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Inflammation Differential Srgap1 Expression During Lung Repulsive Neutrophil Chemotaxis Through Slit2 Regulates Attractive Eosin

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Inflammation Differential Srgap1 Expression During Lung Repulsive Neutrophil Chemotaxis Through Slit2 Regulates Attractive Eosin The Journal of Immunology Slit2 Regulates Attractive Eosinophil and Repulsive Neutrophil Chemotaxis through Differential srGAP1 Expression during Lung Inflammation Bu-Qing Ye,* Zhen H. Geng,† Li Ma,† and Jian-Guo Geng† Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the mo- lecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitiza- tion elicited a Slit2 gradient at so-called bronchus–alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1a–induced che- motaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus–alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation. The Journal of Immunology, 2010, 185: 6294–6305. eukocytes are recruited to the site of infection or tissue of cell adhesion molecules (4), whereas chemotaxis is mediated by injury as part of the inflammatory response of the innate several large families of chemokines and chemoattractants (5–7). L immune system. Directional migration of leukocytes Slit2, a member of the Slit family of secreted migratory cues, involves cellular interactions that are precisely regulated by tem- binds to Robo1, a prototype of the Roundabout family (Robo1–4) poral and spatial presentation of molecules on the surface of mi- of transmembrane cell-surface receptors. Engagement of Robo1 grating cells and their substrates. The cellular interactions that by Slit2 functions as a repellent in axon guidance and neuronal define the different steps of leukocyte recruitment include tether- migration (8–11), an endogenous inhibitor of leukocyte chemo- ing (initial attachment), rolling, weak and firm adhesion (arrest), taxis (12–17), and a chemoattractant of vascular endothelial cells transendothelial migration, and chemotaxis (1, 2). Among these, during vasculogenesis and angiogenesis (18–21). In addition, Slit2 tethering, rolling, and weak adhesion of leukocytes are thought to modulates migration of malignant tumor cells (22–24). be mediated mainly by the binding of selectins (CD62) to their Intracellular molecules downstream of Robo signaling have cognate glycoprotein ligands (3). Firm adhesion is mediated emerged as key regulators for determining the repulsive or at- mainly by the interaction of integrins with the Ig superfamily tractive effect of Slit on the targeting cells. Robo utilizes a variety of different signaling components in different cell types, such as Mena/Ab1 (25) and Calmodulin and Son of sevenless (26). The particular repertoire of intracellular signaling components deter- *Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shang- mines the unique migratory response to Slit2 in cells of the same hai, China; and †Vascular Biology Center and Division of Hematology, Oncology and or different type. One signaling component that has emerged re- Transplantation, Department of Medicine, University of Minnesota School of Med- cently is the family of small Rho GTPases, particularly RhoA, icine, Minneapolis, MN 55455 Rac1, and Cdc42, which critically regulates actin cytoskeleton in Received for publication May 18, 2010. Accepted for publication September 11, 2010. guiding the directional migration of mammalian cells (27). The This work was supported by grants from the National Science Foundation of GTP-bound forms of Rho GTPases are active, whereas the GDP- China (30901302 to B.-Q.Y.), the Ministry of Science and Technology of China bound forms are inactive. The activities of Rho GTPases are (2010CB529702 to B.-Q.Y.), and the National Institutes of Health (RO1AI064743 modulated by GTPase-activating proteins (GAPs), which increase and RO1CA126897 to J.