Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly

Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils This information is current as of September 24, 2021. Darrell Pilling, Luis E. Chinea, Kristen M. Consalvo and Richard H. Gomer J Immunol 2019; 202:239-248; Prepublished online 3 December 2018; doi: 10.4049/jimmunol.1800681 Downloaded from http://www.jimmunol.org/content/202/1/239

Supplementary http://www.jimmunol.org/content/suppl/2018/11/30/jimmunol.180068 http://www.jimmunol.org/ Material 1.DCSupplemental References This article cites 69 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/202/1/239.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils

Darrell Pilling,1 Luis E. Chinea,1 Kristen M. Consalvo, and Richard H. Gomer

The movement of neutrophils between blood and tissues appears to be regulated by chemoattractants and chemorepellents. Compared with neutrophil chemoattractants, relatively little is known about neutrophil chemorepellents. Slit proteins are endog- enously cleaved into a variety of N- and C-terminal fragments, and these fragments are neuronal chemorepellents and inhibit chemoattraction of many cell types, including neutrophils. In this report, we show that the ∼140-kDa N-terminal Slit2 fragment (Slit2-N) is a chemoattractant and the ∼110-kDa N-terminal Slit2 fragment (Slit2-S) is a chemorepellent for human neutrophils. The effects of both Slit2 fragments were blocked by Abs to the Slit2 receptor Roundabout homolog 1 or the Slit2 coreceptor Downloaded from Syndecan-4. Slit2-N did not appear to activate Ras but increased phosphatidylinositol 3,4,5-triphosphate levels. Slit2-N–induced chemoattraction was unaffected by Ras inhibitors, reversed by PI3K inhibitors, and blocked by Cdc42 and Rac inhibitors. In contrast, Slit2-S activated Ras but did not increase phosphatidylinositol 3,4,5-triphosphate levels. Slit2-S–induced chemorepulsion was blocked by Ras and Rac inhibitors, not affected by PI3K inhibitors, and reversed by Cdc42 inhibitors. Slit2-N, but not Slit2-S, increased neutrophil adhesion, myosin L chain 2 phosphorylation, and polarized actin formation and single pseudopods at the leading edge of cells. Slit2-S induced multiple pseudopods. These data suggest that Slit2 isoforms use similar receptors but http://www.jimmunol.org/ different intracellular signaling pathways and have different effects on the cytoskeleton and pseudopods to induce neutrophil chemoattraction or chemorepulsion. The Journal of Immunology, 2019, 202: 239–248.

hemoattraction is the directed movement of cells toward between mice and humans. A truncated ∼110-kDa Slit2 N- an attractant and is the mechanism by which, for terminal fragment (Slit2-S) containing only the four N-terminal C instance, immune cells move to sites of infection and leucine-rich repeat domains is 97.2% identical (98.9% similar) inflammation (1, 2). Although much is known about chemo- between mouse and human. Altered fragmentation patterns of attraction in eukaryotic cells, relatively little is known about how Slit2 have been observed in cancer, inflammation, fibrosis, and eukaryotic cells move away from a repellent (chemorepulsion). obesity (6–11), and we found abnormally low levels of Slit2 by guest on September 24, 2021 Slit was originally identified as an extracellular neuronal che- fragments in the bronchoalveolar lavage of mice with pulmonary morepellent protein sensed by Robo receptors in Drosophila and fibrosis (12). has since been found in Caenorhabditis elegans and vertebrates Immune cells such as neutrophils also express Robo re- (3, 4). Mammals have Slit1, Slit2, and Slit3, which are cleaved ceptors (13–15). Slit2 inhibits the chemotaxis of a variety of into ∼110–140-kDa N-terminal and ∼55-kDa C-terminal frag- immune cells toward attractants, but whether Slit can act as a ments (3, 5). The ∼140-kDa Slit2 N-terminal fragment (Slit2-N) repellent for immune cells is unknown (12–21). In this report, contains four N-terminal leucine-rich repeat domains and five we show that, for neutrophils, Slit2-N is a chemoattractant EGF-like domains and shows 96.8% identity (98.8% similarity) and Slit2-S is a chemorepellent and, although the two fragments use similar receptors, they use different signal transduction pathways. Department of Biology, Texas A&M University, College Station, TX 77843-3474 1D.P. and L.E.C. contributed equally. Materials and Methods ORCIDs: 0000-0002-7413-2773 (D.P.); 0000-0003-3511-2830 (L.E.C.); 0000-0001- 9791-7445 (K.M.C.); 0000-0003-2361-4307 (R.H.G.). Cell isolation and culture Received for publication May 14, 2018. Accepted for publication November 2, 2018. Human venous blood was collected from healthy volunteers who gave This work was supported by National Institutes of Health Grants HL118507 and written consent, with approval from the Texas A&M University GM118355. Institutional Review Board. Neutrophils were isolated as previously described (22), with the exceptions that Polymorphprep gradients Address correspondence and reprint requests to Dr. Darrell Pilling and Dr. Richard H. Gomer, Department of Biology, Texas A&M University, 301 Old Main Drive, (Axis-Shield; Oslo, Norway) were used following the manufacturer’s Interdisciplinary Life Sciences Building, 3474 TAMU, College Station, TX 77843- instructions, and centrifugation of gradients was done for 40 min. Cells 3474. E-mail addresses: [email protected] (D.P.) and [email protected] were then resuspended in RPMI 1640 (Lonza, Walkersville, MD) (R.H.G.) containing 2% BSA (Amresco, Solon, OH) (RPMI-BSA). To check the The online version of this article contains supplemental material. purity of the neutrophil isolation, cell spots were prepared as described previously (22) and stained with eosin and methylene blue. Isolated Abbreviations used in this article: FMI, forward migration index; MLC2, myosin L cell preparations were 95.6 6 0.5% neutrophils, 2.2 6 0.4% mono- chain 2; PBS-T, PBS/0.1% Triton X-100; PIP2, phosphatidylinositol 4,5-bisphosphate; cytes, 1.6 6 0.4% eosinophils, and 0.6 6 0.2% lymphocytes (mean 6 PIP3, phosphatidylinositol 3,4,5-triphosphate; RIP, Ras inhibitory peptide; Robo1, Roundabout homolog 1; RPMI-BSA, RPMI 1640 containing 2% BSA; Slit2-N, SEM, n = 16). In addition, cells attached to fibronectin-coated cover- ∼140-kDa Slit2 N-terminal fragment; Slit2-S, ∼110-kDa Slit2 N-terminal fragment; slips were air dried, fixed in methanol, and stained with methylene blue Sos, Son of sevenless. and eosin. These were 95.8 6 1.2% neutrophils, 2.0 6 1.0% mono- cytes, 1.8 6 0.8% eosinophils, and 0.3 6 0.2% lymphocytes (mean 6 Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 SEM, n =5). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800681 240 Slit2 DIRECTLY REGULATES NEUTROPHIL MIGRATION

