Ly6G ligation blocks recruitment of neutrophils via a β2-integrin–dependent mechanism

Abstract

Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice.In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB 4 and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and ␤2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of ␤2-integrins in LTB 4 -stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of ␤2-integrindeficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a ␤2-integrindependent mechanism. (Blood. 2012; 120(7):1489-1498) IntroductionNeutrophils are one of the first cell types to reach sites of infection or acute inflammation. Recruitment involves an orchestrated sequence of events in which circulating neutrophils respond to chemotactic signals to become firmly adherent to activated endothelium, followed by transendothelial migration via either a paracellular or transcellular route. 1,2 Once at a site of inflammation, neutrophils contribute to ongoing leukocyte recruitment and tissue injury by releasing lipid mediators, proteases, reactive oxygen species (ROS), and other factors. [3][4][5] Whereas they are critical to immune defense, neutrophils can, also play a pathogenic role in chronic inflammatory diseases and therefore their recruitment is subject to numerous levels of control, not all of which are understood completely. 1 Delineating these regulatory pathways will provide insights into the mechanisms of tissue injury in inflammatory disorders and novel targets for drug development.Much of the experimental evidence implicating neutrophils in disease has been obtained through murine studies in which this lineage was selectively depleted using Abs that bind the neutrophil surface antigen Ly6G, such as RB6-8C5 (more typically termed anti-Gr-1). 6,7 However, the function of Ly6G remains unknown. Ly6G is a small protein of approximately 25 kDa that is tethered to the cell surface via a GPI linker. 7 In bone marrow (BM), peripheral blood, and wound exudates, the expression of Ly6G is limited to cells with granulocyte morphology. 7,8 Structurally, Ly6G belongs to the Ly6/urokinase pl...

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“…4A). These results are in agreement with previous reports, where high doses of anti-Ly6G antibody were needed to ameliorate inflammatory edema (24). Neither treatment with anti-Ly6G nor the isotype control modified the response latencies in mice without inflammation subjected to mechanical or thermal stimuli (Fig.…”

“…to block the effects of the EOPs. An anti-Ly6G antibody was administered intraperitoneally to inhibit neutrophil infiltration (24).…”

“…3B), the precursor of β-endorphin (16). Ly6G is selectively present in neutrophils and is needed for migration and recruitment of these immune cells (24). The actions of Ly6G can be inhibited in vivo by the systemic administration of an anti-Ly6G antibody (24).…”

“…Although neutrophils (and other immune cells) can produce EOPs and therefore may participate in decreasing pain during inflammation (16), it is conventionally accepted that they promote pain by synthesizing and releasing algogenic chemicals (15) and also participate in the development of edema (24). In turn, edema can increase pressure on nociceptive nerve endings also involved in pain during inflammation (25).…”

“…Ly6G is selectively present in neutrophils and is needed for migration and recruitment of these immune cells (24). The actions of Ly6G can be inhibited in vivo by the systemic administration of an anti-Ly6G antibody (24). The in vivo administration of antiLy6G antibody (7.5-20 μg) resulted in complete, dose-dependent inhibition of neutrophil infiltration in the inflamed paw, whereas the administration of its isotype control (20 μg) had no effect on neutrophil levels (Fig.…”