type mice 24 h after administration of LPS. Livers from Pilra−/− mouse exhibited obvious massive hemorrhage, disorganization of the hepatic cords and karyolysis of hepatocytes (Fig. 1c). These results imply that severe organ damage might contribute to the high mortality in Pilra−/− mice after LPS administration. Neutrophils are known to accumulate at inflamed tissue sites and to have pivotal roles in tissue injury in septic shock19. Immunohistochemical analysis revealed more neutrophils in Pilra−/− mice than in wildtype mice (Fig. 1d). We quantified neutrophil infiltration into liver and lungs of LPS-challenged mice by measuring myeloperoxidase (MPO) activity20. Tissues from Pilra−/− mice had higher MPO activity compared with wild-type mice, consistent with the histological analyses (Fig. 1e). These data suggest that neutrophil recruitment

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PILRa regulates neutrophil responses to chemotactic stimuli In vitro assays testing the migratory capacity of neutrophils toward chemotactic factors showed that, compared with wild-type neutro phils, Pilra−/− cells exhibited increased transmigration toward the chemoattractant fMLF (Fig. 3a). Pilra−/− neutrophils also exhibited increased transmigration to chemokine CXCL1 (Fig. 3b). Actin polymerization is considered to be the driving force for cell migration22. However, we observed no notable difference in fMLF-induced actin polymerization between wild-type and Pilra−/− neutrophils (Supplementary Fig. 4a). Therefore, PILRα seems not to be involved in regulation of cell motility. Intracellular oxidant production induced by fMLF was also comparable between both genotypes (Supplementary Fig. 4b). Because adhesive events are required for neutrophil migration through the blood vessels, we examined fMLFinduced adhesion. Static adhesion to immobilized ICAM-1 after fMLF stimulation was increased in Pilra−/− neutrophils (Fig. 3c). A monoclonal antibody (mAb) to β2 integrin (GAME-46) completely blocked

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Pilra−/− mice have increased neutrophil recruitment in vivo To directly examine the role of PILRα in neutrophil recruitment to sites of inflammation, we used the thioglycollate-induced sterile peritonitis model. Thioglycollate elicits a sequential recruitment of leukocytes into the peritoneum, starting with an influx of neutrophils (1–24 h after treatment with thioglycollate) followed by an influx of macrophages. Using this model, we counted neutrophils in the peritoneal exudate from Pilra−/− mice and found that their number was increased relative to that in wild-type mice at early time points (Fig. 2a). However, there was no difference in macrophage recruitment 72 h after administration of thioglycollate (Fig. 2b). To exclude the effects of different environmental factors, we performed a competitive recruitment assay21. We labeled purified wild-type and Pilra−/− neutrophils with different fluorescent cell tracers and mixed them in a 1:1 ratio before intravenously injecting them into wild-type mice 2 h after thioglycollate injection. After another 2 h, we collected blood and peritoneal cells, and counted labeled neutrophils. Although the original ratio of Pilra−/− to wild-type neutrophils was essentially maintained in blood, we observed remarkably more Pilra−/− neutrophils in the peritoneum (Fig. 2c). This suggests that PILRα regulates neutrophil infiltration into sites of inflammation.

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Figure 1 Increased mortality and higher neutrophil infiltration in LPS-induced lethal shock in Pilra−/− mice. (a) Survival of wild-type (WT) and Pilra−/− mice (n = 11 each) intraperitoneally challenged with LPS (30 mg per kg body weight). Data are presented as a Kaplan-Meier plot. P = 0.0071 (log-rank test). (b) Serum concentrations of aspartate aminotransferase (AST), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in wild-type and Pilra−/− mice (n = 9 per group) 24 h after administration of LPS. Serum samples from mice given PBS (n = 5 per group) were used to determine the basal concentrations. IU, international units. (c) Hematoxylinand-eosin staining of liver sections from wild-type and Pilra−/− mice 24 h after LPS injection. (d) Cryostat sections of livers from wild-type and Pilra−/− mice stained with FITC-conjugated anti-mouse Gr-1 to visualize the aggregation of neutrophils. Original magnification (c,d), ×200; scale bars, 100 µm. (e) MPO activity in liver and lung samples at indicated time points after LPS administration. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three independent experiments (error bars (b,e), s.d.).

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Articles Figure 2 Pilra−/− mice have increased neutrophil recruitment in thioglycollate-induced peritonitis. (a) Numbers of Gr-1+ cells in the Peritoneal Blood 30 ** 10 104 WT peritoneal cavity at the indicated time points after thioglycollate 25 –/– 8 Pilra 3 + 10 20 injection (n = 5 per group). (b) Numbers of F4/80 cells in the 2.2 4.5 6 ** 15 2 10 peritoneal cavity 72 h after thioglycollate injection (n = 5 per group). * 4 10 4.3 4.6 1 Each symbol represents an individual mouse; small horizontal lines 10 2 5 0 indicate the mean. (c) Representative flow cytometry analysis of 0 0 10 0 1 2 3 4 WT Pilra–/– 10 10 10 10 10 12 24 4 8 blood and peritoneal exudates from a thioglycollate-treated mouse –/– Time (h) FarRed (Pilra ) −/− receiving a mixture of labeled wild-type and Pilra neutrophils. Numbers adjacent to outlined areas indicate percentage of fluorescence-labeled cells in total peritoneal or blood cells. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from one representative of three independent experiments (error bars (a), s.d.). +

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PILRa negatively regulates integrin inside-out signaling β2 integrins have key roles in mediating neutrophil adhesion and transmigration. Because Pilra−/− neutrophils exhibited enhanced adhesion, we tested whether PILRα inhibits chemoattractant-induced activation of β2 integrin in neutrophils. Activation of β2 integrin is accompanied by a conformational change from a low-affinity to a high-affinity state for its ligands. Pilra−/− neutrophils exhibited increased binding to