Insall chamber assays with 1:2000 phalloidin-Alexa 555 (ab176756; Abcam) in PBS-T for 30 min at room temperature. Cells were washed with PBS-T, and cover- Insall chambers (23) were used to generate gradients of Slit2 and slips were mounted with fluorescent mounting medium containing DAPI other compounds to observe the movement of neutrophils on glass (Vector Laboratories). Immunofluorescence images were captured on an m coverslips coated with 10 g/ml human plasma fibronectin (Corning, Olympus FV1000 confocal microscope and analyzed using Olympus Bedford, MA), as previously described (22, 24, 25). Cells were Fluoview and ImageJ software as described previously (12, 26). Cells were allowed to adhere to coverslips for 30 min before the coverslip was scored as having polarized phalloidin staining (one region of staining, placed onto the Insall chamber. Recombinant mouse Slit2 Gln26- either toward or away from the corner of the well where material was ∼ Gln900 ( 110 kDa; R&D Systems, Minneapolis, MN), recombinant added), multiple staining (having two or more regions of phalloidin human Slit2 Gln26-Val1118 (120–140 kDa; PeproTech, Rocky Hill, staining), or circumferential/uniform staining (in which the majority of NJ), and recombinant human Slit2 C-terminal fragment Thr1122- the cell was stained with phalloidin). Ser1529 (∼50 kDa; R&D Systems) were resuspended in RPMI-BSA. Slit2 proteins were checked for purity and size by PAGE and silver Adhesion assays staining, as described previously (12, 26). The neutrophil chemoattractant fMLF (Alfa Aesar, Ward Hill, MA) was diluted in RPMI- 96-well polystyrene plates (353072; BD Biosciences) were coated with BSA and used at 10 nM. 10 mg/ml human plasma fibronectin in PBS for 1 h at 37˚C. The plate Rho GTPase Cdc42 inhibitor ML141, Ras inhibitory peptide (RIP), and was then washed three times with PBS, and 1 3 105 neutrophils in Rac inhibitor NSC 23766 (all from Cayman Chemical, Ann Arbor, MI) 100 ml of RPMI-BSA were allowed to attach to the coated wells for and the PI3K inhibitor LY294002 (BioVision, Milpitas, CA) were 30 min at 37˚C. A total of 100 ml of 10 nM fMLF, 500 ng/ml Slit2-N, reconstituted to 10 mM in DMSO (Amresco) according to the manu- 500 ng/ml Slit2-S, or 10 ng/ml TNF-a (BioLegend) was then added to facturer’s directions. The DMSO stocks were then diluted in RPMI-BSA the wells and incubated for a further 30 min at 37˚C. Plates were then and used at 10 mM. Cells were incubated with inhibitors for 30 min at washed three times with PBS, and adherent neutrophils were counted m 37˚C in a humidified 5% CO2 incubator before placing on the coverslip. in three 900- m-diameter fields of view per well as described previ- Downloaded from Where indicated, cells were incubated with 10 mg/ml sheep anti-human ously (31). In addition, neutrophils were preincubated with 500 ng/ml Roundabout homolog 1 (Robo1) Abs (R&D Systems) or 3 mg/ml sheep Slit2-N or Slit2-S for 15 min at 37˚C before incubation with buffer, anti-human Syndecan-4 Abs (R&D Systems) for 30 min as described fMLF, or TNF-a for an additional 15 min. Cells were then added to above. After placing coverslips with adhered neutrophils on the Insall fibronectin-coated wells, and cell adhesion was measured as described chamber, we waited 20 min for gradients to form before tracking neu- above. trophils as previously described (22, 24). At least 10 neutrophils per experiment were tracked for 40 min. Only cells that could be tracked for Phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol the full 40 min were analyzed. For each donor, neutrophils were tracked 3,4,5-triphosphate, and Ras assays http://www.jimmunol.org/ in a no-gradient control. Neutrophils were never used past 5 h from the 6 end of the isolation step. We used two Insall chamber/microscope/camera 10 3 10 neutrophils in 0.4 ml of RPMI-BSA were incubated at 37˚C in setups in parallel, allowing six to eight experimental conditions to be the presence or absence of 500 ng/ml Slit2 or 10 nM fMLF. For phos- measured per set of donor neutrophils (Supplemental Videos 1–6). The re- phatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5- sults are expressed as the mean 6 SEM of the movement of neutrophils triphosphate (PIP3) assays, after 5 min, 0.4 ml of ice-cold 1000 mM from three or more different donors. We never used the same donor TCAwasaddedtothecellsandthenincubatedonicefor5min.PIP2and twice for a given experiment. PIP3 were extracted and detected using ELISA kits following the manufacturer’s instructions (PIP2 K-4500, PIP3 K-2500s; Echelon, Salt Lake Isolation of cytoskeletal proteins City, UT). For Ras activation, cells were incubated with Slit2 or fMLF for 6 min, then 1.2 ml of ice-cold PBS was added to the cells, and the Preparation of cytoskeletons and gel electrophoresis to visualize F-actin cells were then collected by centrifugation at 500 3 g for 5 min at 4˚C. by guest on September 24, 2021 5 was done as previously described (27–29). Briefly, 5 3 10 cells in 100 ml Cells were then resuspended in 0.2 ml of kit lysis buffer, and the active RPMI-BSA were incubated at 37˚C in the presence or absence of 500 ng/ml GTP-bound form of Ras proteins was isolated and detected following the Slit2 or 10 nM fMLF. At the indicated time points, 1.4 ml of ice-cold manufacturer’s instructions (BK008-S; Cytoskeleton, Denver, CO). PBS was added to the cells, and cells were collected by centrifugation at Briefly, eluate from beads bearing the Ras-binding domain of the Ras 500 3 g for 5 min at 4˚C. Cells were lysed with 100 ml of 100 mM effector kinase Raf1, which isolates GTP-Ras, was analyzed by Western PIPES (pH 6.8), 1 mM MgCl2, 2.5 mM EGTA, and 0.5% Triton X-100 blotting using 4–20% Tris/glycine gels (Lonza, Allendale, NJ) (12, 26). with 43 protease and phosphatase inhibitor (Cell Signaling Technology) After blotting to PVDF membranes (Immobilon P; MilliporeSigma, on ice for 20 min. Triton-insoluble cytoskeletons were collected by Burlington, MA), protein transfer was assessed by Ponceau red staining centrifugation at 12,000 3 g for 5 min at 4˚C. Cytoskeleton pellets were (32). Blots were blocked and then incubated with anti-Ras Abs following resuspended in SDS/DTT sample buffer, heated to 98˚C for 5 min, the manufacturer’s instructions (BK008-S; Cytoskeleton). Cell lysates separated by PAGE, and then stained with Coomassie as described were also analyzed by Western blotting with 200 ng/ml anti-GAPDH previously (29). mouse mAb (Proteintech, Rosemont, IL) to confirm effective protein transfer. Confocal microscopy Statistics 1 3 106 cells in 200 ml of RPMI-BSA were allowed to attach to fibronectin-coated 8-well slides (Corning) for 30 min at 37˚C. Two Statistical analyses with t tests or one-way or two-way ANOVA with microliters of prewarmed RPMI-BSA (control), prewarmed RPMI-BSA Dunnett posttest were done using GraphPad Prism 7 (GraphPad, San plus 100 mg/ml Slit2-N or Slit2-S, or prewarmed RPMI-BSA plus Diego, CA). Significance was defined as p , 0.05. 200 nM fMLF was then added to the corner of the well, taking care not to disturb the cells or the medium. After 10 min, the cells were fixed by carefully adding 200 ml of prewarmed 4% paraformaldehyde (Electron Results Microscopy Sciences, Hatfield, PA) in PBS to the wells for 20 min at 37˚C. Different isoforms of Slit2 induce chemoattraction or Cells were then washed with PBS and permeabilized with PBS/0.1% chemorepulsion of neutrophils Triton X-100 (PBS-T) for 5 min at room temperature. Cells were then incubated with 1:1000 anti–phospho-AKT substrate (proteins containing In the developing nervous system, Slit2 acts as a chemorepellent phospho-serine or phospho-threonine preceded by arginine at the -3 for neurons (3–5). Slit2 concentrations between 400 ng/ml and position; RXXS*/T*) (30) Ab (rabbit IgG, no. 9614; Cell Signaling 100 mg/ml inhibit the chemotaxis of human and murine neu- Technology, Danvers, MA) in PBS-T containing 2% BSA or 5 mg/ml anti-CD11b (mouse IgG, ICRF44; BioLegend, San Diego, CA) in trophils toward the chemoattractants fMLF, CXCL12, IL-8, and PBS-T containing 2% BSA overnight at 4˚C. Cells were washed with the complement component C5a (15, 16, 18). An intriguing PBS-T before the addition of either 1 mg/ml goat anti-rabbit IgG Alexa possibility is that Slit2 might act directly as a chemorepellent 647 (Thermo Fisher Scientific, Waltham, MA) or donkey anti-mouse IgG for neutrophils. To test this, we used a gradient chamber to Alexa 488 (Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature. Cells were washed with PBS-T, and coverslips examine the effect of Slit2 gradients on human neutrophil were mounted with fluorescent mounting medium containing DAPI movement. We first examined the effects of a 110-kDa fragment (Vector Laboratories). Alternatively, after fixation, cells were incubated of mouse Slit2, henceforth designated Slit2-S. Compared with The Journal of Immunology 241

