Inhibitory Functions of Siglec-8 in Transfected RBL-2H3 Cells

RATIONALE: Siglec-8 is expressed on human eosinophils, mast cells and basophils. It has two tyrosine motifs, a proximal motif and a distal motif, which have some inhibitory functions in immune systems. We used the rat mast cell (RBL: rat basophilic leukemia) model to study signaling mechanisms and the inhibitory role of Siglec-8. We examined that Siglec-8 is able to inhibit the IgE receptor-mediated B-hexosaminidase release from RBL cells following co-crosslinking. METHODS: The RBL-2H3 cells were cultured in MEM supplemented with 15% FBS, penicillin(100IU/ml), and streptomycin(100 μg/ml). Stable cell lines and mutant forms of Siglec-8 were obtained by selection with geneticin at 1 mg/ml. The Hexosaminidase release assay was performed by incubation the RBL cells with anti-mouse IgE(0.05 μg/ml) in the absence or presence of anti-Siglec-8 mAbs. After washing, cells were incubated for 30 min at 37°C with goat anti-mouse IgG F(ab')2(at 5, 10, 50 μg/ml). Supernatents were collected, then incubated for 1 hr at 37°C prior to analysis using the microplate reader at 400 nm. RESULTS: Co-crosslinking of Siglec-8 reduced the hexosaminidase release of transfected RBL cells. These results show that Siglec-8 is as potent as Siglec-7 and -9 in delivering inhibitory signals to RBL cells. CONCLUSIONS: We have demonstrated that Siglec-8 should be a new member of the inhibitory receptor superfamily and that the membrane-proximal ITIM is essential for the inhibitory function of Siglec-8 molecules.