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PILRa associates in cis with sialylated glycoprotein ligands The intrinsic differences between wild-type and Pilra−/− neutrophils suggested that downstream signals from PILRα might be initiated without exogenous ligand-induced dimerization or cross-linking. To investigate whether PILRα could associate with an exogenous ligand, we stained neutrophils with PILRα-ligand-Fc chimeric proteins. Although neutrophils highly express PILRα, as detected by mAb to PILRα, these cells could not be stained by either of the PILRα-ligandFc chimera proteins (Supplementary Fig. 5). In contrast, neutrophils were stained with PILRα-Fc (Fig. 4a). This suggested that abundant endogenous PILRα ligands are expressed on the neutrophil cell surface, and they might mask the ligand-binding site of PILRα. Specific sialylated O-glycan structures are required for PILRα ligand recognition15,17. Therefore, we treated neutrophils with sialidase, which cleaves sialic acid from cell surface glycoproteins, thereby abrogating the binding of PILRα to its ligands17. We confirmed the efficiency of removal of sialic acid by sialidase by loss of PILRα-Fc binding to sialidase-treated neutrophils (Fig. 4a). Desialylation markedly increased binding of PILRα-ligand-Fc chimera protein to wild-type

neutrophils but not to Pilra−/− neutrophils. Sialidase treatment had no remarkable effects on cell-surface expression of PILRα (Fig. 4a). These results indicate that PILRα associates in cis with sialylated glycoprotein ligands expressed on neutrophils. We next investigated whether the interaction of PILRα with its ligands in cis would modulate downstream signaling during chemotactic stimulation. We immunoprecipated PILRα from neutrophils and assessed its phosphorylation status using antibodies to phosphotyrosine or phosphatases. The ITIM of PILRα was constitutively phosphorylated and associated with the tyrosine phosphatases SHP-1 and SHP-2 in the resting state (Fig. 4b). Tyrosine phosphorylation of PILRα increased transiently after stimulation with fMLF, which led to recruitment of SHP-1 and SHP-2 (Fig. 4b). The ITIM of PILRα on sialidase-treated neutrophils, which lack association in cis with its ligands, was also constitutively phosphorylated, but stimulation with fMLF did not further enhance the phosphorylation of the ITIM of PILRα or the recruitment of phosphatases (Fig. 4c). These results suggest that the cis interaction of PILRα with its ligands does not affect the constitutive tyrosine phosphorylation of PILRα and the association with phosphatases, but these interactions regulate the dynamic, temporal activation of the downstream signaling of PILRα during chemotactic stimulation.

Figure 3 Pilra−/− neutrophils have enhanced ** WT WT WT * 50 50 ** responses to chemotactic factors. (a,b) Numbers Pilra–/– Pilra–/– Pilra–/– *** 18 10 −/− of wild-type (WT) or Pilra neutrophils crossing ** Transwell inserts coated with mouse ICAM-1– 12 ** 25 25 Fc in response to fMLF (a) or CXCL1 (b). 5 ** (c) Adhesion of wild-type or Pilra−/− neutrophils 6 stimulated with fMLF at the indicated doses 0 0 0 0 and then transferred to plates coated with IgG 0 10 100 0 1 3 0 1 3 ICAM-1–Fc and incubated for 10 min. CXCL1 (ng/ml) fMLF (µm) fMLF (µm) (d) Blocking of neutrophil adhesion by β2 integrin–blocking antibody, GAME-46 WT fMLF (µm) 0 0.25 0.75 2.5 7.5 25 50 Pilra–/– (neutrophils were incubated with 10 µg/ml +/+ –/– +/+ –/– +/+ –/– +/+ –/– +/+ –/– +/+ –/– GAME-46 before stimulation with 3 µM fMLF). p-Erk (e) Adhesion of wild-type or Pilra−/− neutrophils Erk 25 stimulated with fMLF at the indicated doses Time (s) 0 10 30 60 90 180 and then transferred to plates coated with +/+ –/– +/+ –/– +/+ –/– +/+ –/– +/+ –/– +/+ –/– hyaluronic acid and incubated for 5 min. p-Erk 0 (f) Immunoblots of phosphorylated Erk (p-Erk) 0 1 3 Erk and total Erk in lysates of wild-type (+/+) or fMLF (µm) Pilra−/− (–/–) neutrophils stimulated with the indicated doses of fMLF for 45 s or with 3 µM fMLF for the indicated durations. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test). Data are from one representative of five independent experiments (error bars (a–e), s.d.).

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the adhesion of both wild-type and Pilra−/− neutrophils (Fig. 3d). Equal numbers of wild-type and Pilra−/− neutrophils bound to immobilized hyaluronic acid, which is a ligand for adhesion molecule CD44 (Fig. 3e). These results suggest that PILRα specifically regulates integrin-mediated adhesion. Next, we investigated the activation of intracellular signaling molecules upon fMLF stimulation. Pilra−/− neutrophils exhibited a greater dose-dependent and time-dependent increase in Erk phosphorylation than wild-type neutrophils (Fig. 3f). These results indicate that a deficiency of PILRα results in augmented signaling and functional responses of neutrophils to fMLF.

