Abstract
Sulfatides have been established recently as ligands for L-selectin, and we investigated whether they trigger transmembrane signals through ligation of L-selectin. We found that sulfatides trigger the increase of cytosolic free calcium in neutrophils and that this effect was strictly dependent on sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory. Chymotrypsin and phorbol 12-myristate 13-acetate treatment of neutrophils caused shedding of L-selectin, but not of class I major histocompatibility complex antigens or beta 2 integrins, and blunted the capability of neutrophils to respond to sulfatides with an increase of cytosolic free calcium. Four different anti-L-selectin antibodies (DREG-200, LAM1/3, LAM1/6, and LAM1/10), but not four control antibodies directed against different surface molecules of neutrophils, also triggered an increase of cytosolic free calcium. The anti-L-selectin antibodies were stimulatory both if used in a soluble form, after cross-linking with anti-mouse F(ab')2 fragments, and immobilized to protein A of Staphylococcus aureus through the Fc fragment. With immobilized antibodies, an increase of cytosolic free calcium was found also by plating neutrophils on antibodies bound to protein A-coated coverslips and monitoring the increase of cytosolic free calcium by fluorescence microscopy. Both sulfatides and anti-L-selectin antibody effects were not inhibited by pertussis toxin, thus indicating that a pertussis toxin-sensitive GTP-binding protein was not involved in signal transduction. Sulfatides also triggered an increase of tumor necrosis factor-alpha and interleukin-8 mRNAs in neutrophils. Also to act as stimuli of cytokine mRNA expression, sulfatides required sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory, and depended on expression of L-selectin, as shedding of this molecules induced by chymotrypsin blunted their effects. These findings suggest that L-selectin can transduce signals activating selective cell function.
Highlights
Sulfatides TriggerIncrease of Cytosolic Free Calcium and Enhanced Expression of Tumor Necrosis Factor-a and Interleukin-8 mRNA in Human Neutrophils
We found that sulfatides trigger the increase of cytosolic distinct leukocyte surface molecules which recognize counterfree calcium in neutrophils and that this effectwas receptors expressed by the endothelial cell
We show in this study that sulfatides increase cytosolic free calcium in neutrophils, and their effects are blunted by shedding of L-selectin; soluble and cross-linked, or immobilized, anti-L-selectin antibodies have a triggering effect
Summary
Antibodies and Other Reagents-Anti-L-selectin antibodies were kindly donated by Dr T.K. Kishimoto, BoehringerMannheim B9.12.1, directed against HLA-A,B, C antigens (26), a s was kindly donated by Dr R. The anti-FcRyIII antibody, 3G8,was obtained from American Q p e Culture Collection, Bethesda, MD, and purified from ascitic fluid.Goat anti-mouse Fab fragments were from Organon Teknika Corporation, Cappel Research Products, Durham, NC. Antibodieswere used as soluble reagents, immobilized to Staphylococcus aureus (Pansorbin; Calbiochem) or protein A purified from culture medium of a protein. Aureus (Sigma).In the former case, 50 pl of a 10%suspension of S . Before use they were dissolvedin PBS at 5 mg/ml and sonicatedfor 3 min. All of the reagents used for these studies were prepared in endotoxin-free water for clinical use
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