FIGURE 1. Neutrophils show bi- ased movement away from Slit2-S and toward Slit2-N. Neutrophils in (A) control, (B) 0–500 ng/ml Slit2-S, (C) 0–500 ng/ml Slit2-N, or (D) 0–5000 ng/ml Slit2-N gradients were ﬁlmed and tracked. Orientation is such that the source of Slit2 is on the left. Graphs are data from 1 of 10 independent experiments. The number of tracks analyzed is indicated in the top left corner. Red dots repre- sent the average center of mass for the ending positions of all cells. Downloaded from (E–G) Human neutrophils were placed in gradients of the indicated concentrations in nanograms per milliliter of (E) Slit2-S in the presence or absence of 10 nM fMLF, (F) Slit2-N, or (G) Slit2-C using Insall chambers, and video- http://www.jimmunol.org/ microscopy was used to record cell movement. A positive FMI indicates chemorepulsion, and a negative FMI indicates chemoattraction. At least 10 cells per experimental group for each individual donor were tracked for 40 min. Values are means 6 SEM for neutrophils from at least ﬁve different donors. *p , 0.05, **p , 0.01, by guest on September 24, 2021 ***p , 0.001 compared with the no- gradient control (one-way ANOVA, Dunnett test) or for the indicated comparison between two sets (t test).

