Sulfatide-induced L-selectin activation generates intracellular oxygen radicals in human neutrophils: modulation by extracellular adenosine.
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 chemiluminescence (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 chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP). This effect was primarily due to an increased (up to 10-fold) intracellular 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-acetate 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 ionomycin-stimulated neutrophils. Sulfatides also triggered a transient rise in the intracellular free calcium concentration, [Ca2+]i. Consequently, L-selectin activation through sulfatides appear to affect oxidase activity through a Ca(2+)-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, 10-100 times higher concentration of exogenous adenosine was required to inhibit the CL response induced by sulfatides to the same extent as the adenosine-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-nonyl)-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.
Bengtsson
T
T
Department of Medical Microbiology, Faculty of Health Sciences, Linköping University, Sweden.
Grenegård
M
M
Olsson
A
A
Sjögren
F
F
Stendahl
O
O
Zalavary
S
S
D000241:Adenosine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D005609:Free Radicals; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D019041:L-Selectin / Q000378:metabolism*; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology; D018000:Receptors, Peptide / Q000502:physiology; D016897:Respiratory Burst; D013433:Sulfoglycosphingolipids / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005609:Free Radicals; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D013433:Sulfoglycosphingolipids; D019041:L-Selectin; D019000:Phosphotyrosine; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D000241:Adenosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium