Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular 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 sensitization 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-1α–induced chemotaxis 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.

Leukocytes are recruited to the site of infection or tissue injury as part of the inflammatory response of the innate immune system. Directional migration of leukocytes involves cellular interactions that are precisely regulated by temporal and spatial presentation of molecules on the surface of migrating cells and their substrates. The cellular interactions that define the different steps of leukocyte recruitment include tethering (initial attachment), rolling, weak and firm adhesion (arrest), transendothelial migration, and chemotaxis (1, 2). Among these, tethering, rolling, and weak adhesion of leukocytes are thought to be mediated mainly by the binding of selectins (CD62) to their cognate glycoprotein ligands (3). Firm adhesion is mediated mainly by the interaction of integrins with the Ig superfamily of cell adhesion molecules (4), whereas chemotaxis is mediated by several large families of chemokines and chemoattractants (57).

Slit2, a member of the Slit family of secreted migratory cues, binds to Robo1, a prototype of the Roundabout family (Robo1–4) of transmembrane cell-surface receptors. Engagement of Robo1 by Slit2 functions as a repellent in axon guidance and neuronal migration (811), an endogenous inhibitor of leukocyte chemotaxis (1217), and a chemoattractant of vascular endothelial cells during vasculogenesis and angiogenesis (1821). In addition, Slit2 modulates migration of malignant tumor cells (2224).

Intracellular molecules downstream of Robo signaling have emerged as key regulators for determining the repulsive or attractive 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 determines the unique migratory response to Slit2 in cells of the same or different type. One signaling component that has emerged recently is the family of small Rho GTPases, particularly RhoA, Rac1, and Cdc42, which critically regulates actin cytoskeleton in guiding the directional migration of mammalian cells (27). The GTP-bound forms of Rho GTPases are active, whereas the GDP-bound forms are inactive. The activities of Rho GTPases are modulated by GTPase-activating proteins (GAPs), which increase intrinsic GTPase activities, or guanine nucleotide exchange factors, which exchange the GDP on a GTPase for GTP. Upon engagement of Robo1 with Slit2, srGAP1, the prototype of a GAP family that includes srGAP1, -2 and -3, directly binds to the intracellular CC3 motif of Robo1, which inactivates Cdc42 and RhoA, but does not affect the activity of Rac1. This molecular mechanism induces repulsion of migratory neuronal cells from the anterior subventricular zone of the forebrain (28).

Another downstream effector of Robo is the family of PI3Ks. Upon recruitment to the inner leaflet of the plasma membrane, p110 phosphorylates phosphatidylinositol 4,5-bisphosphate on the D3 position to yield phosphatidylinositol 3-5-trisphosphate. There is a diverse set of proteins with pleckstrin homology domains that bind to phosphatidylinositol 3-5-trisphosphate and, consequently, are recruited to the plasma membrane upon activation of PI3Ks (29). These molecules are responsible for activation of a cohort of different signal transduction pathways controlling cell growth, differentiation, proliferation, apoptosis, metabolism, migration, and intracellular trafficking. We have previously shown that PI3K inhibitors, WT and LY294002, inhibit Slit2-induced attractive migration of vascular endothelial cells in a dose-dependent manner (18).

Although the steps of leukocyte recruitment are well defined, the molecular mechanisms underlying attractive versus repulsive migration and directional, nondirectional, or random migration remain obscure (57). Much effort has been directed at identifying exogenous factors that can be used therapeutically to control inflammation (30, 31). Notably, Slit2 has been shown to inhibit migration of neutrophils, lymphocytes, and macrophages in inflammatory responses (1217). In this study, we unexpectedly found that Slit-Robo signaling activated not only repulsive chemotaxis of neutrophils during endotoxin-induced lung inflammation, but also attractive chemotaxis of eosinophils during allergic airway inflammation. To understand whether and how distinctive intracellular pathways downstream of Slit-Robo signaling differentially modulate leukocyte migratory responses to directional stimuli, we investigated the molecular mechanisms of how Slit-Robo signaling regulates directional migration of eosinophils and neutrophils during allergic airway inflammation and endotoxin-induced lung inflammation.

The Slit2 and Robo1 mAbs and polyclonal Abs were prepared and characterized as described before (18). The Abs against Cdc42, srGAP1, GFP, hemagglutinin (HA), CC10, eosinophils peroxidase (EPO), and neutrophils elastase (NE) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Abs of His tag and α-tubulin and bacterial LPSs (serotype 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO). The GAPDH Ab was purchased from Proteintech Group (Chicago, IL). The p85 pAb was purchased from Upstate Biotechnology (Lake Placid, NY). The anti-Akt and anti-phosphorylated Akt Abs were purchased from Cell Signaling Technology (Danvers, MA). The primers and premix reagents for quantitative PCR were purchased from SA Biosciences (Frederick, MD). The anti-rabbit IgG-Cy5 and anti-goat IgG-Cy3 were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Recombinant mouse SDF-1α was purchased from RDI (Fitzgerald Industries, Concord, MA). Recombinant mouse eotaxin and the mouse CXCL1/CXCL2 quantitative ELISA kit were purchased from R&D Systems (Minneapolis, MN). The mouse IgE quantitative ELISA kit was purchased from Bethyl Laboratories (Montgomery, TX). Wortmannin (WT) and 5-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethylene)- thiazolidine-2,4-dioe (AS605240, or AS) were purchased from EMD Calbiochem (Gibbstown, NJ). The EasySep human eosinophil enrichment kit was purchased from StemCell Technologies (Vancouver, British Columbia, Canada).

C57BL6/J (C57) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). IL-5-transgenic (Tg) mice (NJ1638 strain; 32) were generously provided by Dr. James J. Lee (Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, AZ). The Slit2-Tg mice were generated according to standard procedures and characterized as described (33). Mouse experiments were approved by the Institutional Animal Committees of Shanghai Institutes for Biological Sciences (Shanghai, China).

The human Slit2 cDNA (1–2670 bp) was amplified using the forward primer 5′-ACCTTCTAGAATGCGCGGCGTTGGCTGGC-3′ and the reverse primer 5′-GCAGCGGCCGCTCAGTGATGATGATGATGATGATCTGCC ATTTCTCCAGGACC-3′. Postdigestion with XbaI/NotI, the insert was ligated into the pVL1393 vector (BD Pharmingen, San Diego, CA), which was then verified by DNA sequencing. Recombinant human Slit2 with an His tag was expressed in Sf9 insect cells and purified by Talon metal affinity chromatography (BD Clontech, Mountain View, CA), as described (3335). Eluted Slit2 was concentrated with Ultracon (10 kDa cutoff; Millipore, Bedford, MA) and further purified by gel-filtration chromatography (ÄKTA FPLC; GE Healthcare Life Sciences, Piscataway, NJ) on a Superdex 200 column (GE Healthcare Life Sciences), using PBS (pH 7.4) as the running buffer. Isolated Slit2 was eluted as a single sharp peak that was homogenous when subjected to silver staining (data not shown). Contaminated endotoxin in our preparations of Slit2 was routinely removed by Detoxi-Gel Endotoxin Removing Gel (Thermo Scientific, Rockford, IL) until they were <0.03 EU/ml determined by the Limulus amebocyte lysate method. More than three separate Slit2 preparations were used in our experimentation.

HL-60 cells (CCL-240, American Type Culture Collection, Manassas, VA) and Aml14.3D10 (Aml) cells (kindly provided by Dr. Arne Slungaard, University of Minnesota, Minneapolis, MN) were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS, 4 mM l-glutamine, 50 μM 2-ME (Aml cells; 36), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in the presence of 5% CO2.

Mouse neutrophils and eosinophils were isolated from bone marrows or peripheral blood of C57 and IL-5-Tg mice as described (32, 34, 3739). Human neutrophils were isolated from peripheral blood of healthy volunteers as described (34). Human circulating eosinophils were isolated by negative magnetic selection from peripheral blood according to the manufacturer’s protocol. The purity of mouse neutrophils and eosinophils (∼90–92%) and human eosinophils and neutrophils (∼95%) was determined by Wright’s-Giemsa staining (Supplemental Fig. 1). The endotoxin levels in all buffers used were <0.03 EU/ml determined by the Limulus amebocyte lysate method. The use of human blood was approved by the Institutional Review Board of the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences.

For comparing the levels of srGAP1 expression, human Aml and HL-60 cells (both at 4 × 106 cells/aliquot), mouse eosinophils and neutrophils (both at 7 × 106 cells/aliquot), or human eosinophils and neutrophils (both at 2 × 106 cells/aliquot) were lysed with the ice-cold lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10% glycerol, 2 mM PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstanin A, and 150 mM benzamidine) on ice for 30 min. After brief centrifugation, the lysates were immunoblotted with the srGAP1 Ab.

Alternatively, human Aml and HL-60 cells or mouse eosinophils and neutrophils were starved with serum-free RPMI 1640 medium at 37°C for 1 h. They were then stimulated with Slit2 (0.4 μg/ml in PBS) or PBS alone at 37°C for 2 min for immunoprecipitation (5 × 106 cells/aliquot) and for 5 min for determining Akt phosphorylation (3 × 106 cells/aliquot), without or with preincubation with R5 or isotype-matched irrelevant mouse IgG (mIgG; both at 0.5 μM) for 30 min. The lysates were used for coimmunoprecipitation followed by immunoblotting with their respective Abs, as previously described (34).

In addition, mouse lung tissues were homogenized in 1 ml RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.5% deoxycholid acid, 0.1% SDS, 5 mM EDTA, 2 mM PMSF, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 10 μg/ml pepstanin A, 150 mM benzamidine, and 1% Nonidet P-40) in a Dounce tissue homogenizer, followed by centrifugation at 12,000 × g at 4°C for 10 min to remove tissue debris and immunoblotting as described above.

We designed polypeptides encoding the Src homology (SH) 3 region of wild-type srGAP1 (amino acid residues of 674–1022) and its mutant, in which the amino acid residues corresponding to Ser758, Arg760, Glu761, Trp780, and Leu791 in the P3 loop of the SH3 domain were mutated to Ala (40). They were expressed and isolated using methods identical to those described before (34).

Recombinant human Robo1 CC3 region (encoding the aa sequence 1454–1657) fused with GST (GST-Robo1-CC3) and GST alone in the vector of pGEX-4T-1 (GE Healthcare Life Sciences) were expressed in Escherichia coli and purified using Glutathione Sepharose 4B beads (GE Healthcare Life Sciences). Isolated GST-Robo1-CC3 or GST was incubated with the polypeptides of TAT-SH3 and TAT-SH3M. After washing extensively, the beads were boiled in the SDS sample loading buffer and subjected to 12% SDS-PAGE followed by immunoblotting.

The microfluidic chamber (μ-slide VI, Ibidi, München, Germany) for generation and maintenance of a stable gradient of any chemokines for up to 30 min was employed to monitor neutrophil and eosinophil chemotaxis in real time (41). The condition medium of stable HEK293 cell lines expressing human Slit2 or the plain vector (28) was collected by culturing these confluent cell monolayers with serum-free DMEM medium at 37°C for 12 h. Following precoating with 10% murine plasma for 30 min and washing three times with HBSS, mouse neutrophils or eosinophils (1 × 106/aliquot) were loaded into each channel. The Slit2 medium or the control medium was added into the input port, with or without an aliquot (1 μl) of mouse SDF-1α (25 μg/ml) or eotaxin (100 μg/ml) in the presence of mIgG or R5 (both at 0.5 μM), DMSO, WT, or AS (both at 150 nM). The chamber was transferred to a multidimensional live cell imaging workstation (Leica AS MDW, Leica Microsystems, Wetzlar, Germany) equipped with a heated enclosure to keep the chamber at 37°C. The migration of cells was recorded at ×40 original magnification with images taken every 5 s over a period of 20 min for neutrophils and 30 min for eosinophils. The resulting images were imported into Image J software (National Institutes of Health, Bethesda, MD) and processed using a cell-tracking protocol.

A 24-well Transwell plate (5 μm in pore size; Corning, Corning, NY) was coated with 2.5 μg/ml mouse fibrinogen and incubated at 37°C for 1 h. Freshly isolated mouse neutrophils (4 × 106/ml) were suspended in 50% DMEM and 50% M199 medium supplemented with 5% heat-inactivated FBS. An aliquot (0.1 ml) of neutrophils, with or without preincubation with the polypeptide of His-SH3, TAT-SH3, or TAT-SH3M (all at 50 μg/ml) at 37°C for 20 min in the presence of 5% CO2, was transferred into the insert. The inserts were then placed into the wells containing 0.6 ml medium and SDF-1α (50 ng/ml), Slit2 (0.4 μg/ml), WT or AS (both at 150 nM). Alternatively, the plate was pretreated with 0.1% BSA in the RPMI 1640 medium at 37°C for 1 h. An aliquot (0.1 ml) of mouse eosinophils (1 × 107/ml) suspended in the same medium was transferred into the insert. The inserts were then placed into the wells containing 0.6 ml medium and eotaxin (0.5 μg/ml), Slit2 (0.4 μg/ml), WT, or AS (both at 150 nM). Postincubation at 37°C for 2 h (for neutrophils) or 4 h (for eosinophils), cells that had migrated through the filter into the lower wells were collected and counted (12, 42).

An aliquot of cells (3 × 106 cells) were incubated with PBS alone or Slit2 (0.4 μg/ml in PBS) at 37°C for 20 min and then lysed with 500 μl lysis buffer on ice for 30 min. Postcentrifugation at 12,000 rpm at 4°C for 10 min, supernatants were incubated with GST-PBD fusion protein that had bound to the Glutathione Sepharose beads at 4°C for 4 h. After washing three times, the bound proteins were boiled in the SDS sample loading buffer for 5 min followed by immunoblotting with the Cdc42 Ab (28).

Mouse leukocytes in bronchial airway lavage fluids (BALFs) were coated to glass slides. Mouse lung tissues were fixed with 10% formalin, paraffin embedded, and sectioned (5-μm thick). Prior to immunostaining, samples were incubated with 10% BSA in 50 mM Tris-HCl (pH 7.6) and 0.15 M NaCl (TBS) at 37°C for 30 min to reduce nonspecific binding. They were then incubated with appropriate primary Abs in TBS containing 1% BSA at 4°C overnight in a humidified chamber, followed by incubation with respective fluorescence-conjugated secondary Abs. The nuclei were stained with DAPI (blue). All incubations were followed by washing three times in TBS over 15 min. The immunofluorescent staining was observed under a Leica-SP2 laser scanning confocal microscope (Leica Microsystems).

Total RNAs from mouse lung tissues, isolated primary leukocytes, and cultured cell lines were extracted with the Absolutely RNA miniprep Kit (Stratagene, La Jolla, CA). For comparing the level of srGAP1 expression, Aml and HL-60 cells (5 × 106 cells/aliquot), human eosinophils and neutrophils (2 × 106 cells/aliquot), or mouse eosinophils and neutrophils (1 × 107 cells/aliquot) were lysed and 2 μg (Aml and HL-60 cells or mouse eosinophils and neutrophils) and 50 ng (human eosinophils and neutrophils) total RNA per aliquot were used for quantitative and real-time RT-PCR analysis. cDNAs were synthesized using the ThermoScript RT-PCR system (Invitrogen). Quantitative PCR analysis and data collection were performed on the Mx3000 qPCR System (Stratagene) using the primer pairs and the RT2 SYBR Green PCR Master Mix from SA Biosciences. All quantitations were normalized to an endogenous β-actin or GAPDH control. The relative quantitation value for each target gene compared with the calibrator for that target is expressed as 2 − (Ct − Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to β-actin/GAPDH). The relative expression levels of samples are presented using a semilog plot.