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Clustering of PILRa during neutrophil activation Neutrophils exhibit polarized morphology rapidly upon stimulation with chemokines or chemoattractants, with β2 integrins polarized at the leading edge. To further unravel the function of PILRα in activation of β2 integrin, we examined the subcellular localization of PILRα in resting and activated neutrophils. In unstimulated neutrophils,

PILRα was uniformly distributed on the cell surface (Fig. 6a). After permealibization of cells with Triton X-100, PILRα was observed in perinuclear regions (Fig. 6a). Cell-surface staining of PILRα increased rapidly after stimulation with fMLF (Fig. 6b). In contrast, subcellular staining of PILRα consistently decreased upon stimulation with fMLF (data not shown). Thus, PILRα seems to be mobilized to the cell surface from the intracellular pool after stimulation through chemokine receptors. Because stimulation with a chemoattractant induces neutrophil polarization and redistribution of cell-surface receptors, we next examined the distribution of PILRα after stimulation with fMLF. Neutrophils are polarized with the formation of a leading edge and uropod upon fMLF stimulation. CD44 was concentrated at uropod in polarized neutrophils and therefore was used as a uropod marker (Fig. 6c). Compared with its uniform distribution in nonpolarized resting cells, PILRα was preferentially concentrated at the leading edge of the cell in stimulated neutrophils (Fig. 6c,d). We also treated neutrophils with sialidase, which disturbs PILRα cis association with ligands, and examined the translocation of PILRα. Treatment with sialidase resulted in normal redistribution of CD44 to the uropod

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Figure 5 Interactions of PILRα and cis ligands modulate neutrophil 4 integrin inside-out activation after chemoattractant stimulation. 1 (a) Binding of ICAM-1–Fc to wild-type (WT) or Pilra−/− neutrophils 20 40 60 80 100 stimulated with 3 µM fMLF for the indicated durations. For integrin Time (s) blocking, neutrophils were incubated with 10 µg/ml GAME-46 before stimulation. (b) Binding of ICAM-1–Fc to sialidase-treated or control vehicle (PBS)-treated wild-type and Pilra−/− neutrophils stimulated as in a. (c) Intracellular calcium influx in purified wild-type and Pilra−/− neutrophils loaded with Fluo-4 AM and Fura red, measured by flow cytometry before and after addition of indicated doses of fMLF or ionomycin. (d) Total Rap1 and GTP-bound Rap1 protein in wild-type and Pilra−/− neutrophils stimulated with 3 µM fMLF. *P < 0.01 (Student’s t-test). Data are from one representative of five experiments (error bars (a,b), s.d.).

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s oluble ICAM-1–Fc after stimulation with fMLF (Fig. 5a). There was no marked difference in the expression of cell-surface β2 integrins (Supplementary Fig. 6a). ICAM-1–Fc binding to wild-type and Pilra−/− neutrophils was completely blocked by a β2 integrin–blocking mAb (Fig. 5a). Sialidase treatment, which disrupts the cis interaction of PILRα with its ligands, reduced the enhanced binding of Pilra−/− neutrophils to ICAM-1 and resulted in wild-type neutrophils and Pilra−/− neutrophils binding with similar affinity to ICAM-1 (Fig. 5b). Thus, PILRα seems to regulate the conformational change of β2 integrin to its activated state during the neutrophil response to chemotactic stimulation, although the possibility that sialidase treatment affects integrin activation in a PILRα-independent manner cannot be excluded23. Because integrin activation is known to be mediated by an early increase in intracellular Ca2+ followed by a late activation of the small GTPase Rap1 (refs. 2,24), we examined the fMLF-induced intra cellular calcium influx. There was no difference in calcium response between wild-type and Pilra−/− neutrophils upon ionomycin stimulation. When we incubated cells with different doses of fMLF, Pilra−/− neutrophils showed higher calcium responses than wild-type neutro phils at any fMLF concentration, especially at lower doses of fMLF (Fig. 5c), suggesting that Pilra−/− neutrophils were more sensitive to fMLF-induced calcium influx than wild-type neutrophils. Activation of Rap1 (GTP-bound Rap1) after fMLF stimulation of Pilra−/− neutro phils was increased and prolonged compared to that for wild-type neutrophils (Fig. 5d). Other small GTPases, such as Cdc42, RhoA and Rac-1, which are also reported to be involved in integrin activation25, were activated similarly by fMLF in wild-type and Pilra−/− neutrophils (Supplementary Fig. 6b). These results suggest that PILRα restrains integrin activation mediated by G protein–coupled receptor (GPCR) by regulating GPCR-induced calcium response and Rap1 activation.

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Ctrl Figure 4 PILRα associates with ligands in cis –/– WT + fMLF WT + fMLFPilra Sialidase on neutrophils. (a) Flow cytometry analysis of Time (s): Ctrl 30 60 120 Ctrl Time (s): Ctrl 30 60 120 IB: p-Tyr IB: p-Tyr PILRα-Fc chimera proteins binding to wild-type WT 1.0 0.9 1.0 0.9 1.0 1.8 1.5 1.0 (WT) or Pilra−/− neutrophils treated with sialidase SHP-1 SHP-1 or a vehicle control (Ctrl). (b) Immunoblot (IB) 1.0 1.3 1.5 2.0 1.0 1.0 1.1 0.7 50 of tyrosine phosphorylation status of PILRα in SHP-2 SHP-2 40 wild-type (WT) or Pilra−/− neutrophils (1 × 107) –/– 30 1.0 1.0 0.8 1.0 1.0 1.8 2.3 1.8 Pilra 20 left resting (Ctrl) or stimulated with fMLF for 10 PILRα PILRα indicated times, followed by immunoprecipitated 0 0 1 2 3 4 10 10 10 10 10 (IP) with polyclonal antibody to PILRα IP: anti-PILRα IP: anti-PILRα PILRα-Fc PILRα gB300-Fc CD99-Fc (anti-PILRα) and immunoblotted with antibodies to indicated proteins. Numbers under blots indicate fold induction of phosphorylated proteins or phosphatases over unstimulated cells (Ctrl in first lane) after normalizing to total PILRα proteins. (c) Analysis of the tyrosine phosphatases of PILRα in sialidase-treated WT neutrophils stimulated and processed as described in b. Data are representative of five independent experiments.

