Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice

Abstract

The anti-granulocyte receptor-1 (Gr-1) mAb, RB6-8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6-8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6-8C5 may deplete not only neutrophils but also other Gr-l⁺ (Ly6C⁺) cells. This study describes the use of an Ly6G-specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6-8C5 reduced blood neutrophils and Gr-1⁺ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr-1⁺ monocytes. Plasma TNF-α in endotoxemia was increased 20-fold by RB6-8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF-α staining in brefeldin-treated wound leukocytes was increased by pretreatment with RB6-8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6-8C5, as it preserves non-neutrophil Gr-1⁺ cells depleted by the anti-Gr-1 antibody. The loss of non-neutrophil Gr-1⁺ populations in RB6-8C5-treated animals is associated with increased TNF-α responses, suggesting these cells may function to suppress TNF-α production.