C57 and Slit2-Tg mice (∼7 wk old) were sensitized with 20 μg OVA (Sigma-Aldrich) and 2 mg alum i.p. on days 0 and 5. Sham-immunized mice were received alum alone. From days 12 to 14, mice were aerosol challenged with 1% OVA in saline (each time for 1 h, twice per day separated for 4 h) for 3 d (43), with or without i.v. injection of R5 or mIgG or i.p. injection of WT or AS (all at 1 μg/g mouse body weight) prior to aerosol challenge. Alternatively, C57 mice were exposed to Slit2 aerosol (8 μg/ml in PBS) for 1 h prior to OVA aerosol challenge for 3 d or given i.v. Slit2 (5 ng/g mouse body weight) prior to the OVA aerosol challenge on day 13 (day 2 of aerosol challenge; 13). On day 15 (24 h after the last aerosol challenge), mice were anesthetized followed by surgical exposure of the lungs and hearts. Tracheas were cannulated, and each lung was lavaged with 1.5 ml PBS. Leukocytes in BALFs and blood were counted and stained with Wright’s dye, and cell differentials were enumerated based on morphology and staining profile. For histologic studies, mice were perfused with 10 ml PBS through the right ventricle to remove all blood followed by fixation with 3 ml 10% formalin, paraffin embedding, and sectioning (5-μm thick).

C57 and Slit2-Tg mice (8–10 wk) were aerosol challenged with 300 μg/ml LPS in saline for 20 min (44), with or without prior i.v. injection of R5 or mIgG (1 μg/g mouse body weight). Eight hours after aerosol challenge, mice were anesthetized, cannulated, and lavaged. Leukocytes and their differentials were counted as described above.

Statistical significance was determined by Student t test. For multiple comparisons, ANOVA test (Bonferroni post hoc) was employed. The p values <0.05 and <0.01 were considered statistically significant and very significant, respectively.

Slit2 reportedly inhibits chemotaxis of neutrophils, macrophages, and T lymphocytes (1217). Although the mechanism by which Slit2 attenuates chemotaxis of these leukocytes is unknown, Slit2 has been shown to induce repulsion of migratory neuronal cells within the developing brain through a pathway involving Robo1, srGAP1, and Cdc42. We therefore investigated the activity of this pathway during leukocyte chemotaxis using neutrophils and eosinophils. Using RT-PCR and quantitative RT-PCR, we found that human eosinophilic Aml cells and mouse eosinophils freshly isolated from IL-5-Tg mice (32) expressed almost undetectable srGAP1 mRNA (Fig. 1A, 1B) and protein (Fig. 1C) compared with human promyeloid HL-60 cells and primary mouse neutrophils. Consistently, a significantly lower level of srGAP1 mRNA and protein expression was also detected in human circulating eosinophils as compared with human circulating neutrophils (Fig. 1D, 1E). As predicted, recombinant human Slit2 induced srGAP1 binding to Robo1 (Fig. 1F) and resulted in inactivation of Cdc42 (Fig. 1G) in HL-60 cells, but not in Aml cells.

FIGURE 1.

srGAP1 expression and Robo1 downstream signaling in eosinophils and neutrophils. RT-PCR (A) and quantitative real-time RT-PCR (B) analysis of srGAP1 mRNA in human eosinophilic Aml cells, human promyeloid HL-60 cells, mouse eosinophils, and mouse neutrophils. C, Immunoblotting of srGAP1 and α-tubulin in Aml cells, HL-60 cells, mouse eosinophils, and neutrophils. D and E, The quantitative real-time PCR and immunoblotting analysis of srGAP1 expression in human eosinophils and neutrophils. F, Effects of Slit2 on srGAP1 binding to Robo1. Endogenous srGAP1 was coimmunoprecipitated with the Robo1 Ab from the lysates of HL-60 cells preincubated with PBS (–) or Slit2, followed by immunoblotting with the Abs srGAP1 or Robo1. G, Measurement of Cdc42 activity in Aml and HL-60 cells preincubated with PBS or Slit2. H, Effects of Slit2 on p85 binding to Robo1. Endogenous p85 was coimmunoprecipitated with the Robo1 Ab from the lysates of Aml cells, HL-60 cells, mouse eosinophils, and neutrophils that had pretreated with PBS (–) or Slit2, followed by immunoblotting with the Abs to p85 and Robo1. I, Immunoprecipitation of p85–Robo1 complex. Endogenous Robo1 was coimmunoprecipitated with the p85 mAb from the lysates of Aml cells preincubated with PBS or Slit2, followed by immunoblotting with the Abs to Robo1 and p85. J, Determination of Slit2-induced PI3K activation. Total and phosphorylated Akt in Aml and HL-60 cells preincubated with PBS or Slit2 was detected by immunoblotting with their respective Abs. K, The time course of Slit2-induced Akt phosphorylation in Aml cells. L, Akt phosphorylation in mouse eosinophils and neutrophils preincubated with PBS or Slit2. M, Inhibitory effect of R5 or mIgG (both at 0.5 μM) on Slit2-induced Akt phosphorylation in Aml cells. N, Measurements of Cdc42 activity and srGAP1 expression in DMSO or WT-pretreated Aml cells. Data represent two or three experiments or the mean ± SD of two to four independent experiments. *p < 0.05; **p < 0.01.

FIGURE 1.

srGAP1 expression and Robo1 downstream signaling in eosinophils and neutrophils. RT-PCR (A) and quantitative real-time RT-PCR (B) analysis of srGAP1 mRNA in human eosinophilic Aml cells, human promyeloid HL-60 cells, mouse eosinophils, and mouse neutrophils. C, Immunoblotting of srGAP1 and α-tubulin in Aml cells, HL-60 cells, mouse eosinophils, and neutrophils. D and E, The quantitative real-time PCR and immunoblotting analysis of srGAP1 expression in human eosinophils and neutrophils. F, Effects of Slit2 on srGAP1 binding to Robo1. Endogenous srGAP1 was coimmunoprecipitated with the Robo1 Ab from the lysates of HL-60 cells preincubated with PBS (–) or Slit2, followed by immunoblotting with the Abs srGAP1 or Robo1. G, Measurement of Cdc42 activity in Aml and HL-60 cells preincubated with PBS or Slit2. H, Effects of Slit2 on p85 binding to Robo1. Endogenous p85 was coimmunoprecipitated with the Robo1 Ab from the lysates of Aml cells, HL-60 cells, mouse eosinophils, and neutrophils that had pretreated with PBS (–) or Slit2, followed by immunoblotting with the Abs to p85 and Robo1. I, Immunoprecipitation of p85–Robo1 complex. Endogenous Robo1 was coimmunoprecipitated with the p85 mAb from the lysates of Aml cells preincubated with PBS or Slit2, followed by immunoblotting with the Abs to Robo1 and p85. J, Determination of Slit2-induced PI3K activation. Total and phosphorylated Akt in Aml and HL-60 cells preincubated with PBS or Slit2 was detected by immunoblotting with their respective Abs. K, The time course of Slit2-induced Akt phosphorylation in Aml cells. L, Akt phosphorylation in mouse eosinophils and neutrophils preincubated with PBS or Slit2. M, Inhibitory effect of R5 or mIgG (both at 0.5 μM) on Slit2-induced Akt phosphorylation in Aml cells. N, Measurements of Cdc42 activity and srGAP1 expression in DMSO or WT-pretreated Aml cells. Data represent two or three experiments or the mean ± SD of two to four independent experiments. *p < 0.05; **p < 0.01.