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Figure 6 PILRα aggregates at the leading edge of polarized neutrophils. (a) Confocal microscopy of PILRα in purified neutrophils that were fixed in suspension and treated with or without 0.3% Triton X-100. (b) Flow cytometry analysis of PILRα expression on neutrophils stimulated for 0–5 min with 3 µM fMLF. MFI, mean fluorescence intensity. (c) PILRα distribution on cell surface after neutrophils were stimulated with 3 µM fMLF (+) or not stimulated (–) in suspension and stained with anti-PILRα and anti-CD44. (d) Quantitative analysis of PILRα distribution on polarized neutrophils described in c showing PILRα accumulation at the opposite side to the location of CD44. Results are presented as a percentage of total polarized cells as defined by the presence of CD44-positive uropods (100 cells; mean ± s.d. of three independent experiments). Scale bars, 10 µm (a,c).

upon stimulation with fMLF. However, PILRα maintained a diffuse distribution similar to that in the resting state in sialidase-treated cells (Fig. 6c,d). This suggested that in the absence of cis association with ligands, PILRα could not polarize in response to chemotactic stimulation. We next asked whether the redistribution of PILRα affected the subcellular localization of phosphatases SHP-1 and SHP-2. We observed no marked differences in the distribution of SHP-1 and SHP-2 in wild-type or Pilra−/− neutrophils in the resting state; both SHP-1 and SHP-2 were distributed throughout the plasma membrane and the cytoplasm (Fig. 7a,b). Upon stimulation with fMLF, SHP-2 was recruited to the perinuclear region in neutrophils of both genotypes, indicating that redistribution of SHP-2 was independent of PILRα. After stimulation with fMLF, however, SHP-1 was preferentially redistributed to the leading edge in wild-type neutrophils but not Pilra−/− neutrophils (Fig. 7c,d). These results suggests that the redistribution of SHP-1 in activated neutrophils is PILRα-dependent. Collectively, these findings demonstrate that PILRα is spatially clustered at the leading edge upon chemotactic stimulation and concequently affects the redistribution of SHP-1. DISCUSSION Here we found that an ITIM-bearing inhibitory receptor, PILRα, which is expressed on neutrophils, is important in regulating integrin activation. Pilra−/− neutrophils had increased binding capacity to the integrin ligand ICAM-1 after chemotactic stimulation. Pilra−/− mice were more highly susceptible to LPS-induced endotoxin shock than wild-type mice were. Increased accumulation of neutrophils and consequent severe damage to the inflamed tissues contributed to the high mortality of Pilra−/− mice. Neutrophils are one of the first responders in systemic inflammation caused by infection or injury3. In systemic inflammation induced by 38

endotoxin challenge, neutrophils are sequestered at microcapillaries in several organs26,27. Depletion of neutrophils protects against LPS hepatotoxicity28, and neutrophil elastase-deficient and cathepsin G–deficient mice are resistant to endotoxin shock29. These reports suggested that neutrophils are the major effector cells in endotoxemia. Although neutrophil accumulation in liver sinusoids is mediated by mechanical processes at the early stage after LPS administration30, subsequent accumulation of neutrophils in postsinusoidal venules or portal venules and the migration of neutrophils into the parenchyma requires β2 integrin–ICAM-1 interaction31. After extravasation, proteases and reactive oxygen radicals are released from activated neutrophils and participate in tissue injury. Therefore, appropriate regulation of neutrophil infiltration is important for protecting against inflammation-associated damage. Although several other ITIMbearing receptors are also expressed on neutrophils32,33, none have been implicated in protecting against excessive neutrophil infiltration in lethal inflammation. Therefore, PILRα has crucial and nonredundant roles in regulating neutrophil recruitment in inflammation. Neutrophil extravasation and chemotaxis require spatiotemporal regulation of intracellular signaling events. Therefore, we propose that cellular localization is also crucial for determining the function of PILRα. Cell-surface PILRα was increased rapidly after fMLF stimulation, whereas subcellular PILRα was consistently decreased upon fMLF stimulation. Although we did not directly observe PILRα mobilization, PILRα seems to be recruited from the intracellular pool to the cell surface. PILRα was also clustered at the leading edge of the cell after chemoattractant stimulation. PILRα clustering facilitated phosphorylation of intracellular ITIM. Without an association in cis with its ligands, PILRα retained uniform distribution in activated neutrophils, and consequently, ITIM phosphorylation was essentially no greater than in resting neutrophils. Therefore, PILRα in activated neutrophils undergoes spatial regulation to modulate downstream signal transduction. Both SHP-1 and SHP-2 are recruited by PILRα. These two phosphatases regulate the neutrophil functional response in opposite ways. SHP-2 has a positive role in activating Erk and chemotactic migration34–36. Because other receptors such as Ly49Q have also been

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Figure 7 SHP-1 redistribution in activated neutrophils is mediated by PILRα. (a–d) Confocal microscopy of SHP-1 (a,c) or SHP-2 (b,d) in wild-type (WT) and Pilra−/− neutrophils left unstimulated (–) or stimulated with 3 µM fMLF (+) for 3 min before intracellular staining with anti-CD44 in combination with anti–SHP-1 or anti–SHP-2 in suspension. Asterisks in c indicate the leading edge. Scale bars, 10 µm. Over 100 cells were analyzed in three independent experiments and representative images are shown.