cells in no gradient and gradients of 0–5 and 0–50 ng/ml, Slit2-S significantly lower than the percentage of cells going the did not significantly affect neutrophil movement (Fig. 1A, 1E, “wrong way” in the Slit2-S gradient (p , 0.05, t test), sug- Supplemental Fig. 1A, 1B, 1G, and Supplemental Video 1). A gesting that the fMLF gradient affects more neutrophils than gradient of 0–500 ng/ml (0–5 nM) Slit2-S caused neutrophils the Slit2-S gradient. An fMLF gradient alongside a 0–500 ng/ml to move away from the source of the Slit2-S (Fig. 1B, 1E, Slit2-S gradient showed a dominance of the fMLF chemoattraction Supplemental Fig. 1G, and Supplemental Video 2), with a over the Slit2-S chemorepulsion (Fig. 1E, Supplemental Fig. 1G). forward migration index (FMI) comparable to the FMI induced Some chemoattractants affect the speed and/or directionality (the by the chemorepellent DPPIV for neutrophils (22) or AprA for distance between the starting and ending point of a cell divided by Dictyostelium cells (24). We previously observed that ∼17% of the distance along the cell’s path) of cells (34–36). fMLF with cells in DPPIV or AprA chemorepellent gradients move toward 0 and 50 ng/ml Slit2-S increased the speed of the neutrophils the source of the chemorepellent (22, 24), and we observed that along their tracks (Supplemental Fig. 1A). fMLF and fMLF plus 29 6 5% of cells (mean 6 SEM, n = 6 donors) of the neu- 500 ng/ml Slit2-S caused the directionality to increase compared trophils in the 0–500 ng/ml Slit2-S gradient moved toward the with control (Supplemental Fig. 1B). Other than 0–5 ng/ml Slit2-S source. A gradient of 0–5000 ng/ml Slit2-S, however, did not decreasing speed for female neutrophils (Supplemental Fig. 2C), cause chemorepulsion (Fig. 1E). As previously observed there were no significant differences in the response of neutro- (15, 33), 1 nM fMLF caused chemoattraction of neutrophils phils from male and female donors to Slit2-S (Supplemental (Fig. 1E, Supplemental Fig. 1G, and Supplemental Video 3). In Fig. 2). Together, these data suggest that Slit2-S can act as a the fMLF gradient, only 6 6 4% (mean 6 SEM, n = 6 donors) chemorepellent for human male and female neutrophils but that of neutrophils moved away from the fMLF source. This is this can be overridden by an fMLF chemoattraction gradient. 242 Slit2 DIRECTLY REGULATES NEUTROPHIL MIGRATION

FIGURE 2. Anti-Robo1 and anti–Syndecan-4 Abs inhibit Slit-2 chemotaxis. Human neutrophils were preincubated with anti-Robo1 (A) or anti–Syndecan-4 (B) Abs before videomicroscopy in gradients of Slit2 as in Fig. 1. At least 10 cells per experimental group for each individual donor were tracked for 40 min. Values are means 6 SEM for neutrophils from at least ﬁve different donors. *p , 0.05, **p , 0.01, ***p , 0.001 compared with the no-gradient control (one-way ANOVA, Dunnett test) or for the indicated Downloaded from comparison between two sets (t test). http://www.jimmunol.org/

