SULFATIDE-INDUCED L-SELECTIN ACTIVATION GENERATES INTRACELLULAR OXYGEN RADICALS IN HUMAN NEUTROPHILS - MODULATION BY EXTRACELLULAR ADENOSINE

The sulfated form of galactocerebrosides (sulfatides) have recently be en established as ligands for L-selectin. In this study we show that e xposure of human neutrophils to sulfatides induces a transient generat ion of oxygen radicals, revealed by the luminol-enhanced chemiluminesc ence (CL) technique. The CL response was mainly located intracellularl y, and was dependent on sulfation of the galactose ring, since non-sul fated galactocerebrosides had no effect. Sulfatides also dramatically amplified the CL response triggered by the chemotactic peptide formylm ethionyl-leucyl-phenylalanine (fMLP). This effect was primarily due to an increased (up to 10-fold) intracellular generation of oxygen metab olites. Removal or blocking of L-selectin with chymotrypsin and monocl onal antibodies, respectively, markedly reduced the effects of sulfati des. Furthermore, sulfatides amplified the CL response triggered by io nomycin, whereas the response induced by phorbol-12-myristate-13-aceta te was slightly reduced. The tyrosine kinase inhibitor, genistein, mar kedly inhibited the oxygen radical production induced by sulfatides, a nd totally abolished the potentiating effects of sulfatides in fMLP- a nd ionomycin-stimulated neutrophils. Sulfatides also triggered a trans ient rise in the intracellular free calcium concentration, [Ca2+](i). Consequently, L-selectin activation through sulfatides appear to affec t oxidase activity through a Ca2+-dependent pathway involving tyrosine phosphorylation. Adenosine is an anti-inflammatory agent predominatel y released from the vascular endothelium which might suppress an inapp ropriate activation of the oxidase during L-selectin-mediated rolling of neutrophils. Indeed, we found that adenosine inhibited the oxidativ e burst induced by sulfatides, mainly by attenuating the intracellular generation of oxygen radicals. However, 10-100 times higher concentra tion of exogenous adenosine was required to inhibit the CL response in duced by sulfatides to the same extent as the adenosine-mediated inhib ition of the fMLP-induced response. This difference in sensitivity to adenosine could be explained by various expression of extracellular ad enosine deaminase (ADA), since we found that the ADA-inhibitor erythro -9-(2-hydroxy-3-nonyl)-adenine (EHNA) markedly reduced the oxygen radi cal production caused by sulfatides and almost totally abolished the p otentiating effects of sulfatides on the fMLP-induced respiratory burs t. In contrary, EHNA only slightly reduced the fMLP-triggered CL respo nse. We suggest that the initial activation of L-selectin prepare the neutrophil for an effective microbicidal activity in the extravascular space. This process might be dependent on a L-selectin-mediated incre ase in the expression and activity of ADA, which locally reduces the e xtracellular level of adenosine.