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Articles reported to recruit SHP-2, PILRα might have a redundant role in regulating SHP-2 translocation. In contrast to SHP-2, SHP-1 is generally a negative regulator in neutrophils37. Neutrophils from SHP-1–deficient ‘motheaten’ mice are hyperadherent to integrin ligands even in the absence of chemotactic stimulation38. We observed fMLF-induced redistribution of SHP-1 to the leading edge of the cell in wild-type neutrophils but not in Pilra−/− neutrophils. Notably, signal components that activate integrins are also redistributed to the leading edge39,40. Our observations are also consistent with a previous report of SHP-1 localization at the leading edge after stimulation with IL-8 (ref. 41). Our results showed that PILRα suppresses the fMLF-induced elevation of intracellular Ca2+, resulting from the direct stimulation of phospho lipase C beta (PLCβ) by GPCRs42. SHP-1 has been shown to directly associate with PLC-β3 in mast cells43. Hence, we propose that SHP-1 is recruited to the leading edge via interaction with PILRα, which then regulates upstream signal molecules that activate integrin. The cis ligands for PILRα on neutrophils have not yet been identified. A previously reported ligand, CD99, is expressed on many cell types including neutrophils. Homophilic CD99 interactions with endothelial cells are important in neutrophil transmigration in both mouse and human44,45. However, PILRα-Fc binds neutrophils better than would be expected from the amount of CD99 expression on the cells. Therefore, PILRα might associate with other, thus far unidentified, ligands on the neutrophil cell surface. In addition, PILRα is phosphorylated in the resting state in the absence of any interactions with cis ligands. The fact that PILRα expressed in cell lines is not phosphorylated (data not shown) suggests that PILRα expression on the cell surface in itself is insufficient for ITIM phosphorylation. Therefore, PILRα might associate with other molecules via interactions at sites other than the ligand-binding domain. Sialic acids are the most abundant terminal carbohydrate moieties on the cell surface. Sialic acid–binding immunoglobulin-like lectins, known as Siglecs, are commonly masked by sialic acids expressed on the same cell surface46. PILRα has low homology with Siglecs but has the distinguishing feature of binding to a structural domain involving both sialic acid and protein determinants13,17. Siglec masking can be overcome by high-density and/or high-affinity ligand presentation in trans47. Although PILRα interacts with its ligands with a higher affinity than Siglecs do with their glycan ligands13, herpes simplex virus infection through PILRα expressed on human monocytes suggests that the trans interaction could also occur when PILRα encounters a higher-avidity ligand14. This finding suggests that pathogens might have evolved to acquire high-avidity ligands to bind to PILRα in trans, which may facilitate their invasion or survival in the host. It also suggests that PILRα binding to trans ligands is likely to have critical functions other than modulating signals for integrin activation. In conclusion, our results indicate that PILRα has a major role in controlling neutrophil recruitment via regulating chemoattractantmediated integrin activation. We demonstrated that PILRα interacts with cis ligands on neutrophils and this interaction defines the spatial and temporal downstream signal transduction of PILRα. Our findings not only provide insights into the functional complexity of ITIMbearing inhibitory receptors but also reveal a novel inhibitory pathway for regulating integrin inside-out activation of neutrophils. Because pharmacological inhibition of integrins is of great interest for treating inflammatory disease, PILRα might be a potential therapeutic target in acute and chronic inflammatory disorders. Methods Methods and any associated references are available in the online version of the paper. nature immunology VOLUME 14 NUMBER 1 JANUARY 2013

Note: Supplementary information is available in the online version of the paper. Acknowledgments We thank M. Matsumoto, K. Shida and R. Hirohata for technical assistance, T. Suenaga, M. Kohyama, K. Hirayasu and F. Saito for discussions, and M. Okabe, A. Kawai and M. Tanaka for advice and technical assistance with the generation of the gene-targeted mice. This work was supported by research grants from JST, CREST, a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan and The Osaka Foundation for Promotion of Clinical Immunology (H.A.). AUTHOR CONTRIBUTIONS J.W. and H.A. designed the experiments, analyzed the data and wrote the manuscript. J.W. did most of the experiments. I.S., J.U. and M.I. generated Pilra−/− mice. I.S. established a mAb to PILRα. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/doifinder/10.1038/ni.2456. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Cohen, J. The immunopathogenesis of sepsis. Nature 420, 885–891 (2002). 2. Bergmeier, W. et al. Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. J. Clin. Invest. 117, 1699–1707 (2007). 3. Nathan, C. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6, 173–182 (2006). 4. Ley, K., Laudanna, C., Cybulsky, M.I. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678–689 (2007). 5. Choi, E.Y. et al. Del-1, an endogenous leukocyte-endothelial adhesion inhibitor, limits inflammatory cell recruitment. Science 322, 1101–1104 (2008). 6. Shattil, S.J., Kim, C. & Ginsberg, M.H. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300 (2010). 7. Hogg, N., Patzak, I. & Willenbrock, F. The insider’s guide to leukocyte integrin signalling and function. Nat. Rev. Immunol. 11, 416–426 (2011). 8. Shiratori, I., Ogasawara, K., Saito, T., Lanier, L.L. & Arase, H. Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor. J. Exp. Med. 199, 525–533 (2004). 9. Fan, Q. & Longnecker, R. The Ig-like v-type domain of paired Ig-like type 2 receptor alpha is critical for herpes simplex virus type 1-mediated membrane fusion. J. Virol. 84, 8664–8672 (2010). 10. Wilson, M.D., Cheung, J., Martindale, D.W., Scherer, S.W. & Koop, B.F. Comparative analysis of the paired immunoglobulin-like receptor (PILR) locus in six mammalian genomes: duplication, conversion, and the birth of new genes. Physiol. Genomics 27, 201–218 (2006). 11. Fournier, N. et al. FDF03, a novel inhibitory receptor of the Immunoglobulin superfamily, Is expressed by human dendritic and myeloid cells. J. Immunol. 165, 1197–1209 (2000). 12. Kogure, A., Shiratori, I., Wang, J., Lanier, L.L. & Arase, H. PANP is a novel O-glycosylated PILRα ligand expressed in neural tissues. Biochem. Biophys. Res. Commun. 405, 428–433 (2011). 13. Sun, Y. et al. Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands. J. Biol. Chem. 287, 15837–15850 (2012). 14. Satoh, T. et al. PILRα Is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein B. Cell 132, 935–944 (2008). 15. Wang, J. et al. Binding of herpes simplex virus glycoprotein B (gB) to paired immunoglobulin-like type 2 receptor α depends on specific sialylated O-linked glycans on gB. J. Virol. 83, 13042–13045 (2009). 16. Arii, J. et al. A single-amino-acid substitution in herpes simplex virus 1 envelope glycoprotein B at a site required for binding to the paired immunoglobulin-like type 2 receptor α (PILRα) abrogates PILRα-dependent viral entry and reduces pathogenesis. J. Virol. 84, 10773–10783 (2010). 17. Wang, J., Shiratori, I., Satoh, T., Lanier, L.L. & Arase, H. An essential role of sialylated O-linked sugar chains in the recognition of mouse CD99 by paired Ig-like type 2 receptor (PILR). J. Immunol. 180, 1686–1693 (2008). 18. Banerjee, A. et al. Modulation of paired immunoglobulin-like type 2 receptor signaling alters the host response to Staphylococcus aureus-induced pneumonia. Infect. Immun. 78, 1353–1363 (2010). 19. Brown, K.A. et al. Neutrophils in development of multiple organ failure in sepsis. Lancet 368, 157–169 (2006). 20. Gonzalez-Rey, E., Chorny, A., Robledo, G. & Delgado, M. Cortistatin, a new antiinflammatory peptide with therapeutic effect on lethal endotoxemia. J. Exp. Med. 203, 563–571 (2006). 21. Xu, W. et al. Integrin-induced PIP5K1C kinase polarization regulates neutrophil polarization, directionality, and in vivo infiltration. Immunity 33, 340–350 (2010). 22. Pollard, T.D. & Borisy, G.G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 (2003).