We also examined whether gradients of a commercially available of Slit2 to inhibit neutrophil chemoattraction (15, 18). Neutrophils ∼140-kDa fragment of human Slit2, henceforth designated Slit2-N, preincubated with anti-Robo1 Abs did not respond to either a can affect neutrophil movement. Gradients of 0–5 or 0–50 ng/ml 0–500 ng/ml Slit2-S gradient or a 0–500 ng/ml Slit2-N gradient by guest on September 24, 2021 Slit2-N did not significantly affect neutrophil movement (Fig. 1F, (Fig. 2A). The anti-Robo1 Abs did not significantly affect cell Supplemental Fig. 1H), 0–500 ng/ml (0–3.6 nM) gradients caused speed (Supplemental Fig. 3A), but anti-Robo1 Ab pretreat- neutrophils to move toward the Slit2-N (Fig. 1C, 1F, Supplemental ment with a Slit2-S gradient increased directness (Supplemental Fig. 1H, and Supplemental Video 4), and 0–5000 ng/ml gradients Fig. 3B). These data suggest that Robo1 is necessary for Slit2 caused female but not male cells to move away from the Slit2-N chemorepulsion or chemoattraction of human neutrophils. (Fig. 1D, 1F, Supplemental Fig. 2B). In the 0–500 ng/ml gradient of Syndecans are proteoglycans that can act as coreceptors for Robo1 Slit2-N, 14 6 6% (mean 6 SEM, n = 6) of the neutrophils moved signaling (37, 38). Of the four human syndecans, Syndecan-4 ap- away from the source; this was not significantly different from the pears to mediate human neutrophil chemotaxis (39). Preincubation percentage of wrong-way cells in an fMLF gradient (t test). In the of human neutrophils with anti–Syndecan-4 Abs inhibited the re- 0–5000 ng/ml gradient of Slit2-N, 38 6 6% (mean 6 SEM, n =6) sponse of neutrophils to 0–500 ng/ml gradients of both Slit2-S and of the neutrophils moved toward the source; this was significantly Slit2-N (Fig. 2B). In the presence of Slit2-S or Slit2-N gradients, the higher (p = 0.0042, one-way ANOVA, Dunnett test) than the anti-Syndecan Abs increased cell speed (Supplemental Fig. 3C) but percentages of wrong-way cells in fMLF or 0–500 ng/ml Slit2-N did not significantly affect directness (Supplemental Fig. 3D). These gradients (p = 0.0309; one-way ANOVA, Dunnett test). None of data suggest that Syndecan-4 is necessary for Slit2 chemorepulsion the Slit2-N concentrations significantly affected neutrophil speed or chemoattraction of human neutrophils. or directness (Supplemental Fig. 1C, 1D). Other than males not Slit2-N generates polarized F-actin in neutrophils responding to the 0–5000 ng/ml Slit2-N gradients, there were no significant differences in the response of neutrophils to Slit2-N Neutrophil migration to chemoattractants leads to cytoskeletal from male and female donors (Supplemental Fig. 2B, 2D, 2F). reorganization and formation of F-actin at the leading edge of the The data suggest that 500 ng/ml Slit2-N acts as a chemoattractant. cell, which can be visualized by phalloidin staining (2, 40). To Although gradients of 0–50, 0–500, or 0–5000 ng/ml of a determine if Slit2-induced migration induces F-actin reorganiza- 55-kDa human Slit2-C fragment did increase neutrophil speed tion, neutrophils were allowed to adhere to fibronectin-coated (Supplemental Fig. 1E), they did not significantly affect the 8-well slides and were then stimulated with a point source of direction or directionality of neutrophil movement (Fig. 1G, buffer, Slit2, or fMLF for 10 min and then fixed, permeabilized, Supplemental Fig. 1F, 1I). and stained with phalloidin. Cells were then analyzed for the location of the F-actin. The addition of buffer to one corner of Robo and Syndecan-4 Abs block the effects of the well did not cause any significant asymmetry of F-actin Slit2-induced migration localization toward or away from the source of the buffer, To induce chemorepulsion of growing axons, neurons use the many cells had F-actin around the periphery of the cell, and Robo1 receptor to sense Slit2 (3–5). Robo1 also mediates the ability some cells had two or more (multiple) spots of F-actin at the The Journal of Immunology 243 Downloaded from http://www.jimmunol.org/

