A Novel Function Of ASGM1in Inducing Anaphylaxis-like Shock Via Activating Basophils In Mice

Background:Anaphylaxis usually occurs in an allergen or specific antibody dependent fashion. Classical forms of anaphylaxis are mediated by histamines released from mast cells that are activated by allergens via crosslinking of surface-bound specific IgE. However, recent human and animal studies have indicated that this classical pathway does not account for all anaphylactic responses. Various human and animal studies have indicated that IgG and FcyR pathways also mediate anaphylaxis in an IgE-independent fashion. Although basophils are known to be involved in anaphylactic process, it remains largely unclear how basophils are activated and contributed to the development of anaphylaxis.Objective:To determine the role of ASGM1in basophil activation and assess its functional implication in triggering anaphylaxis-like shocks.Methods:1. Inducing anaphylaxis-like shockC57BL/6mice were injected intraperitoneally with200ng of PTX on day0and day2. On day7, the mice were injected intraperitoneally or intravenously with of anti-ASGM1antibody, PK136, PTX, flagellin, or PAF, respectively. 2. In vivo blocking and depletionMice were treated with CV-3988for inhibition of PAF, with triprolidine to inhibit histamine, with anti-mouse-FcεRI (MAR-1) for basophils depletion, with anti-NKl.1(PK136) for NK cell or/and NKT cell depletion, and with gadolinium chloride (GdCl3) to inhibit monocyte/macrophage function.3. Other detection content1) The expression pattern of ASGM1on immune cells, the expression of FcεRI on basophils and the frequency of basophils in the spleen were examined by flow cytometry.2) Basophils were derived from bone marrow, and in which intracellular calcium, Phosphorylation ERK and mRNA levels of LysoPAFAT/LPCAT2were measured.3) Vascular leakage were measured by intravenous injection with Evans blue dye in vivo and F-actin staining with phalloidin.4) The effects of PTX treatment were determined in vitro and in vivo.Results:1. Ligation of ASGM1, with anti-ASGM1antibodies or flagellin, triggered anaphylaxis-like shock in PTX-pretreated mice. 2. PAF was the pivotal mediator in the shock. ASGM1expressed on basophils contributed to the shock by allowing opportunity for the activation of the basophils and synthesis of the PAF.3. ASGM1ligation lead to the activation of murine basophils with Ca2+mobilization and ERK1/2phosphorylation whereas PAF is synthesized and released from the activated basophils.4. PTX augmented the susceptibility of mice ASGM1-ligation-induced-shock by enhancing the vascular permeability5. PTX elevated the level of FcεRI on basophils and serum IgE as well as release of IL-33.Conclusion:Ligation of ASGM1can activate basophils, causing them to release PAF and triggering anaphylaxis-like shocks. Damage of endothelial cells can promote the release of IL-33from nucleus and augment the susceptibility to anaphylaxis.