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We have previously shown that the PI3K inhibitors, WT and LY294002, inhibit Slit2-induced attractive migration of vascular endothelial cells in a dose-dependent manner (18). We thus hypothesized that Slit2 might also regulate eosinophil migration through PI3K signaling. Indeed, Slit2 induced the Robo1 Ab coimmunoprecipitation of the p85 subunit of PI3K in Aml cells or mouse eosinophils, but not in HL-60 cells or mouse neutrophils (Fig. 1H, 1I). We next examined whether Slit2 might activate Akt, as evidenced by Akt phosphorylation at the amino acid residue of Ser473. Preincubation of Aml cells with Slit2 triggered Ser473 phosphorylation of Akt (Fig. 1J, upper left panel), which occurred in a time-dependent manner (Fig. 1K, upper panel). Slit2 also elicited Ser473 phosphorylation of Akt in mouse eosinophils (Fig. 1L, upper left panel). In contrast, Slit2 failed to induce Ser473 phosphorylation of Akt in HL-60 cells (Fig. 1J, upper right panel) or mouse neutrophils (Fig. 1L, upper right panel). Total Akt was also determined as a sample loading control (Fig. 1J–L, lower panels). Preincubation of Aml cells with R5, a mouse mAb to the first Ig domain of Robo1 that neutralizes the interaction of Robo1 with Slit2 (1821), but not its isotype-matched irrelevant control mIgG, prevented Slit2-induced Ser473 phosphorylation of Akt in Aml cells (Fig. 1M). Interestingly, the srGAP1 level in Aml cells was apparently so low that inhibition of PI3K by WT was not enough to rescue the srGAP1-mediated Cdc42 inactivation (Fig. 1N). It is thus apparent that due to the differential level of srGAP1 expression in leukocytes—that is, a higher level of srGAP1 expression in neutrophils versus a lower level of srGAP1 expression in eosinophils—Slit2 recruits srGAP1 to Robo1 for inactivation of Cdc42 in neutrophils while recruiting PI3K to Robo1 for activation of PI3K signaling in eosinophils. As Cdc42 and PI3K activities are known to critically determine Slit2-induced cell migration (18, 28), our results implicate that Slit2, by modulation of Cdc42 and PI3K activities, may differentially alter chemotaxis of eosinophils and neutrophils in response to chemokines and chemoattractants.

On the basis of above biochemical studies, we employed a microfluidic chamber (μ-slide, Ibidi), which generates a stable gradient for up to 30 min, for real-time monitoring of chemotactic migration of leukocytes in response to a Slit2 gradient (41). As expected, conditioned medium from 293 cells overexpressing human Slit2 (the Slit2 medium), but not conditioned medium from 293 cells transfected with the plain vector (the control medium) (28), polarized mouse and human eosinophils, causing them to spread and migrate in a random and nondirectional manner (Fig. 2A, Supplemental Table I, Supplemental Videos 1, 3). In contrast, mouse and human neutrophils retained their quiescent state when exposed to Slit2 (Fig. 2A, Supplemental Table I, Supplemental Videos 2, 3).

FIGURE 2.

Chemotactic modulation of Slit2 on eosinophils and neutrophils. A, Real-time monitoring of activated mouse eosinophils (30 out of 34) and mouse neutrophils (2 out of 25) in response to a Slit2 gradient. B, Real-time monitoring of migrating mouse eosinophils (15 out of 29 for eotaxin alone and 8 out of 12 for eotaxin plus Slit2) and mouse neutrophils (16 out of 22 for SDF-1α alone and 3 out of 26 for SDF-1α plus Slit2) in a μ-Slide chemotaxis assay in response to the indicated gradients. Eotaxin and SDF-1α gradients were higher in the left side and lower in the right side of the image. Scale bars, 20 μm for A and B. Migration of eosinophils (C) and neutrophils (D) in a Transwell chamber. E, Preventive effects of R5 (2 out of 13), WT (2 out of 10), mIgG (10 out of 20), DMSO (9 out of 15) or AS (3 out of 18) on Slit2-triggered activation of mouse eosinophils. Scale bar, 20 μm. Effects of WT and AS on eotaxin-mediated eosinophil chemotaxis potentiated by Slit2 (F) and SDF-1α–mediated neutrophil chemotaxis suppressed by Slit2 (G) in a Transwell chamber. Data represent two or three experiments or the mean ± SD of two to four independent experiments. *p < 0.05; **p < 0.01.

FIGURE 2.

Chemotactic modulation of Slit2 on eosinophils and neutrophils. A, Real-time monitoring of activated mouse eosinophils (30 out of 34) and mouse neutrophils (2 out of 25) in response to a Slit2 gradient. B, Real-time monitoring of migrating mouse eosinophils (15 out of 29 for eotaxin alone and 8 out of 12 for eotaxin plus Slit2) and mouse neutrophils (16 out of 22 for SDF-1α alone and 3 out of 26 for SDF-1α plus Slit2) in a μ-Slide chemotaxis assay in response to the indicated gradients. Eotaxin and SDF-1α gradients were higher in the left side and lower in the right side of the image. Scale bars, 20 μm for A and B. Migration of eosinophils (C) and neutrophils (D) in a Transwell chamber. E, Preventive effects of R5 (2 out of 13), WT (2 out of 10), mIgG (10 out of 20), DMSO (9 out of 15) or AS (3 out of 18) on Slit2-triggered activation of mouse eosinophils. Scale bar, 20 μm. Effects of WT and AS on eotaxin-mediated eosinophil chemotaxis potentiated by Slit2 (F) and SDF-1α–mediated neutrophil chemotaxis suppressed by Slit2 (G) in a Transwell chamber. Data represent two or three experiments or the mean ± SD of two to four independent experiments. *p < 0.05; **p < 0.01.

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We next tested whether Slit2 could act with certain well-characterized chemokines for modulation of leukocyte chemotaxis. Indeed, mouse eosinophils migrated toward an eotaxin gradient, whereas mouse neutrophils migrated toward an SDF-1α gradient (Fig. 2B, Supplemental Table I, Supplemental Videos 1, 2). Notably, the speed of eotaxin-mediated eosinophil migration was slower than that of SDF-1α–mediated neutrophil migration. Importantly, the Slit2 medium, but not the control medium, increased eotaxin-mediated eosinophil migration while decreasing SDF-1α–mediated neutrophil migration.

To determine the net results of leukocyte chemotaxis over a period of 1 h, we used a traditional Transwell assay consisting of two chambers separated by a semipermeable membrane. Eosinophils or neutrophils were added to the upper chamber while eotaxin or SDF-1α was added to the lower chamber. Leukocytes that had migrated from the upper chambers to the lower chambers were then analyzed. As expected, eotaxin augmented eosinophil chemotaxis (Fig. 2C), whereas SDF-1α enhanced neutrophil chemotaxis (Fig. 2D). Although it alone did not induce chemotaxis of eosinophils or neutrophils, Slit2 significantly enhanced eotaxin-induced eosinophil chemotaxis and mitigated SDF-1α–induced neutrophil chemotaxis. These in vitro findings support to our hypothesis that srGAP1, which is downstream of Slit-Robo signaling, may determine chemokine-mediated directional migration of eosinophils and neutrophils in response to Slit2.