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23. Feng, C. et al. Endogenous PMN sialidase activity exposes activation epitope on CD11b/ CD18 which enhances its binding interaction with ICAM-1. J. Leukoc. Biol. 90, 313–321 (2011). 24. Hyduk, S.J. et al. Phospholipase C, calcium, and calmodulin are critical for α4β1 integrin affinity up-regulation and monocyte arrest triggered by chemoattractants. Blood 109, 176–184 (2007). 25. Bolomini-Vittori, M. et al. Regulation of conformer-specific activation of the integrin LFA-1 by a chemokine-triggered Rho signaling module. Nat. Immunol. 10, 185–194 (2009). 26. Stearns-Kurosawa, D.J., Osuchowski, M.F., Valentine, C., Kurosawa, S. & Remick, D.G. The pathogenesis of sepsis. Annu. Rev. Pathol. 6, 19–48 (2011). 27. Wagner, J.G. & Roth, R.A. Neutrophil migration during endotoxemia. J. Leukoc. Biol. 66, 10–24 (1999). 28. Hewett, J.A., Schultze, A.E., VanCise, S. & Roth, R.A. Neutrophil depletion protects against liver injury from bacterial endotoxin. Lab. Invest. 66, 347–361 (1992). 29. Tkalcevic, J. et al. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12, 201–210 (2000). 30. McDonald, B. et al. Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids. J. Exp. Med. 205, 915–927 (2008). 31. Jaeschke, H. & Hasegawa, T. Role of neutrophils in acute inflammatory liver injury. Liver Int. 26, 912–919 (2006). 32. Zhang, H., Meng, F., Chu, C.L., Takai, T. & Lowell, C.A. The Src family kinases Hck and Fgr negatively regulate neutrophil and dendritic cell chemokine signaling via PIR-B. Immunity 22, 235–246 (2005). 33. Cross, A.S. et al. Recruitment of murine neutrophils in Vivo through endogenous sialidase activity. J. Biol. Chem. 278, 4112–4120 (2003). 34. Zhang, S.Q. et al. Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol. Cell 13, 341–355 (2004). 35. Lacalle, R.A. et al. Specific SHP-2 partitioning in raft domains triggers integrinmediated signaling via Rho activation. J. Cell Biol. 157, 277–289 (2002).

36. Sasawatari, S. et al. The Ly49Q receptor plays a crucial role in neutrophil polarization and migration by regulating raft trafficking. Immunity 32, 200–213 (2010). 37. Kim, C.H. et al. Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses. J. Exp. Med. 190, 681–690 (1999). 38. Kruger, J. et al. Deficiency of Src homology 2-containing phosphatase 1 results in abnormalities in murine neutrophil function: studies in motheaten mice. J. Immunol. 165, 5847–5859 (2000). 39. Katagiri, K., Maeda, A., Shimonaka, M. & Kinashi, T. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat. Immunol. 4, 741–748 (2003). 40. Katagiri, K., Imamura, M. & Kinashi, T. Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7, 919–928 (2006). 41. Wu, Y., Stabach, P., Michaud, M. & Madri, J.A. Neutrophils lacking plateletendothelial cell adhesion molecule-1 exhibit loss of directionality and motility in CXCR2-mediated chemotaxis. J. Immunol. 175, 3484–3491 (2005). 42. Wu, D., Huang, C.K. & Jiang, H. Roles of phospholipid signaling in chemoattractantinduced responses. J. Cell Sci. 113, 2935–2940 (2000). 43. Xiao, W. et al. Phospholipase C-β3 regulates FcεRI-mediated mast cell activation by recruiting the protein phosphatase SHP-1. Immunity 34, 893–904 (2011). 44. Lou, O., Alcaide, P., Luscinskas, F.W. & Muller, W.A. CD99 is a key mediator of the transendothelial migration of neutrophils. J. Immunol. 178, 1136–1143 (2007). 45. Bixel, M.G. et al. A CD99-related antigen on endothelial cells mediates neutrophil but not lymphocyte extravasation in vivo. Blood 109, 5327–5336 (2007). 46. Held, W. & Mariuzza, R.A. Cis interactions of immunoreceptors with MHC and non-MHC ligands. Nat. Rev. Immunol. 8, 269–278 (2008). 47. Collins, B.E. et al. High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J. Immunol. 177, 2994–3003 (2006).