FIGURE 3. Only Slit2-N and fMLF generate polarized F-actin. Neutrophils were incubated in the presence or absence of a single point source (asterisk) of (A) buffer control, (B) fMLF, (C) Slit2-N, or (D) Slit2-S for 10 min. Cells were then fixed and stained for F-actin with phalloidin-Alexa 555 (red) and counterstained with DAPI (blue). Images are from one of three different donors. Scale bar, 20 mm. (E) Quantification of phalloidin staining location, indicating the percentage of cells with F-actin at the edge of the cell either toward or away from the stimulus, or cells showing multipolar or uniform staining. (F and G) Neutrophils were incubated with fMLF, Slit2-N, or Slit2-S for 20, 40, 60, 90, 120, and 300 s. Cells were then lysed in Triton X-100 buffer to isolate cytoskeletal and cytoplasmic proteins. (F) Triton X-100 insoluble cytoskeletal proteins were analyzed by PAGE and stained with by guest on September 24, 2021 Coomassie to quantify F-actin. (G) Cytoskeletal proteins were also analyzed for pMLC2 by Western blotting. All values are mean 6 SEM for neutrophils from three to four different donors. *p , 0.05, **p , 0.01, ***p , 0.001 compared with the t = 0 buffer control (one-way ANOVA, Dunnett test). edges of cells (Fig. 3A, 3E). Neutrophils in gradients of fMLF fibronectin-coated plates before the addition of Slit2 proteins, the or Slit2-N showed a decrease in the percentage of cells with a chemoattractant fMLF, or TNF-a, a cytokine that promotes neu- uniform distribution of F-actin at the edge of the cell and an trophil adhesion (31). Compared with unstimulated neutrophils, increase in the percentage of cells with F-actin localized to the cells incubated with fMLF, Slit2-N, or TNF-a had significantly edge of the cell toward the attractant (Fig. 3B, 3C, 3E). Gra- increased adhesion (Fig. 4A), whereas the chemorepellent Slit2-S dients of the chemorepellent Slit2-S also decreased the per- did not significantly affect adhesion (Fig. 4A). Preincubation with centage of cells with F-actin all around the cell and increased Slit2 isoforms inhibits the ability of fMLF or TNF-a to increase the percentage with multiple regions of F-actin at the edge but adhesion of neutrophils to activated endothelial cells (19, 42, 43). did not significantly affect the percentage of cells with F-actin Both Slit2-N and Slit2-S significantly inhibited fMLF-induced at the edge of the cell toward or away from the source of Slit2-S adhesion to fibronectin, and Slit2-S but not Slit2-N inhibited (Fig. 3D, 3E). As previously observed (27, 28, 41), fMLF TNF-a–induced adhesion (Fig. 4B). Cell migration leads to a transiently increased total F-actin polymerization at 90 s change in cell shape, with an elongation of the cell with pseu- (Fig. 3F). Slit2-S and Slit2-N did not cause significant changes dopods forming at the leading edge of the cell and a more flattened in F-actin over 300 s (Fig. 3F). Compared with control cells, cell as it attaches to the underlying matrix (1, 2). Compared with both fMLF and Slit2-N induced phosphorylation of myosin L unstimulated neutrophils, cells in gradients of fMLF or Slit2-N chain 2 (MLC2) at 60 s, and Slit2-N also led to reduced pMLC2 had an increase in cell length and cell area, whereas cells in levels at 5 min (Fig. 3G). Slit2-S had no significant effect on Slit2-S gradients were not significantly different from control cells pMLC2 levels (Fig. 3G). These data suggest that Slit2-S affects (Fig. 4C, 4D). These data suggest that, unlike the chemoattractants the cytoskeleton to induce chemorepulsion in a manner that is fMLF and Slit2-N, which increase adhesion to fibronectin, cell different from how fMLF and Slit2-N affect the cytoskeleton to elongation, and cell flattening, the chemorepulsive Slit2-S does induce chemoattraction. not affect these parameters. Slit2 isoforms differentially regulate neutrophil adhesion Slit2 isoforms differentially regulate surface receptor and Cell migration requires binding to a surface to allow traction but intracellular signaling protein localization not with too low or too high an adhesive force, which pre- Neutrophil recruitment to an inflamed tissue involves initial at- vents migration (34–36). To determine if the Slit2 isoforms affect tachment to endothelial cells, followed by rolling across the endo- cell–substratum adhesion, we allowed neutrophils to adhere to thelial cell surface, and then firm adhesion to the endothelium, 244 Slit2 DIRECTLY REGULATES NEUTROPHIL MIGRATION Downloaded from

FIGURE 4. Slit2-N and Slit2-S have different effects on cell adhesion and cell area. (A) Neutrophils were allowed to adhere to fibronectin for 30 min, and Slit2 proteins, fMLF, TNF-a, or an equal volume of buffer (control) was added for an additional 30 min. Plates were then washed, and adherent cells were air dried, stained, and counted. Values are mean 6 SEM, n =4.(B) Cells were preincubated for 15 min with either Slit2-N or Slit2-S before the addition of fMLF or TNF-a for an additional 15 min. Cells were then allowed to adhere to fibronectin for 30 min. Adherent cells http://www.jimmunol.org/ were air-dried, stained, and counted. Values are mean 6 SEM, n =4.#,p , 0.05 compared with the no-stimulus control (t test). (C and D) Neutrophils were incubated with a point source of buffer, Slit2 proteins, or fMLF for 10 min, then fixed and (C) cell length and (D) cell area were measured with ImageJ. The results are mean 6 interquartile range of 20 cells analyzed from three different donors. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way ANOVA, Dunnett test). transendothelial migration, and chemotaxis through the tissue to differentially reorganize cell surface integrins and differentially the inflammatory source (1, 44). Adhesion of neutrophils to either regulate AKT activity. endothelial cells or tissue extracellular matrix is mediated by integrins, including CD11b/CD18 (45, 46), which relocate to the PI3K, Cdc42, Rac, and Ras mediate the effect of Slit by guest on September 24, 2021 front and rear of the cell during adhesion and migration (47, 48). on neutrophils To determine if Slit2 proteins affect integrin relocation, neutro- PI3K mediates the movement of neutrophils toward some phils were incubated on fibronectin in Slit2 gradients, fixed, and chemoattractants (35). LY294002 is an inhibitor of PI3K (54) stained for CD11b, and the staining intensity was then scanned and has been used at 10–50 mM to inhibit PI3K signaling and along an axis running through the middle of the cell parallel to the migration in human neutrophils and endothelial cells (35, 55). gradient. Compared with unstimulated cells, which show peaks of To determine if the effects of Slit2 on neutrophils are mediated CD11b staining at the edges of the cell, fMLF did not significantly by PI3K, neutrophils were preincubated with LY294002 and alter the staining of CD11b at the edge of the cell closest to the then observed in Slit2 gradients. LY294002 had no significant stimulus, increased staining in a region immediately behind the effect on the ability of a 0–500 ng/ml gradient of Slit2-S to edge, and decreased staining at the rear (Fig. 5A). Cells incubated induce chemorepulsion (Fig. 6A). However, LY294002 caused in a Slit2-S gradient had significantly less CD11b staining at neutrophils in a 0–500 ng/ml Slit2-N gradient to be repelled the side of the cell closest to the Slit2-S and had decreased by the Slit2-N (Fig. 6A, Supplemental Video 5). LY294002 staining throughout the cell (Fig. 5A). Slit2-N decreased caused neutrophil speed to increase in a Slit2-S gradient staining of CD11b in the part of the cell farthest from the (Supplemental Fig. 4A), but LY294002 did not significantly Slit2-N. affect directness (Supplemental Fig. 4B). To confirm the role of Cell migration is driven by the detection of chemostimulants PI3K in Slit2-N mediated chemoattraction, PIP2 and PIP3 through a diverse number of receptors, including G protein– levels were measured in neutrophils incubated with Slit2-N, coupled receptors, such as the fMLF receptor (49), and single- Slit2-S, or fMLF. After 5 min of incubation, none of the pass transmembrane receptors, such as Robo (3, 5). Although the treatments significantly affected PIP2 levels, but compared intracellular domains of the fMLF and Robo receptors bind with unstimulated cells, Slit2-N and fMLF increased levels different signaling molecules, both receptors appear to regulate of PIP3, whereas Slit2-S did not significantly affect PIP3 pathways downstream from the AKT kinase (14, 50–53). To levels (Fig. 7A). These data suggest that Slit2-S does not in- determine if the isoforms of Slit2 differentially regulate AKT crease PIP3 and does not need PI3K to cause chemorepulsion, activity, we used an Ab that detects phosphorylation of AKT whereas Slit2-N increases PIP3 and needs PI3K to cause substrates (30). Compared with unstimulated cells, cells exposed chemoattraction. to a gradient of fMLF had no discernable difference in pAKT Cdc42 is a small GTPase that mediates the effects of many substrate staining (Fig. 5B). Cells incubated with Slit2-N had chemoattractants on the cytoskeleton (56). In the developing increased staining at the front and rear of the cell, whereas cells nervous system, Slit2 activation of Robo causes an inactiva- incubated with Slit2-S had reduced staining in the middle of tion of Cdc42, and this inactivation is necessary for the ability the cells (Fig. 5B). These data suggest that the Slit2 fragments of Slit2 to cause chemorepulsion of neuronal cells (57). The Journal of Immunology 245

