Sulfatide-induced L-selectin activation generates intracellular oxygen radicals in human neutrophils: modulation by extracellular adenosine

摘要

The sulfated form of galactocerebrosides (sulfatides) have recently been established as ligands for L-selectin. In this study we show that exposure of human neutrophils to sulfatides induces a transient generation of oxygen radicals, revealed by the luminol-enhanced cheniiluminescence (CL) technique. The CL response was mainly located intracellularly, and was dependent on sulfation of the galactose ring, since non-sulfated galactocerebrosides had no effect. Sulfatides also dramatically amplified the CL response triggered by the dieinotaelie peptide formylmethionyl-leucyl-phenylahinine (fMLP). This effect was primarily due to an increased (up to 10-fold) inimcellular generation of oxygen metabolites. Removal or blocking of L-selectin with chymotrypsin and monoclonal antibodies, respectively, markedly reduced the effects of sulfatides. Furthermore, sulfatides amplified the CL response triggered by ionomycin, whereas the response induced by phorbol-12-myristate-13-acetute was slightly reduced. The tyrosine kinase inhibitor, genistein, markedly inhibited the oxygen radical production induced by sulfatides, and totally abolished the potentiating effects of sulfatides in fMLP- and ionoinyein-stimulated neutrophils. Sulfatides also triggered a transient rise in the intracellular free calcium concentration, [Ca24] Consequently, L-selectin activation through sulfatides appear to affect oxidase activity through a Ca~+-dependent pathway involving tyrosine phosphorylation. Adenosine is an anti-inflammatory agent predominately released from the vascular endothelium which might suppress an inappropriate activation of the oxidase during L-selectin-mediated rolling of neutrophils. Indeed, we found that adenosine inhibited the oxidative burst induced by sulfatides, mainly by attenuating the intracellular generation of oxygen radicals. However, 10-IOC) times higher concentration of exogenous adenosine was required to inhibit the CL response induced by sulfatides to the same extent as the aclenosine-mediated inhibition of the fMLP-induced response. This difference in sensitivity to adenosine could be explained by various expression of extracellular adenosine deaminase (ADA), since we found that the ADA-inhibitor erythro-9-(2-hydroxy-3-nonyD-adenine (EHNA) markedly reduced the oxygen radical production caused by sulfatides and almost totally abolished the potentiating effects of sulfatides on the fMLP-induced respiratory burst. In contrary, EHNA only slightly reduced the fMLP-triggered CL response. 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 increase in the expression and activity of ADA, which locally reduces the extracellular level of adenosine.