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ONLINE METHODS

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Generation of Pilra-deficient mice. The Pilra-targeting vector was constructed by replacing the second exon (encoding the extracellular domain of PILRα) with a neomycin-resistance gene cassette. This vector was transfected by electroporation into embryonic strain 129 stem cells. Embryonic stem (ES) cells were selected using G418, and clones with normal karyotypes were injected into C57BL/6 blastocysts, which were then transferred into pseudopregnant females. Offspring were screened by PCR and Southern blotting of DNA from tails. Pilra−/− mice were backcrossed to C57BL/6 mice for >12 generations and were bred in the same cages with wild-type mice under specific pathogen–free conditions. We confirmed that the genetic background of Pilra−/− mice was identical to that of the C57BL/6 mouse strain by analyzing microsatellite markers that distinguish differences between C57BL/6 and 129 mouse strains48. Mice were used at 8–12 weeks of age. All experiments were done according to the guidelines of the Animal Research Committee of the Research Institute for Microbial Diseases, Osaka University. Antibodies and reagents. Fc chimera proteins were prepared by fusing the extracellular domains of PILRα, CD99 or N-terminal glycoprotein B fragment (amino acid residues 30–150) with the Fc region of human IgG as described15,17. PILRα monoclonal antibody was prepared as described17. In brief, Wistar rats (Japan SLC) were immunized with mouse PILRα-Fc protein using TiterMax Gold (TiterMax) adjuvant. Two weeks after immunization, lymph node cells were fused with SP2/0, and clones recognizing mouse PILRαtransfected Ba/F3 cells were selected, followed by selection of those clones that did not recognize mouse PILRβ and blocked binding of PILRα to CD99. One clone was selected for flow cytometry experiments. Goat polyclonal antibody to PILRα (AF4318, R&D Systems) was used for immunoprecipitation, immunoblotting and immunostaining. The following mouse mAbs from eBioscience were used: fluorescent isothiocyanate (FITC)-conjugated Gr-1 (RB6-8C5), CD11b (M1/70), CD11a (M17/4), CD18 (M18/2) and Phycoerythrin (PE)-conjugated Gr-1 (RB6-8C5), F4/80 (BM8) and Ly-6C (HK1.4). Rabbit polyclonal antibodies to SH-PTP1 (C-19), SH-PTP2 (C-18) and β2 integrin–blocking antibody (GAME-46, sc-19624) were from Santa Cruz. Mouse mAb to phosphorylated tyrosine (4G10) and rabbit polyclonal antibodies to p-Erk (Thr202/Tyr204, #4370), Erk (#4695), Cdc42 (#2466), Rac1 (#2465) and RhoA (#2117) were from Cell Signaling Technology. Rabbit polyclonal antibody to Rap1 (A01047) was from GenScript. Allophycocyanin (APC)-conjugated streptavidin (016-130-084), anti-rat IgG (712-136-153) and anti-human IgG (109-366-098) were from Jackson ImmunoResearch. Recombinant mouse ICAM-1–Fc (796-IC) was from R&D Systems. fMLF was from Peptide Institute. Ionomycin was from Calbiochem. Rhotekin RBD agarose, PAK1 PBD agarose and RalGDS RBD agarose were from Cell Biolabs. Lipopolysaccharide-induced endotoxin shock. LPS from Escherichia coli (serotype 055:B5; Sigma-Aldrich) was diluted in sterile PBS and injected intraperitoneally into mice. Mice were monitored every 12 h. During endotoxic shock, blood was collected by retro-orbital sinus bleeding at the indicated times. Serum samples were sent to Oriental Yeast for determination of the levels of alanine aminotransferase, blood urea nitrogen and lactate dehydrogenase. Serum cytokine levels were determined with enzyme-linked immuno sorbent assay (ELISA) kits (eBioscience). Liver tissues from LPS-treated mice were fixed with 4% PFA and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) and assessed microscopically (Axioplan, Carl Zeiss). Cryostat sections of frozen livers (5 µm) were fixed with ice-cold acetone for 10 min and air-dried. Sections were incubated with blocking solution (1% BSA in PBS) for 1 h at 25 °C and stained with FITC-conjugated anti-mouse Gr-1. Sections were mounted in Softmount (Wako) and examined using an Axiovert microscope (Carl Zeiss) with a Nikon G3 digital camera. Myeloperoxidase assay. Livers and lungs were collected at different times, weighed and homogenized in 20 mM phosphate buffer (50 mg/ml), then centrifuged at 10,000g for 15 min. Pellets were resuspended in 50 mM phosphate buffer (pH 6.0) with 10 mM EDTA and 0.5% hexadecyltrimethylammonium bromide. Samples were frozen-thawed and incubated at 60 °C for 2 h, then centrifuged at 10,000g for 30 min at 4 °C. Supernatant was mixed with