We next tested whether PI3K inhibitors, WT (a pan-inhibitor; 18) or AS (a selective inhibitor for p110γ; 45), might suppress Slit2-induced eosinophil polarization and chemotaxis. We found that in addition to R5, WT and AS, but not mIgG and DMSO, also attenuated Slit2-induced eosinophil activation, such as spreading, motility, and random and nondirectional migration (Fig. 2E, Supplemental Table I, Supplemental Video 4). Additionally, WT and AS neutralized of the ability of Slit2 to augment eotaxin-mediated chemotaxis of mouse eosinophils (Fig. 2F). In contrast, WT and AS failed to prevent the ability of Slit2 to inhibit SDF-1α–mediated chemotaxis of mouse neutrophils (Fig. 2G). Our results indicate that, upon Slit2 binding to Robo1, PI3K is recruited to Robo1, which phosphorylates Akt and consequently increases eotaxin-mediated eosinophil chemotaxis. Taken together, these in vitro results suggest that Slit2 has opposite biological effects on leukocyte chemotaxis depending on the intracellular machinery, with Slit2 signaling through PI3K for increasing eotaxin-mediated eosinophil chemotaxis, whereas through srGAP1 for decreasing SDF-1α–mediated neutrophils chemotaxis.

To verify our hypothesis regarding to srGAP1, we decided to test whether blockade of srGAP1 binding to the intracellular CC3 motif of Robo1 could neutralize the inhibitory effect of Slit2 on SDF-1α–mediated chemotaxis of neutrophils. The crystal structure of the srGAP1-Robo1 complex indicates that the srGAP1 amino acid residues of Ser758, Arg760, Glu761, Trp780, and Leu791 in the P3 loop of the SH3 domain are essential for recognition of the Robo1 intracellular CC3 motif (40). We thus designed the polypeptides encoding the wild-type SH3 domain (674–1022 aa residules) of srGAP1 (TAT-SH3) or an SH3 domain mutant, srGAP1S758A, R760A, E761A, W780A, L791A (TAT-SH3M), both of which were fused with a TAT sequence to facilitate incorpotation into the cytoplasm of mammalian cells (Fig. 3A) (34). The polypeptides were also fused with the His tag for purification with the nickle beads. We also designed a polypeptide encoding the wild-type SH3 domain without TAT (His-SH3) as a negative control. Following expression and isolation of these polypeptides, we found that, compared with the GST beads, the GST-Robo1 CC3 motif protein-bound beads pulled down TAT-SH3, but not TAT-SH3M, as detected by the anti-His tag Ab (Fig. 3B, lower panel). The loaded polypeptides of TAT-SH3 and TAT-SH3M were also detected by the anti-His tag Ab (Fig. 3B, upper panel). These polypeptides did not affect the survival of isolated mouse neutrophils as measured by the MTT assay (data not shown) (18, 21, 46). As expected, TAT-SH3, but not TAT-SH3M, inhibited srGAP1 binding to HA-Robo1 (Fig. 3C) and prevented Slit2-induced Cdc42 inactivation (Fig. 3D). Direct immunoblotting of HA-Robo1, GFP-srGAP1 (Fig. 3C), and total Cdc42 (Fig. 3D) was used as sample loading controls. These polypeptides were directly conjugated with FITC followed by incubation with mouse neutrophils. Compared to the FITC-conjugated His-SH3, the cytoplasmic localization of FITC-conjugated TAT-SH3 and TAT-SH3M was evident (Fig. 3E). Importantly, TAT-SH3, but not TAT-SH3M or His-SH3, prevented Slit2 from inhibiting SDF-1α–mediated neutrophil chemotaxis (Fig. 3F). Our results indicate that, upon Slit2 binding to Robo1, srGAP1 inactivates Cdc42 and consequently inhibits SDF-1α–mediated chemotaxis of neutrophils.

FIGURE 3.

Role of srGAP1 in Slit2 regulation of neutrophils chemotaxis. A, Design of the polypeptides encoding the srGAP1 SH3 domain and its mutant fused with the His tag and TAT sequence. B, GST-Robo1-CC3 pulldown of TAT-SH3, but not TAT-SH3M. C, Inhibitory effects of TAT-SH3 or TAT-SH3M on GFP-srGAP1 binding to HA-Robo1. Neutralizing effect of TAT-SH3, TAT-SH3M, or His-SH3 on Slit2-induced inactivation of Cdc42 (D) and inhibition of SDF-1α–mediated chemotaxis (F) of mouse neutrophils. E, Cytoplasmic incorporation of the FITC-conjugated TAT-SH3, TAT-SH3M, or His-SH3 in mouse neutrophils. Data are the mean ± SD of three independent experiments. **p < 0.01.

FIGURE 3.

Role of srGAP1 in Slit2 regulation of neutrophils chemotaxis. A, Design of the polypeptides encoding the srGAP1 SH3 domain and its mutant fused with the His tag and TAT sequence. B, GST-Robo1-CC3 pulldown of TAT-SH3, but not TAT-SH3M. C, Inhibitory effects of TAT-SH3 or TAT-SH3M on GFP-srGAP1 binding to HA-Robo1. Neutralizing effect of TAT-SH3, TAT-SH3M, or His-SH3 on Slit2-induced inactivation of Cdc42 (D) and inhibition of SDF-1α–mediated chemotaxis (F) of mouse neutrophils. E, Cytoplasmic incorporation of the FITC-conjugated TAT-SH3, TAT-SH3M, or His-SH3 in mouse neutrophils. Data are the mean ± SD of three independent experiments. **p < 0.01.

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As eosinophils and neutrophils are essential to the pathogenesis of OVA-induced allergic airway inflammation and endotoxin-induced lung inflammation (43, 44), we examined the expression profiles of Slit2 and Robo1 in normal and inflammatory murine tissues and leukocytes. Consistent with the previous reports of Slit2 expression in developing mouse lung (47, 48), we detected Slit2 expression on nonciliated secretory Clara cells (CC10-positive), but not on cilia cells (tubulin IV-positive), of normal bronchial epithelium (Fig. 4A). Robo1 expression was detected on the cell surfaces of freshly isolated mouse eosinophils (positive for EPO) and neutrophils (positive for NE) (Fig. 4B). Importantly, the expression of Slit2 mRNA and protein was dramatically upregulated, peaking at 12–36 h following aerosol challenge with OVA (Fig. 4C, 4E). In contrast, no obvious upregulation of Slit2 mRNA and protein expression was detected in lung tissues of endotoxin-induced lung inflammation, even though the basal level of Slit2 mRNA and protein expression was clearly visible (Fig. 4D, 4F). In addition, no clear upregulation of Robo1 was detected in eosinophils isolated from OVA-sensitized mice or neutrophils isolated from LPS-challenged mice (data not shown). Given that Slit2 acts synergistically with eotaxin for promoting eosinophil chemotaxis while suppressing SDF-1α–mediated neutrophil chemotaxis in vitro (Fig. 2), our findings of Slit2 expression in nonciliated secretory Clara cells and Robo1 expression on eosinophils and neutrophils suggest that Slit-Robo signaling may regulate chemotaxis of eosinophils and neutrophils in vivo, especially during allergic airway inflammation when Slit2 expression is upregulated.

FIGURE 4.

Expression of Slit2 and Robo1 in lung tissues and leukocytes. A, The expression of Slit2 in CC10-positive Clara cells, but not tubulin IV-positive cilia cells, of the bronchial epithelium. B, Robo1 expression (green) in mouse eosinophils (EPO-positive; red) and mouse neutrophils (NE-positive; red). DAPI (blue) was used for staining of cell nuclei. qRT-PCR (C, D) and immunoblotting (E, F) analysis of Slit2 mRNA and protein. Lysates were extracted from lung tissues after aerosol challenge with OVA (C, E) or LPS (D, F) at the indicated time points. G, Immunofluorescent staining of Slit2 in OVA-sensitized mouse lung tissues at the indicated time points. A, B, and G, Original magnification ×630; scale bar, 10 μm. Data are representative of two to three experiments.