-G.G.). intrinsic GTPase activities, or guanine nucleotide exchange fac- Address correspondence and reprint requests to Dr. Jian-Guo Geng at the current address: Department of Biologic and Materials Sciences, University of Michigan tors, which exchange the GDP on a GTPase for GTP. Upon en- School of Dentistry, A323 MSRB III, 1150 West Medical Center Drive, Ann Arbor, gagement of Robo1 with Slit2, srGAP1, the prototype of a GAP MI 48109-0632. E-mail address: [email protected] family that includes srGAP1, -2 and -3, directly binds to the in- The online version of this article contains supplemental material. tracellular CC3 motif of Robo1, which inactivates Cdc42 and Abbreviations used in this paper: Aml, Aml14.3D10; AS, 5-(2,2-difluoro-benzo[1,3] RhoA, but does not affect the activity of Rac1. This molecular dioxol-5-ylmethylene)-thiazolidine-2,4-dione; BALF, bronchial airway lavage fluid; mechanism induces repulsion of migratory neuronal cells from the EPO, eosinophils peroxidase; GAP, GTPase-activating protein; HA, hemagglutinin; mIgG, mouse IgG; NE, neutrophils elastase; SDF-1a, stromal cell-derived factor-1a; anterior subventricular zone of the forebrain (28). SH, Src homology; srGAP1, Slit-Robo GTPase-activating protein 1; Tg, transgenic; Another downstream effector of Robo is the family of PI3Ks. WT, wortmannin. Upon recruitment to the inner leaflet of the plasma membrane, p110 Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 phosphorylates phosphatidylinositol 4,5-bisphosphate on the D3 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001648 The Journal of Immunology 6295 position to yield phosphatidylinositol 3-5-trisphosphate. There is verse primer 59-GCAGCGGCCGCTCAGTGATGATGATGATGATGATC- a diverse set of proteins with pleckstrin homology domains that TGCC ATTTCTCCAGGACC-39. Postdigestion with XbaI/NotI, the insert bind to phosphatidylinositol 3-5-trisphosphate and, consequently, was ligated into the pVL1393 vector (BD Pharmingen, San Diego, CA), which was then verified by DNA sequencing. Recombinant human Slit2 are recruited to the plasma membrane upon activation of PI3Ks with an His tag was expressed in Sf9 insect cells and purified by Talon (29). These molecules are responsible for activation of a cohort of metal affinity chromatography (BD Clontech, Mountain View, CA), as different signal transduction pathways controlling cell growth, dif- described (33–35). Eluted Slit2 was concentrated with Ultracon (10 kDa ferentiation, proliferation, apoptosis, metabolism, migration, and cutoff; Millipore, Bedford, MA) and further purified by gel-filtration chromatography (A¨ KTA FPLC; GE Healthcare Life Sciences, Piscat- intracellular trafficking. We have previously shown that PI3K in- away, NJ) on a Superdex 200 column (GE Healthcare Life Sciences), using hibitors, WT and LY294002, inhibit Slit2-induced attractive mi- PBS (pH 7.4) as the running buffer. Isolated Slit2 was eluted as a single gration of vascular endothelial cells in a dose-dependent manner sharp peak that was homogenous when subjected to silver staining (data (18). not shown). Contaminated endotoxin in our preparations of Slit2 was Although the steps of leukocyte recruitment are well defined, the routinely removed by Detoxi-Gel Endotoxin Removing Gel (Thermo Scientific, Rockford, IL) until they were ,0.03 EU/ml determined by the molecular mechanisms underlying attractive versus repulsive mi- Limulus amebocyte lysate method. More than three separate Slit2 prepa- gration and directional, nondirectional, or random migration re- rations were used in our experimentation. main obscure (5–7). Much effort has been directed at identifying exogenous factors that can be used therapeutically to control in- Cell lines flammation (30, 31). Notably, Slit2 has been shown to inhibit HL-60 cells (CCL-240, American Type Culture Collection, Manassas, VA) migration of neutrophils, lymphocytes, and macrophages in in- and Aml14.3D10 (Aml) cells (kindly provided by Dr. Arne Slungaard, flammatory responses (12–17). In this study, we unexpectedly University of Minnesota, Minneapolis, MN) were cultured in RPMI found that Slit-Robo signaling activated not only repulsive 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat- chemotaxis of neutrophils during endotoxin-induced lung in- inactivated FBS, 4 mM L-glutamine, 50 mM 2-ME (Aml cells; 36), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37˚C in the presence of flammation, but also attractive chemotaxis of eosinophils during 5% CO2. allergic airway inflammation. To understand whether and how distinctive intracellular pathways downstream of Slit-Robo sig- Isolation of murine and human leukocytes naling differentially modulate leukocyte migratory
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