The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.

Abstract

In inflammatory diseases circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not effectively prevent neutrophil recruitment. The Slit family of secreted proteins, and their transmembrane receptor, Roundabout (Robo), repel axonal migration during central nervous system development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types towards a variety of chemotactic factors, in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow, and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration, but not random movement, of neutrophils towards formyl-methionyl-leucyl-phenylalanine (fMLP). Slit2 also inhibited neutrophil migration towards other chemoattractants, namely C5a and interleukin (IL)-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2, but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and macrophage inflammatory protein-2 (MIP-2). In all instances, Slit2 effectively reduced neutrophil recruitment (p < 0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis, but not random motion, of circulating neutrophils, and point to Slit2 as a potential new therapeutic for preventing localized inflammation.