FIGURE 4.

Expression of Slit2 and Robo1 in lung tissues and leukocytes. A, The expression of Slit2 in CC10-positive Clara cells, but not tubulin IV-positive cilia cells, of the bronchial epithelium. B, Robo1 expression (green) in mouse eosinophils (EPO-positive; red) and mouse neutrophils (NE-positive; red). DAPI (blue) was used for staining of cell nuclei. qRT-PCR (C, D) and immunoblotting (E, F) analysis of Slit2 mRNA and protein. Lysates were extracted from lung tissues after aerosol challenge with OVA (C, E) or LPS (D, F) at the indicated time points. G, Immunofluorescent staining of Slit2 in OVA-sensitized mouse lung tissues at the indicated time points. A, B, and G, Original magnification ×630; scale bar, 10 μm. Data are representative of two to three experiments.

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As nonciliated secretory Clara cells in the bronchial epithelium secrete Slit2 (Fig. 4A), and aerosol challenge with OVA markedly increases Slit2 expression (Fig. 4C, 4E), we suspected that OVA-sensitization might elicit a Slit2 gradient at the bronchus–alveoli axis—that is, the amount of secreted Slit2 might be higher in the bronchial epithelium and lower in the alveolar tissues and their supplying blood vessels. To test this hypothesis, we examined Slit2 expression in OVA-sensitized lung tissues. Immunofluorescent staining of Slit2 was elevated at ∼12 h, peaking at ∼36 h and gradually declining at ∼60 to ∼80 h (Fig. 4G), which is consistent with the expression profile of Slit2 determined by quantitative RT-PCR (Fig. 4C) and immunoblotting (Fig. 4E). Importantly, Slit2 expression was higher in the bronchi and lower in surrounding alveolar tissues, providing experimental evidence for the suspected Slit2 gradient at the bronchus–alveoli axis following aerosol challenge with OVA (Fig. 4G).

Slit2 knockout mice are either embryonic lethal or die within 1 or 2 wk postbirth (49). We therefore used Slit2-Tg mice with the pCMV promoter for efficient, but nonselective, expression of human Slit2 in mice (33). The availability of Slit2-Tg mice allows us to investigate the functional importance of Slit2 in systemic inflammation in vivo. Compared to C57 mice, the Flag tag in the lung extracts of Slit2-Tg mice was detected by PCR, using its specific primer pair (Supplemental Fig. 2A, left upper panel) (33). β-actin was used as a sample loading control (Supplemental Fig. 2A, left lower panel). For detection of the Slit2 transgene protein, detergent extracts of whole lungs from C57 mice and Slit2-Tg mice were immunoprecipitated with S1 (an anti-Slit2 mAb) or mIgG and immunoblotted with M2 (an anti-Flag mAb). Compared to C57 mice, the expression of Flag-Slit2 fusion protein was detected in the lung extracts of Slit2-Tg mice (Supplemental Fig. 2B, right panel). These data attest to the successful overexpression of the Slit2 transgene in the lungs of Slit2-Tg mice.

To investigate the roles of Slit2 in eosinophil chemotaxis in vivo, we compared leukocyte infiltration in C57 mice and Slit2-Tg mice subjected to OVA-induced allergic airway inflammation. Aerosol challenge of C57 mice with OVA (43) clearly triggered the infiltration of leukocytes, mainly eosinophils, into the peribronchial and perivascular regions of the lungs (Fig. 5A, left upper panels) and BALFs (Fig. 5B). Slit2-Tg mice manifested massive infiltration of eosinophils into the peribronchial and perivascular regions (Fig. 5A, left middle panels) and displayed significantly increased deposition (∼3-fold) of eosinophils in BALFs as compared with C57 mice (Fig. 5B). Identical data were obtained from experiments using founder B of Slit2-Tg mice (data not shown). Notably, total leukocytes and leukocyte subpopulations in blood remained unchanged (Fig. 5C), ruling out possibility of a hematopoietic mechanism by which eosinophils more aggressively infiltrate lung tissues in OVA-sensitized Slit2-Tg mice.

FIGURE 5.

Effects of Slit2 on eosinophil chemotaxis and allergic airway inflammation. A, H&E (left panels) and periodic acid-Schiff (Diagnostics Biosystems, Pleasanton, CA; right panels) staining of lung tissues obtained from C57 and Slit2-Tg mice without (Sham) or with (Model) aerosol challenge with OVA in the absence or presence of mIgG or R5 treatment. B, Infiltration of eosinophils in BALFs in C57 and Slit2-Tg mice following OVA sensitization. Leukocyte counts in blood (C) and IgE levels in BALFs (F) and blood (G) of C57 and Slit2-Tg mice with or without aerosol challenge with OVA. Effects of mIgG or R5 on eosinophil deposition in BALFs (D), circulatory leukocytes (E), and serum IgE (H) of OVA-sensitized C57 mice. A, Left panels, Original magnification ×100; scale bar, 20μm. A, Right panels, Original magnification ×200; scale bar, 5μm. Data represent the mean ± SD of two to three independent experiments (n = 5–10 for each group). *p < 0.05; **p < 0.01.

FIGURE 5.

Effects of Slit2 on eosinophil chemotaxis and allergic airway inflammation. A, H&E (left panels) and periodic acid-Schiff (Diagnostics Biosystems, Pleasanton, CA; right panels) staining of lung tissues obtained from C57 and Slit2-Tg mice without (Sham) or with (Model) aerosol challenge with OVA in the absence or presence of mIgG or R5 treatment. B, Infiltration of eosinophils in BALFs in C57 and Slit2-Tg mice following OVA sensitization. Leukocyte counts in blood (C) and IgE levels in BALFs (F) and blood (G) of C57 and Slit2-Tg mice with or without aerosol challenge with OVA. Effects of mIgG or R5 on eosinophil deposition in BALFs (D), circulatory leukocytes (E), and serum IgE (H) of OVA-sensitized C57 mice. A, Left panels, Original magnification ×100; scale bar, 20μm. A, Right panels, Original magnification ×200; scale bar, 5μm. Data represent the mean ± SD of two to three independent experiments (n = 5–10 for each group). *p < 0.05; **p < 0.01.

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To assess the importance of Slit2 in the pathological features of allergic airway inflammation, we compared mucus deposition in bronchial tissues of C57 and Slit2-Tg mice. Mucus staining of bronchial tissues was negative unless mice were aerosol challenged with OVA (Fig. 5A, right upper panels). Compared to C57 mice, OVA-sensitized Slit2-Tg mice had profoundly more accumulation of mucus (dark blue staining) in the bronchi (Fig. 5A, right middle panels), elevated levels of IgE in BALFs (Fig. 5F) and serum (Fig. 5G), and increased amounts of CXCL1 and 2 mRNAs and proteins (Supplemental Fig. 3). These findings provide evidence that Slit-Robo signaling exacerbates allergic airway inflammation and is associated with eosinophil recruitment to lung tissue.