neutrophils (Supplemental Fig. 4D). These results suggest that blocking Cdc42 activity reverses Slit2-S chemorepulsion and inhibits Slit2-N chemoattraction. Rac is another small GTPase involved in neutrophil chemotaxis (15, 59). The Rac inhibitor NSC23766 inhibits Rac activation (60). Although 10 mM NSC23766 only causes a partial inhibition of Rac in neutrophils (61), we used it at 10 mM because higher concentrations of this inhibitor have off- target effects (62). NSC23766 blocked the ability of Slit2-S to act as a chemorepellent (Fig. 6C). Although NSC23766 appeared to reduce the ability of Slit2-N to act as a chemoattractant, the effect was not statistically significant. NSC23766 in the presence or absence of Slit increased cell speed (Supplemental Fig. 4E), and the combination of NSC23766 and a Slit2-N gradient increased directionality (Supplemental Fig. 4F). To- gether, these results suggest that Rac may mediate the ability of Slit2-S to act as a chemorepellent. Son of sevenless (Sos) is a guanine nucleotide exchange factor

that can activate small GTPases such as Ras (63). In Drosophila, Downloaded from Slit activation of Robo in neurons causes recruitment of Sos to the plasma membrane (63). Ras mediates neutrophil chemotaxis (1, 2, 64). Guanine nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signaling (65, 66). RIP corresponds to a region within human Sos1 that interacts with an

