Eosinophils, basophils and mast cells are considered key effector cells in allergy. Their recruitment, activation, and release of preformed and newly generated mediators, granule proteins, and cytokines are regarded as central to the pathophysiology of allergic diseases, including asthma. An improved understanding of the mechanisms involved in these responses is therefore of great relevance to asthma pathogenesis and the development of new therapeutics. The objective of this proposal during the last funding period was to further elucidate the molecular mechanisms for preferential recruitment and activation of eosinophils and basophils in asthma. As part of ongoing efforts to discover novel adhesion molecules, we recently cloned Siglec-8 from a human eosinophil cDNA library. Siglecs (sialic acid binding Ig-like lectins) are a family of transmembrane, I-type lectins characterized by an N-terminal V-set Ig domain that binds sialic acid. We now know that Siglec-8 is expressed only on human eosinophils, basophils and mast cells, giving it a unique expression pattern on effector cells of allergic disease. Our preliminary data suggests that Siglec-8 exists in two isoforms, one of which contains two putative cytoplasmic tyrosine-based signaling motifs, including an ITIM sequence. Because of the ITIM sequence, we hypothesized that Siglec-8 ligation would inhibit functions of eosinophils, basophils, and mast cells. Incubation of eosinophils with specific Siglec-8 binding monoclonals under crosslinking conditions caused rapid and profound caspase-dependent apoptosis, and this response could not be rescued by the survival-promoting cytokine IL-5. In fact, IL-5 appears to enhance the ability of Siglec-8 crosslinking to induce eosinophil apoptosis. Parallel studies with mast cells and basophils also suggest a pro-apoptotic effect of anti-Siglec-8, as well as inhibition of histamine release responses. In this renewal we propose experiments to expand these observations by examining human eosinophils, basophils, and mast cells for inhibitory consequences of Siglec-8 ligation in vitro. Cytokine-primed cells and cells isolated from late phase bronchoalveolar lavage fluids will also be studied. Signal transduction pathways and other mechanisms involved in these responses will be elucidated using normal and activated human cells, as well as full-length and truncated Siglec-8 transfectants. Preliminary results probing cells and tissues with Siglec-8 Ig fusion proteins have detected potential ligands on human respiratory epithelial cells. These studies will be expanded to characterize these ligands. Finally, in order to lay the groundwork to explore the biology of Siglec-8 in vivo, we initiated efforts to discover murine Siglec-8 homologs and have identified several candidates. Experiments are thus proposed to generate and characterize new reagents to study Siglec-8 homologs on murine eosinophils and mast cells in vitro and in vivo. Activation via Siglec-8 by its natural ligands or pharmacologic agonists could potentially be used to inhibit eosinophil, basophil and mast cell function and survival in vivo, providing a novel strategy for reducing or inhibiting these cells in allergic diseases. ? ?
