IntroductionEosinophils, through release of preformed and newly generated mediators, are considered key effector cells in several diseases. Their recruitment and activation are regarded as central to the pathophysiology of allergic disorders, including asthma. [1][2][3] Besides selective migration, increased cell survival and decreased apoptosis have been proposed as mechanisms contributing to tissue-specific accumulation of these inflammatory cells. 4 Hence, therapeutic efforts in the area of allergic inflammation continue to focus on the development of agents to suppress eosinophil recruitment, activation, and survival.As a result of molecular efforts to identify novel, specific markers expressed on eosinophils, our group and others recently discovered Siglec-8, formerly called SAF-2. 5,6 Sialic acid binding immunoglobulin-like lectins (Siglecs) belong to the immunoglobulin (Ig) supergene family and characteristically have an N-terminal V-set domain that binds sialic acid. 7 Although originally isolated from a human eosinophil cDNA library by random expressed sequence tag sequencing, Siglec-8 is also expressed on human basophils and mast cells 5 and exists in 2 isoforms with identical extracellular and transmembrane sequences. One isoform (Siglec-8) has a short cytoplasmic tail with no known signaling sequences, while the other, Siglec-8 long form (Siglec-8L), has a longer cytoplasmic tail containing 2 tyrosine-based signaling motifs. 8,9 Although the function of Siglec-8 and Siglec-8L, and indeed most Siglecs, is unknown, the cytoplasmic region of Siglec-8L contains one consensus immunoreceptor tyrosine-based inhibitory motif (ITIM) and a signaling lymphocyte activation molecule (SLAM)-like motif, suggesting that Siglec-8L may possess signal transduction activity. 7-9 Indeed, for some Siglecs, such as Siglec-3 and Siglec-7 (p75/AIRM-1), antibody crosslinking has been shown to inhibit proliferation and survival of myeloid leukemic cells. 10-12 Therefore we hypothesized that ligation of Siglec-8 would inhibit eosinophil survival, and we present data that show a proapoptotic effect with antibody-mediated cross-linking.
Materials and methods
Antibodies and recombinant proteinsMurine monoclonal IgG 1 isotype antibodies (Abs) recognizing Siglec-8 (2E2, 2C4, and 9G4) were generated using previously described methods 5 and were endotoxin-free (ie, below the detection level of Ͻ 0.01 EU/mL as determined by amebocyte lysate assay; BioWhittaker, Walkersville, MD). Unless noted otherwise, these monoclonal Abs (mAbs) were used at saturating concentrations (2.5 g/mL). Normal sheep serum and goat antisheep horseradish peroxidase-linked Abs were from Calbiochem (La Jolla, CA). Goat antirabbit horseradish peroxidase-linked Ab was from Amersham Pharmacia Biotech (Piscataway, NJ). Polyclonal intact and F(abЈ) 2 goat antimouse IgG (heavy and light chain) were purchased from Caltag Laboratories (Burlingame, CA). Recombinant human interleukin 5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were from R&D Syst...