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tetramethyl-benzidine to measure MPO levels using an MPO standard control (Enzo Life Science). The samples were read at OD450 nm (Molecular Devices). Thioglycollated-induced sterile peritonitis. Mice were injected intraperitoneally with 1 ml 3% thioglycollate broth (BD Difco) and killed at different times thereafter. Peritoneal exudate cells were collected by washing with 5 ml PBS containing 1% BSA and counted by Guava PCA system (Millipore). The percentage of neutrophils and macrophages was determined by flow cytometry. Competitive recruitment assays were performed as described21. Briefly, purified wild-type neutrophils were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE; Molecular Probes), whereas Pilra−/− neutrophils were labeled with Far-Red DDAO SEM (Molecular Probes), mixed 1:1 and injected through the retro-orbital venous sinus into wild-type recipient mice, which had been injected with 2 ml of 3% thioglycollate 2 h earlier. After another 2 h, peritoneal and blood cells were analyzed by flow cytometry. fMLF-induced actin polymerization, respiratory burst and calcium influx. Neutrophils were isolated from the bone marrow of sex- and age-matched mice by discontinuous Percoll gradients (GE Healthcare), as described32. Purified neutrophils with purity >90% were used for functional analyses. Actin poly merization was measured by incubating neutrophils for different durations at 37 °C with fMLF (1 µM), after which they were fixed, permeabilized and stained with Alexa Fluor 488–conjugated phalloidin (Molecular Probes) for flow cytometry. Oxidant production was measured by flow cytometry by loading neutrophils with 2 µM of the oxidant-sensitive dye dihydrorhodamine 123 (Molecular Probes) for 10 min at 37 °C. Cells were then stimulated with fMLF (1 µM), fixed with 4% paraformaldehyde and analyzed by flow cytometry. To determine intracellular free Ca2+ flux, 1 × 107 purified neutrophils in PBS containing 1 mM Ca2+ and 1 mM Mg2+ were incubated with 2.5 µM Fluo-4 and Fluo-5 µM Fura-red (both from Molecular Probes) at 37 °C for 30 min, washed and resuspended at 5 × 106 cells/ml. The Ca2+ flux was monitored using FACSCalibur flow cytometer with an excitation at 488 nm. Data were analyzed with FlowJo software. Adhesion and transmigration assay. Flat-bottomed 96-well plates were coated with mouse ICAM-1–Fc (2 µg/ml) or hyaluronic acid (Sigma, 1 mg/ml) and blocked with PBS containing 1% BSA for 1 h. fMLF or control buffer was added to purified neutrophils, and cells were immediately plated and incubated at 37 °C for 10 min. Nonadherent cells were removed by washing. The bound cells were stained with 0.1% crystal violet, lysed in 0.5% Triton X-100 and read at OD595 nm. For transmigration assays, transwell inserts (pore size 5.0 µm; Corning) were coated with ICAM-1–Fc (2 µg/ml) and placed in 24-well plates filled with RPMI-1640 containing fMLF or CXCL1. Neutrophils were placed in the upper chambers followed by incubation for 1 h at 37 °C, 5% CO2. Cells in the lower chamber were collected and counted using the Guava PCA system (Millipore). Sialidase treatment. Neutrophils were treated with sialidase (Arthrobacter ureafaciens, 0.1 unit/ml, Roche) or vehicle control for 30 min at 37 °C. After washing away the sialidase, neutrophils were resuspended in RPMI-1640 and used for functional assays. Immunoprecipitation and immunoblotting. Cells were lysed in 1% Nonidet P-40 in 150 mM NaCl, 2 mM EDTA, 25 mM Tris-HCl (pH 7.4) containing protease inhibitor mixture (Sigma), 2 mM sodium orthovanadate and phenyl methylsulfonyl fluoride (100 µg/ml). Lysates were incubated with streptavidinsepharose (GE Healthcare) coupled to biotinylated goat anti-PILRα polyclonal antibody. Immunoprecipitates were separated by 5–20% SDS-PAGE and transferred to PVDF membranes (Millipore). Immunoblots were incubated with primary antibodies and then horseradish peroxidase–conjugated secondary antibodies (Thermo Scientific). Immunoblots were developed using SuperSignal (Thermo Scientific) and detected with an LAS-1000 instrument (Fujifilm). For the detection of tyrosine-phosphorylated proteins in total cell lysate, purified neutrophils (1.5 × 106) were suspended in RPMI-1640 and stimulated with fMLF. Stimulation was terminated by addition of ice-old lysis buffer. After centrifugation, cell lysates were subjected to immunoblotting.

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Small GTPase activity pull-down assay. Purified neutrophils were suspended in RPMI-1640 and stimulated with 3 µM fMLF. After addition of ice-old lysis buffer, lysates were incubated with Rhotekin RBD-agarose (for RhoA-GTP), PAK1 PBD agarose (for Cdc42-GTP and Rac1-GTP) or RalGDS RBDagarose (for Rap1-GTP) for 1 h at 4 °C. Proteins binding the agarose beads were immunoblotted. Total GTPases and activated GTPases were detected with respective antibodies.

Statistical analysis. Statistical significance was analyzed by two-tailed Student’s t-test. P < 0.05 was considered significant. Survival rate differences were analyzed by Kaplan-Meier analysis, and significance was determined using a log-rank test (GraphPad Prism 4.0). 48. Ogonuki, N. et al. A high-speed congenic strategy using first-wave male germ cells. PLoS ONE 4, e4943 (2009).

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ICAM binding assay. To measure integrin affinity, ICAM-1–Fc-F(ab′)2 complexes were generated by incubating phycoerythrin (PE)-conjugated goat antihuman Fcγ fragment–specific IgG F(ab′)2 fragments (Jackson ImmunoResearch) and ICAM-1–Fc (50 µg/ml) for 30 min. Neutrophils were suspended at 1.5 × 107/ml in RPMI-1640 containing 0.1% BSA and mixed with ICAM-1– Fc-F(ab′)2 complexes in the presence or absence of 3 µM fMLF. The reaction was stopped by adding 4% paraformaldehyde. After 15 min, fixed cells were washed in ice-cold 1% BSA in PBS. Cells were pelleted, resuspended in flow cytometry buffer and analyzed by flow cytometry. For antibody blocking, neutrophils were incubated with antibody (20 µg/ml) for 30 min before the binding assay. For sialidase treatment, cells were incubated with sialidase or control buffer for 30 min at 37 °C and washed twice before the binding assay.

Confocal microscopy. Purified neutrophils in steady state or stimulated with 3 µM fMLF for 3 min were fixed with 4% PFA, incubated with 3% BSA in PBS to block nonspecific binding and then with appropriate antibodies. Alexa Fluor 647–conjugated anti-rabbit IgG (H+L), Alexa Fluor 647–conjugated anti-goat IgG (H+L) and Alexa Fluor 555–conjugated streptavidin (Molecular Probes) were used as secondary antibodies. Nuclei were visualized with Hoechst 33258. For intracellular staining, the cells were incubated with 0.3% Triton X-100 in PBS for 5 min before staining. After staining, the cells were affixed on cover glass by cytospin (Shandon). The cover glasses were mounted on glass slides using PermaFluor mounting medium (Thermo-Fisher). Microscopic analysis was performed with a fluorescence microscope (FV10i; Olympus). Images were analyzed using Olympus software.

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doi:10.1038/ni.2456