Analysis of the functional characteristics of L-selectin and its expression on normal and CD18-deficient bovine neutrophils.
In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18-deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly increased (P < 0.05) in four normal calves at 5-20 min after in vivo administration of epinephrine; however, no significant increase of neutrophils was found in three calves with bovine leucocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from three calves with BLAD was 61-77% of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate caused a marked decrease in the expression of L-selectin on neutrophils from both normal and BLAD calves. The sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased. Following stimulation with aggregated IgG, the transient phase of [Ca2+]i in neutrophils from normal and BLAD calves was increased; however, the sustained phase of [Ca2+]i in BLAD neutrophils was significantly lower (P < 0.05) than that of controls. Sulfatide-induced O2- production and chemiluminescent response in neutrophils from calves with BLAD were 48-51% of those of normal calves and were inhibited by genistein and wortmannin, respectively, in a dose-dependent manner. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from BLAD calves stimulated with sulfatides was 57% of that of controls. The degree of L-selectin expression on neutrophils was correlated with the intracellular signalling events and the related superoxide production.
Nagahata
H
H
Department of Animal Health, School of Veterinary Medicine, Laboratory of Biology, Faculty of Environmental Systems, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan. nght-h@rakuno.ac.jp
Higuchi
H
H
Yamashiki
N
N
Yamaguchi
M
M
D000818:Animals; D000831:Animals, Newborn; D018821:CD18 Antigens / Q000032:analysis; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004837:Epinephrine / Q000494:pharmacology; D005434:Flow Cytometry; D019041:L-Selectin / Q000032:analysis / Q000502:physiology*; D018370:Leukocyte-Adhesion Deficiency Syndrome / Q000097:blood / Q000188:drug therapy / Q000276:immunology*; D018613:Microscopy, Confocal; D018375:Neutrophil Activation; D009504:Neutrophils / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010100:Oxygen / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D013433:Sulfoglycosphingolipids; D013481:Superoxides / Q000032:analysis
D018821:CD18 Antigens; D013433:Sulfoglycosphingolipids; D013481:Superoxides; D019041:L-Selectin; D011505:Protein-Tyrosine Kinases; D010100:Oxygen; D002118:Calcium; D004837:Epinephrine