The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors

Abstract

Migration is a basic feature of many cell types in a wide range of species. Since the 1800s, cell migration has been proposed to occur in the nervous and immune systems, and distinct molecular cues for mammalian neurons and leukocytes have been identified. Here we report that Slit, a secreted protein previously known for its role of repulsion in axon guidance and neuronal migration, can also inhibit leukocyte chemotaxis induced by chemotactic factors. Slit inhibition of the chemokine-induced chemotaxis can be reconstituted by the co-expression of a chemokine receptor containing seven transmembrane domains and Roundabout (Robo), a Slit receptor containing a single transmembrane domain. Thus, there is a functional interaction between single and seven transmembrane receptors. Our results reveal the activity of a neuronal guidance cue in regulating leukocyte migration and indicate that there may be a general conservation of guidance mechanisms underlying metazoan cell migration. In addition, we have uncovered an inhibitor of leukocyte chemotaxis, and propose a new therapeutic approach to treat diseases involving leukocyte migration and chemotactic factors.

Figure 1

Expression of Slit and Robo in adult tissues. a–c, RNase protection assays (RPAs) were used to determine the expression of three slit genes in adult rats. Total RNA was prepared from different rat tissues and cell lines, as indicated (lanes 2–13). Total RNA (5 μg) was used in RPAs with probes specific for slit1, slit2 and slit3 genes (Sl). The rat L32 gene probe controlled for RNA input (lower band in each panel). Probes in lane 1 contain polylinker regions and are longer than the protected bands. LN, lymph node; PBMC, peripheral blood mononuclear cells; RAEC, rat endothelial cell line; GEDC, glomerular endothelial cells from the rat kidney; MC, rat kidney mesangial cells; 49F, rat kidney fibroblasts. d, Expression of Robo1 protein detected with anti-Robo1 antibodies. Arrowhead indicates full-length Robo1. Lane 1, HEK cells; lane 2, Robo1-transfected HEK cells; lane 3, a strong band with lower relative molecular mass (M_r) than full-length Robo1 was detected reproducibly in rat thymus, indicating a crossreacting band or a proteolytically cleaved product; lane 4, HL-60 cells; lane 5, neutrophils differentiated from HL-60 cells; lane 6, rat PBMCs: no full-length Robo1 was detected but several lower M_r bands were visible; lane 7, rat lymph node. e, slit2 messenger RNA distribution detected by in situ hybridization in the glomerulus of adult human kidney. Arrowhead indicates mesangial cells; arrow indicates epithelial cells in Bowman’s capsule. In the glomerulus, most of the positive cells are epithelial cells, but some endothelial cells can be seen. f, slit2 mRNA expression in vascular endothelial cells in the human kidney. Arrow indicates endothelial cells in a venule; arrowhead indicates endothelial cells in an arteriole. g, Haemotoxylin–eosin staining of a section of the same kidney as that shown in f. These two sections are not immediately next to each other, but corresponding regions are conveniently identified by landmarks in the sections.

Figure 2

Effect of Slit on leukocyte chemotaxis induced by SDF-1α. a, Transwell migration of rat lymphocytes. Control or SDF-containing media were added to the lower chamber. Cells that migrated to the lower chamber were counted, and are expressed as a percentage of the cells added to the upper chamber. 1, control; 2, 10 nM SDF-1α; 3, 100 pM SLIT2; 4, 10 pM SLIT2; 5, 10 nM of SDF-1α and 100 pM of SLIT2; 6, 10 nM of SDF-1α and 10 pM of SLIT2. b, Rat lymphocytes were examined in transfilter assays in the presence of SDF-1α (10 nM). SLIT2 (100 pM) was added to the lower chamber, the upper chamber or both upper and lower chambers. 1, control; 2, SDF-1α; 3, both SDF-1α and SLIT2 in the lower chamber; 4, SDF-1α in the lower chamber and SLIT2 in the upper chamber; 5, SDF-1α in the lower chamber and SLIT2 in both chambers. c, Slit inhibition of chemotaxis induced by fMLP. HL-60 cells differentiated into neutrophil-like cells after treatment with DMSO. Chemotaxis was observed in transwell assays. 1, control; 2, SLIT2; 3, fMLP; 4, SLIT2 and fMLP.

Figure 3

Robo is involved in mediating Slit inhibition of chemotaxis. a, The effect of Slit is inhibited by RoboN. Chemotaxis was analysed by using rat lymph node cells in transwell assays. SLIT2 (SL) and RoboN (RN) were added to the upper well and SDF-1α was added to the lower well. b, Expression of CXCR4 and Robo in HEK cells can reconstitute Slit inhibition of SDF-1α-induced chemotaxis. HEK migration was measured with the transfilter assay in microchemotaxis chambers using HEK cells expressing CXCR4, or both CXCR4 and rat Robo1 in the presence of different concentrations of SDF-1α. Control vehicle or 100 pM of purified Slit protein was added. Squares, migration of HEK cells expressing CXCR4 in response to SDF-1; diamonds, migration of HEK cells expressing CXCR4 in response to SDF-1α in the presence of Slit; circles, migration of HEK cells expressing Robo and CXCR4 in response to SDF-1α; triangles, migration of HEK cells expressing Robo and CXCR4 in response to SDF-1α in the presence of Slit.

Figure 4

Lack of general inhibition by Slit. a, Neutrophils differentiated from HL-60 were treated with fMLP, or fMLP and SLIT2. Phosphorylation of ERK was examined by an anti-phospho-ERK antibody. Top, anti-ERK staining; bottom, anti-phospho-ERK staining. fMLP could induce ERK phosphorylation, which was not inhibited by the addition of SLIT2. Lane 1, control; lane 2, fMLP; lane 3, SLIT2; lane 4, fMLP and SLIT2. b, Oxidative burst was shown by SOD-inhibitable production of superoxide, and 1 unit was defined as nmol per 2 × 10⁶ cells per 15 min. 1, control; 2, SLIT2; 3, fMLP; 4, SLIT 2 and fMLP. Results shown are representative of three experiments, each with triplicate samples.