Siglec-8 is a member of the CD33 sialic acid-bind immunoglobulin-like lectin (Siglec) family found only on human mast cells, eosinophils and basophils. This transmembrane protein has an extracellular domain that recognizes specific carbohydrate molecules (glycans), and intracellular tyrosine-based inhibition motifs that putatively converts receptor binding into immune suppression. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs. Mouse Siglec-F is expressed predominantly on mouse eosinophils. Incubation of human eosinophils with specific Siglec-8 antibodies, or mouse eosinophils with Siglec-F antibodies, induces apoptosis, and dosing of mice with Siglec-F antibodies reduces eosinophil numbers. In contrast, Siglec-8 engagement on human mast cells by antibodies in vitro does not induce apoptosis but does inhibit mediator release induced via FceRI activation. Both Siglec-8 and Siglec-F bind a unique glycan ligand referred to as 6'-sulfo-sLex, or NeuAca2-3(6-O-sulfo)Ga1S1-4[Fuca1-3]G1cNAc. Therefore, activation of Siglec-8/Siglec-F through its natural glycan ligand, or through antibodies or glycomimetic agonists, may provide a novel means to specifically inhibit and/or deplete eosinophils and mast cells, thereby reducing allergic inflammatory responses. We thus hypothesize that: (i) Siglec-8/Siglec-F on the surface of allergic inflammatory cells binds to its natural carbohydrate ligands, expressed on tissues, to limit allergic inflammation by activating a negative signaling pathway; (ii) Allergic inflammation in vivo can be controlled by regulating expression of natural Siglec-8 ligands in tissues; and (iii) Siglec-8/Siglec-F mAbs or synthetic molecules based on the structure of their ligands (glycomimetics) will be capable of interrupting allergic inflammation. These concepts will be explored through four specific aims.
Aim 1 will characterize natural Siglec-8/Siglec-F ligands and determine their tissue expression.
Aim 2 will determine the ability of Siglec- 8/Siglec-F ligand mimetics to limit allergic responses in eosinophils and mast cells in vitro.
Aim 3 will explore the ability of mAbs or ligand mimetics to limit allergic responses in vivo, and Aim 4 will identify mechanisms by which Siglec-8 engagement induces eosinophil apoptosis and inhibits mast cell mediator release. Our proposed work on Siglec-8/Siglec-F should identify new therapeutic approaches for the treatment of diseases characterized by increased numbers of, or mediators from, eosinophils, basophils and mast cells. ? ? ?
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