To further support our conclusion, we tested whether blockade of Slit2 binding to Robo1 suppresses allergic airway inflammation in vivo. Indeed, i.v. injection of R5, but not mIgG, potently inhibited eosinophil deposition in the peribronchial and perivascular regions of the lungs (Fig. 5A, left lower panels) and BALFs (Fig. 5D) of OVA-sensitized C57 mice, without affecting the number of total leukocytes or leukocyte subpopulations in circulation (Fig. 5E). R5 treatment also significantly decreased mucus accumulation within the bronchial lumens (Fig. 5A, right lower panels) and reduced the level of plasma IgE (Fig. 5H). Our results that Slit2 potentiates eosinophil chemotaxis, promotes mucus deposition, and elevates IgE collectively indicate that Slit-Robo signaling exaggerates allergic airway inflammation. Notably, Slit2-Tg mice displayed accelerated neutrophil infiltration (Fig. 5A) and elevated CXCL1 and -2 (Supplemental Fig. 3), whereas R5 treatment attenuated neutrophil deposition (Fig. 5D), suggesting that the promoting effects of Slit2 in allergic airway inflammation may culminate in amplification of nonallergic inflammation.

To examine the effects of Slit2 on neutrophil chemotaxis in vivo, we employed a murine model of endotoxin-induced lung inflammation to compare the pneumonia-like phenotypic changes between C57 mice and Slit2-Tg mice (44). Aerosol challenge with LPS triggered a dramatic infiltration of leukocytes, mainly neutrophils, in BALFs of C57 and Slit2-Tg mice (Fig. 6A). Importantly, Slit2-Tg mice manifested a decreased deposition of neutrophils (∼50%) as compared with C57 mice. In contrast, neutralization of Slit2 binding to Robo1 by R5, but not mIgG, increased neutrophil infiltration by ∼3-fold in BALFs of C57 mice subjected to LPS challenge (Fig. 6C). Again, total leukocytes and leukocyte subpopulations in blood remained relatively unaltered (Fig. 6B, 6D). The observed inhibitory effect of Slit2 on neutrophil chemotaxis in vivo is fully consistent with our in vitro findings (Fig. 2), further supporting the notion that Slit-Robo signaling induces a chemorepellent response on neutrophils in endotoxin-induced lung inflammation.

FIGURE 6.

Effects of Slit2 on neutrophil chemotaxis and endotoxin-induced lung inflammation. A, Infiltration of neutrophils in BALFs of C57 and Slit2-Tg mice following aerosol challenge with LPS. B, Leukocyte counts and differentials in the blood of C57 and Slit2-Tg mice without (Sham) or with (Model) aerosol challenge with LPS. C, Effects of mIgG or R5 on neutrophil deposition in BALFs of C57 mice following aerosol challenge with LPS. D, Leukocyte counts and differentials in the blood of C57 mice pretreated with R5 or mIgG. Data represent the mean ± SD of two to three independent experiments (n = 6 to 7 for each group). *p < 0.05; **p < 0.01.

FIGURE 6.

Effects of Slit2 on neutrophil chemotaxis and endotoxin-induced lung inflammation. A, Infiltration of neutrophils in BALFs of C57 and Slit2-Tg mice following aerosol challenge with LPS. B, Leukocyte counts and differentials in the blood of C57 and Slit2-Tg mice without (Sham) or with (Model) aerosol challenge with LPS. C, Effects of mIgG or R5 on neutrophil deposition in BALFs of C57 mice following aerosol challenge with LPS. D, Leukocyte counts and differentials in the blood of C57 mice pretreated with R5 or mIgG. Data represent the mean ± SD of two to three independent experiments (n = 6 to 7 for each group). *p < 0.05; **p < 0.01.

Close modal

To mimic the effects of the Slit2 gradient at the bronchus–alveoli axis (Fig. 4G), we hypothesized that, following OVA-sensitization, aerosol inhalation of Slit2 would increase the bronchial pool of Slit2, whereas i.v. administration of Slit2 would decrease it by increasing the circulatory pool of Slit2. As predicted, aerosol administration of Slit2 markedly enhanced eosinophil infiltration into BALFs of OVA-sensitized lungs (Fig. 7A). In contrast, i.v. administration of Slit2 drastically mitigated eosinophil accumulation in BALFs (Fig. 7B). Taken together, these functional findings demonstrate that this newly identified Slit2 gradient at the bronchus–alveoli axis accelerates eosinophil chemotaxis during allergic airway inflammation.

FIGURE 7.

Slit2 gradient at bronchus–alveoli axis modulates eosinophil chemotaxis Eosinophil infiltration in BALFs of OVA-sensitized C57 mice following aerosol inhalation of Slit2 (A) or i.v. injection of Slit2 (B). Data represent the mean ± SD of two independent experiments (n = 3–7 for each group). *p < 0.05; **p < 0.01.

FIGURE 7.

Slit2 gradient at bronchus–alveoli axis modulates eosinophil chemotaxis Eosinophil infiltration in BALFs of OVA-sensitized C57 mice following aerosol inhalation of Slit2 (A) or i.v. injection of Slit2 (B). Data represent the mean ± SD of two independent experiments (n = 3–7 for each group). *p < 0.05; **p < 0.01.

Close modal

To examine the in vivo importance of PI3K signaling in eosinophil infiltration in response to Slit2, C57 and Slit2-Tg mice were aerosol challenged with OVA in the absence or presence of WT (18) or AS (45). Both WT (Fig. 8A) and AS (Fig. 8B) only partially suppressed eosinophil infiltration in BALFs of C57 mice following OVA sensitization, which is consistent with a previous report that WT and LY294002 exhibited low potency against eotaxin-induced chemotactic responses (6). In sharp contrast, both agents potently abolished Slit2-potentiated eosinophil accumulation in Slit2-Tg mice to the levels comparable to those in C57 mice. Our results thus indicate the functional importance of PI3K signaling, a downstream effector of Slit-Robo signaling, for enhancing eosinophil chemotaxis in vivo.

FIGURE 8.

PI3K dependence of Slit2-potentiated eosinophil infiltration. Eosinophil infiltration in BALFs of OVA-sensitized C57 and Slit2-Tg mice in the absence or presence of DMSO, WT (A), or AS (B). Data represent the mean ± SD of two independent experiments (n = 3–7 for each group). **p < 0.01.

FIGURE 8.

PI3K dependence of Slit2-potentiated eosinophil infiltration. Eosinophil infiltration in BALFs of OVA-sensitized C57 and Slit2-Tg mice in the absence or presence of DMSO, WT (A), or AS (B). Data represent the mean ± SD of two independent experiments (n = 3–7 for each group). **p < 0.01.

Close modal

Engagement of Robo1 by Slit2 induces srGAP1 binding to Robo1, which inactivates Cdc42 for repulsive migration of neuronal cells (28). In this study, we show that Slit2, by differential regulation of Cdc42 activity, potentiates eotaxin-mediated chemotaxis of eosinophils (srGAP1low) for exaggeration of allergic airway inflammation while suppressing SDF-1α–mediated chemotaxis of neutrophils (srGAP1high) for attenuation of endotoxin-induced lung inflammation. During leukocyte chemotaxis, srGAP1 signaling acts as a default or dominant mechanism, whereas PI3K signaling acts as an alternative or supplementary mechanism. The balance between repulsive srGAP1 signaling and attractive PI3K signaling thus appears to critically determine directional chemotaxis of leukocytes during allergic and endotoxin-induced lung inflammation (Supplemental Fig. 4). Notably, although Slit2 mediates axon guidance and neuronal migration and induces migration of vascular endothelial cells and cancer cells, it does not directly exert any appreciable action on the chemotactic activity of leukocytes, attesting to the functional role of Slit2 as a chemotactic regulator, but not as a chemokine itself, for leukocytes. To the best of our knowledge, this is the first example of an endogenous factor that regulates both attractive and repulsive chemotaxis among different subtypes of leukocytes.

