Antibody ligation of murine Ly-6G induces neutropenia, blood flow cessation, and death via complement-dependent and independent mechanisms

Abstract

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.

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

“…on May 9, 2018. by guest www.bloodjournal.org From Interestingly, in mice pretreated with TNF␣, administration of either anti-Gr-1 or anti-Ly6G promotes development of leukocyte/ platelet aggregates, leading to disseminated intravascular coagulation. 16,17 Anti-Gr-1-induced adherence between platelets and leukocytes was also found in mice with proteoglycan-induced arthritis, an effect replicated with anti-CD44 and also recombinant P-selectin-Fc fusion protein. 47 Neutropenia induced by these agents could be attenuated by heparin or platelet depletion, suggesting that aggregate formation is an important mechanism of Ab-mediated neutrophil depletion.…”

“…TNF␣-primed mice given anti-Gr-1 (which binds both Ly6G and the structurally related protein Ly6C) 7,8 or the Ly6G-specific Ab 1A8 (hereafter referred to as anti-Ly6G) develop disseminated intravascular coagulation and death. 16,17 In vitro, cross-linking using anti-Gr-1 F(abЈ) 2 fragments and a secondary Ab induces up-regulation of neutrophil CD11b and a modest rise in F-actin, but whether this phenomenon reflects binding of Ly6G, Ly6C, or both is unknown. 17 Anti-Gr-1 administration has also been associated with the appearance of apoptotic-appearing neutrophils in inflammatory infiltrates, giving rise to the hypothesis that anti-Gr-1 induces apoptosis in circulating or inflammatory neutrophils but not in their BM precursors, potentially because of differential expression of the antiapoptotic Bcl-2 family protein Mcl-1.…”

“…TNF␣-primed mice given anti-Gr-1 (which binds both Ly6G and the structurally related protein Ly6C) 7,8 or the Ly6G-specific Ab 1A8 (hereafter referred to as anti-Ly6G) develop disseminated intravascular coagulation and death. 16,17 In vitro, cross-linking using anti-Gr-1 F(abЈ) 2 fragments and a secondary Ab induces up-regulation of neutrophil CD11b and a modest rise in F-actin, but whether this phenomenon reflects binding of Ly6G, Ly6C, or both is unknown. 17 Anti-Gr-1 administration has also There is an Inside Blood commentary on this article in this issue.…”

“…Indeed, cross-linking of anti-Gr-1 F(abЈ)2 fragments induces F-actin and CD11b expression, whereas neutrophil activation might explain the lethal outcome of anti-Ly6G administration into TNF␣-treated mice. 16,17 We therefore used a range of assays to assess neutrophil activation and endothelial adhesion. We found no effect of anti-Ly6G on expression of the activation markers CD11b and CD62L, actin polymerization, generation of ROS, or adherence to TNF␣-activated murine endothelium under low physiologic wall shear stress (Figure 6A-D).…”

C/EBPβ Is Involved in the Amplification of Early Granulocyte Precursors during Candidemia-Induced “Emergency” Granulopoiesis

“…However, the expression of Ly-6C is not restricted to granulocytes, and so, the Ab crossreacts with nongranulocytic lineages such as dendritic cells (28), monocytes (29,30), and a subpopulation of lymphocytes (31). In contrast, Ly-6G, which was cloned as a member of the Ly-6 alloantigen family (32), is expressed exclusively on granulocytes (22,33), and treatment with an anti-Ly-6G Ab specifically depletes granulocytes in vivo (34,35). Although the physiological ligand and biological function of Ly-6G are unclear, the granulocyte-specific expression of Ly-6G makes it an ideal marker of granulocytic differentiation.…”