'Outside-in' signalling mechanisms underlying CD11b/CD18-mediated NADPH oxidase activation in human adherent blood eosinophils.
1 Incubation of human eosinophils in BSA-coated tissue culture plates resulted in time-dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18. 2 SB 203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=-6.57). 3 PP1, an inhibitor of the src-family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18-mediated superoxide anion generation with similar potencies (pEC50s=-5.53 and -5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion. 4 The protein kinase C (PKC) inhibitors Ro-31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion-dependent NADPH oxidase activation (pIC50s=-6.61, -6.05 and -4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCdelta and PKCepsilon are implicated in the suppression of oxidant production. 5 Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns 3-kinase), abolished superoxide anion production in adherent eosinophils (pEC50=-9.06). Similarly, CD11b/CD18-dependent adhesion was suppressed with the same potency (pEC50=-9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion-driven oxidase activation. 6 PD 098059 and piceatannol, inhibitors of MAP kinase kinase-1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18-mediated adhesion or NADPH oxidase activation. 7 The results of this study demonstrate that human eosinophils adhere to BSA-coated plastic by a CD11b/CD18-dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3-kinase.
Lynch
O T
OT
Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse Street, London SW3 6LY.
Giembycz
M A
MA
Barnes
P J
PJ
Hellewell
P G
PG
Lindsay
M A
MA
D000730:Androstadienes / Q000494:pharmacology; D018821:CD18 Antigens / Q000502:physiology*; D002227:Carbazoles / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002452:Cell Count; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004804:Eosinophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D016177:Macrophage-1 Antigen / Q000502:physiology*; D008301:Maleimides / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology
D000730:Androstadienes; D018821:CD18 Antigens; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D007527:Isoenzymes; D016177:Macrophage-1 Antigen; D008301:Maleimides; D020928:Mitogen-Activated Protein Kinases; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases; C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; C081021:Go 6976; C070515:bisindolylmaleimide I; C093642:SB 203580; C064758:Ro 31-8220