The formation of a Slit2 gradient within the subventricular zone of the brain is essential to directional migration of neuronal progenitor cells, the direction of cilia, and the flow of cerebrospinal fluid (50). In addition, solid tumors exhibit a Slit2 gradient, which is high in the center and low in the periphery, for tumor-induced angiogenesis (18). In the current study, we show that nonciliated secretory Clara cells within normal bronchial epithelium secrete Slit2. Although the biological function of Slit2 secreted by Clara cells in the absence of inflammatory stimulation remains to be elucidated, we speculate that it may participate in the maintenance of physiological homeostasis of leukocytes and bronchi (12, 38, 45, 47, 50). Interestingly, aerosol challenge with OVA, but not LPS, dramatically elevated Slit2 expression, which is characteristically high in the bronchi and low in the alveoli, thus forming a Slit2 gradient at the so-called bronchus–alveoli axis. These results collectively suggest that by adhering to heparan sulfate proteoglycans on the cell surface of neighboring cells (51), secreted Slit2 forms a concentration gradient for mediating directional migration of neuronal cells and vascular endothelial cells and for regulating directional chemotaxis of leukocytes.

As Slit2 regulates chemokine-mediated chemotaxis of eosinophils and neutrophils in vitro, we suspected that the newly discovered Slit2 gradient at the bronchus–alveoli axis may play an essential role in guiding leukocyte chemotaxis during allergic airway inflammation. To test this hypothesis, we reasoned that manipulation of the Slit2 gradient would alter the direction of eosinophil chemotaxis in vivo. Remarkably, increasing the Slit2 gradient by aerosol administration of isolated recombinant human Slit2 markedly enhanced infiltration of eosinophils into BALFs, whereas decreasing the Slit2 gradient by i.v. administration of purified Slit2 significantly reduced it. Our results thus indicate the biological significance of the Slit2 gradient at the bronchus–alveoli axis for governing eosinophil chemotaxis during allergic airway inflammation. These data further demonstrate the feasibility, for the first time to the best of our knowledge, of adopting mouse lungs as a powerful assay for in vivo study of leukocyte chemotaxis.

Although this study has been focused on modulation of leukocyte chemotaxis by Slit-Robo signaling and its underlying molecular mechanisms, we have systematically assessed the significance of Slit-Robo signaling in the pathogenesis of allergic and endotoxin-induced lung inflammation. For instance, in vivo expression of exogenous Slit2 exaggerated allergic airway inflammation, but mitigated endotoxin-induced lung inflammation, as evidenced by a comparison between Slit2-Tg and C57 mice. Additionally, neutralization of endogenous Slit2 binding to Robo1 by R5, but not isotype-matched irrelevant mIgG, decreased allergic airway inflammation, but increased endotoxin-induced lung inflammation. Our experimental findings are fully consistent with previously published reports (1217), demonstrating a pathological role for Slit-Robo signaling in modulating chemotaxis of neutrophils, macrophages, and T lymphocytes. These results collectively indicate the pathological importance of Slit-Robo signaling in inflammatory responses in vivo.

As IL-5 critically mediates eosinophil maturation, survival, activation, and chemotaxis (32), IL-5-Tg mice have been used extensively for a variety of studies, such as eosinophil migration and chemotaxis (52) and phagocytosis (53). In this study, we have isolated mouse eosinophils from IL-5-Tg mice for in vitro experiments of eosinophil polarization, migration, and chemotaxis. We have also performed OVA-induced allergic airway inflammation for in vivo investigation of eosinophils functionality. Notably, mouse eosinophils isolated from IL-5-Tg mice phenocopy human eosinophils in srGAP1 expression and Slit2 enhancement of chemotaxis, attesting to the suitability of mouse eosinophils isolated from IL-5-Tg mice to replace human eosinophils isolated from asthma patients in our present study.

In the current study, we have shown that nonsecretory Clara cells express Slit2, whereas neutrophils and eosinophils express Robo1. In addition, rSlit2 inhibits SDF-1α–induced chemotaxis of neutrophils while enhancing eotaxin-induced eosinophil chemotaxis in vitro. Furthermore, Slit-Robo signaling potently exaggerates eosinophil chemotaxis during allergic airway inflammation while drastically suppressing neutrophil chemotaxis during LPS-induced lung inflammation in vivo, as demonstrated by the studies using Slit2-Tg mice, R5 neutralizing mAb, and rSlit2 protein. These results collectively indicate a direct effect of Slit2 on chemotaxis of neutrophils and eosinophils during lung inflammation, which is fully consistent with previous reports (1217).

However, our findings do not eliminate the possibility of an indirect effect of Slit2 on chemotaxis of neutrophils and eosinophils in vivo. As Slit2 potently inhibits neutrophil infiltration in the model of endotoxin-induced lung inflammation (Fig. 6), it appears quite unexpected to observe an increased deposition of neutrophils in the BALFs of OVA-sensitized Slit2-Tg mice (Fig. 5B, 5D). To address this parodoxal finding, we reasoned that Slit-Robo signaling may possibly have other unknown direct and indirect targets during lung inflammation. In this regard, although asthma is traditionally considered as an eosinophilic disease in the airways, increased deposition of neutrophils in the lungs (54), exaggerated accumulation of neutrophils in the sputum (55), and elevated neutrophil-associated chemokines, such as IL-8 in patients and keratinocyte chemoattractant (CXCL1) and MIP-2 (CXCL2) in mice (39, 56, 57), have been well documented in allergic lung inflammation. We thus speculate that the attractive effects of Slit2 on eosinophil chemotaxis may overwhelm its repulsive effects on neutrophil chemotasis in the murine model of OVA-induced allergic airway inflammation, culminating in amplification of the entire inflammatory responses. The findings of increased neutrophil infiltration (Fig. 5B) and elevated CXCL1 and CXCL2 (Supplemental Fig. 3) in OVA-sensitized Slit2-Tg mice but decreased neutrophil deposition following R5 treatment (Fig. 5D) appear to lend a support to this hypothesis. Further studies along this line of investigation are therefore promising to identify other direct and indirect targets of Slit-Robo signaling during the pathogenesis of lung inflammation.

In response to Slit2 binding, leukocytes must send signals from the cell surface to the cytoplasm, which dynamically and reversibly control the cellular machinery for leukocyte chemotaxis. In this study, we show that the balance between repulsive srGAP1 signaling and attractive PI3K signaling may determine the directional chemotaxis of leukocytes during allergic and endotoxin-induced lung inflammation. However, it remains to be determined whether the signal transduction pathways involved in leukocyte chemotaxis are the same as those downstream of Robo and/or other potential receptors in neurons, endothelial cells, and cancer cells. Further pursuit of this line of investigation should enhance our understanding of the biological functions of guidance cues and the molecular mechanisms driving the polarization and the directional migration of these fascinating cells.

We thank James J. Lee (Mayo Clinic Arizona, Scottsdale, AZ) for IL-5-Tg mice and Michael J. Franklin (University of Minnesota, Minneapolis, MN) for editing the manuscript.

Disclosures The authors have no financial conflicts of interest.

This work was supported by grants from the National Science Foundation of China (30901302 to B.-Q.Y.), the Ministry of Science and Technology of China (2010CB529702 to B.-Q.Y.), and the National Institutes of Health (RO1AI064743 and RO1CA126897 to J.-G.G.).

The online version of this article contains supplemental material.

Abbreviations used in this paper:

Aml

Aml14.3D10

AS

5-(2,2-difluoro-benzo[1,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione

BALF

bronchial airway lavage fluid

EPO

eosinophils peroxidase

GAP

GTPase-activating protein

HA

hemagglutinin

mIgG

mouse IgG

NE

neutrophils elastase

SDF-1α

stromal cell-derived factor-1α

SH

Src homology

srGAP1

Slit-Robo GTPase-activating protein 1

Tg

transgenic

WT

wortmannin.

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