SH3 domain of Grb2, preventing an interaction that is essential for http://www.jimmunol.org/ Ras activation (65), and RIP has been used at 10 mM to inhibit Ras in neutrophils (66). RIP blocked the ability of Slit2-S to act as a chemorepellent but did not appear to affect the chemoattraction in a 0–500 ng/ml Slit2-N gradient (Fig. 6D). RIP alone or in the presence of Slit2-S, but not in the presence of Slit2-N, increased neutrophil speed (Supplemental Fig. 4G), and RIP had no significant effect on directness (Supplemental Fig. 4H). To confirm the role of Ras in Slit2-S mediated chemorepulsion, GTP-bound Ras was measured in neutrophils incubated with Slit2-N, Slit2-S, or fMLF. by guest on September 24, 2021 After 6 min, compared with unstimulated cells, Slit2-S and fMLF increased levels of GTP-bound Ras, whereas Slit2-N did not significantly affect levels of GTP-bound Ras (Fig. 7B, 7C). Together, these data suggest that Slit2-S increases levels of GTP-bound Ras and needs Ras to cause chemorepulsion, whereas Slit2-N does not significantly affect levels of GTP-bound Ras and does not need Ras FIGURE 5. Slit2 proteins regulate CD11b and intracellular sig- to cause chemoattraction. naling protein redistribution. Neutrophils were incubated with a point source of Slit2 proteins or fMLF for 10 min, then fixed and Discussion stained with Abs to (A) CD11b or (B) phosphorylated AKT sub- We found that two isoforms of the N-terminal fragment of Slit2 strates. Staining across the cell from the side of the cell closest to differentially regulate neutrophil chemotaxis, with Slit2-N di- the chemo-stimulant (0 on the graph) to the far side of the cell was rectly acting as a chemoattractant, and Slit2-S directly acting as a 6 analyzed by ImageJ. The results are mean SEM of 10 cells analyzed from chemorepellent. Both Slit2 fragments appeared to require Robo1 three different donors (30 cells total). *p , 0.05 compared with the and the coreceptor Syndecan-4 to direct neutrophil movement. control unstimulated cells (two-way ANOVA, Dunnett multiple com- parisons test). Slit2-N chemoattraction appeared to not require activation of Ras and Rac, was blocked by a Cdc42 inhibitor, increased PIP3 levels, and was reversed by a PI3K inhibitor. In contrast, Slit2-S–induced Cdc42 also mediates the ability of Slit2 to inhibit neutrophil chemorepulsion was independent of PI3K, activated Ras, was chemotaxis toward stromal cell–derived factor 1a (18). blocked by Ras and Rac inhibitors, and was reversed by a Cdc42 ML141 is an allosteric inhibitor of Cdc42 and has been used at inhibitor, indicating that, unexpectedly, the two Slit2 fragments 10 mM to inhibit Cdc42-mediated chemotaxis in human neu- activate different signal transduction pathways. Similar to the trophils (58). ML141 caused 500 ng/ml Slit2-S to act as a canonical neutrophil chemoattractant fMLF, a Slit2-N gradient chemoattractant rather than as a chemorepellent (Fig.6B, induced a single zone of F-actin at the leading edge, increased Supplemental Video 6) and blocked the ability of 500 ng/ml cell adhesion, and altered cell morphology, whereas Slit2-S Slit2-N to act as a chemoattractant (Fig. 6B). For unknown rea- gradients caused multiple zones of F-actin accumulation and sons, the 0–500 ng/ml Slit2-S gradient increased the speed did not increase adhesion or alter cell morphology. Slit2-S thus of neutrophils from the donors used for this experiment causes chemorepulsion using an unusual effect on the cyto- (Supplemental Fig. 4C). In the presence or absence of Slit2, skeleton. Although Slit2-C did not directly induce chemotaxis, it ML141 also increased the speed of cells (Supplemental Fig. 4C) increased cell speed, indicating that Slit2-C can modulate neu- and, in the absence of Slit2, slightly increased the directionality of trophil chemotaxis. 246 Slit2 DIRECTLY REGULATES NEUTROPHIL MIGRATION Downloaded from

FIGURE 6. Signaling inhibitors have different effects on the response of neutrophils to Slit2-N and Slit2-S. Human neutrophils were preincubated for A B C D

30 min with inhibitors of ( ) PI3K (LY294002), ( ) Cdc42 (ML141), ( ) Rac (NSC23766), or ( ) Ras (RIP) before videomicroscopy in gradients of http://www.jimmunol.org/ Slit2 as in Fig. 1. A positive FMI indicates chemorepulsion, and a negative FMI indicates chemoattraction. At least 10 cells per experimental group for each individual donor were tracked for 40 min. All values are means 6 SEM for neutrophils from at least six different donors. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 compared with the no-gradient control (one-way ANOVA, Dunnett test) or for the indicated comparison between two sets (t test).

Slit2-S induces chemorepulsion using a pathway involving The ability of Slit2-N to act as a neutrophil chemoattractant Ras and Rac but not PI3K. In agreement with the observation at low concentrations but act as a chemorepellent at high that Slit2-S does not appear to activate PI3K, Slit2S decreases concentrations (5000 ng/ml; ∼36 nM) is similar to the effects by guest on September 24, 2021 the phosphorylation of AKT substrates. The ability of a Cdc42 of many other chemotactic factors, which act as chemo- inhibitor to cause Slit2-S to act as a chemoattractant suggests attractants at low concentrations but either prevent migration that Slit2-S can activate an unknown chemoattraction pathway (by inducing increased adhesion) or act as chemorepellents but preferentially activates a chemorepulsion pathway that (fugetaxis) at high concentrations (36, 67, 68). At concentra- requires Cdc42. This chemoattraction pathway may be the tions of 1000–5000 ng/ml, Slit2 inhibits directed cell migra- Ras- and Rac-independent pathway used by Slit2-N. Con- tion and activation of intracellular signaling pathways in many versely, the ability of a PI3K inhibitor to cause Slit2-N to cell types (13, 15, 69, 70). An intriguing possibility is that a act as a chemorepellent suggests that Slit2-N can activate a gradient of Slit2-N that starts at a high concentration may chemorepulsion pathway but preferentially activates a chemo- attract cells to tissues but create an exclusion zone near the attraction pathway that requires PI3K. source of the Slit2-N.

FIGURE 7. Slit2-N increases PIP3, and Slit2-S activates Ras. Neutrophils were incubated with Slit2 proteins or fMLF for (A) 5 min to measure PIP2 and PIP3 levels, or (B and C) 6 min to detect Ras activation. (B) A Western blot of bead lysate from neutrophils incubated with Slit2 proteins or fMLF was stained with Ponceau red (PS) (upper panel), anti-Ras Abs (middle panel), or anti-GAPDH Abs (lower panel). The quantiﬁcation of Ras staining is indicated below the blot (middle panel). The positions of molecular mass standards in kDa are at left. (C) Quantiﬁcation of Western blots. Values are means 6 SEM for neutrophils from three to six different donors. *p , 0.05 compared with the no-stimulus control (one-way ANOVA, Dunnett test) or for the indicated comparison between two sets (t test). The Journal of Immunology 247

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