Intracellular protein phosphorylation in eosinophils and the functional relevance in cytokine production.
BACKGROUND
Eosinophils play a pivotal role in the pathogenesis of asthma. Thus, it is of paramount importance to investigate the mechanism of eosinophil activation. Although a number of factors including cytokines/chemokines activate eosinophils, the potency of each stimulus to phosphorylate intracellular molecules and activate eosinophils remains to be elucidated. In the present study, we performed inclusive analyses of protein phosphorylation in eosinophils and studied the functional relevance of such phosphorylation in cytokine production.


METHODS
Blood eosinophils were purified using Percoll and anti-CD16 antibody-coated magnetic beads. Purified eosinophils were stimulated with various stimuli. The eosinophil lysates were subjected to phosphoprotein analysis using the Luminex system. In some of these experiments, we studied the effect of a few signaling inhibitors on cytokine production from eosinophils.


RESULTS
We found that several factors such as IL-5, eotaxin, platelet-activating factor (PAF), and PGD2 phosphorylated Akt, ERK1/2, p38 MAPK, and glycogen synthase kinase-3 (GSK-3) in the eosinophils. Because eotaxin most potently induced the production of various cytokines, we performed the inhibition study using eotaxin-stimulated eosinophils. Eotaxin-induced production of IL-1beta, IL-6, and MIP-1beta was significantly reduced by the MEK inhibitor PD98059, p38 MAPK inhibitor SB203580, or PI3K inhibitor LY294002. In contrast, the GSK-3 inhibitor SB216763 blocked only IL-1beta production from the eosinophils.


CONCLUSIONS
In terms of the phosphorylation of intracellular signaling molecules, we could quantify the potency of various stimuli that activate eosinophils. We are the first to demonstrate the role of GSK-3 in cytokine production from eosinophils. The Luminex system aids in examining the mechanism of eosinophil activation.
Yamamura
Koichi
K
Department of Internal Medicine, Teikyo University School of Medicine, Tokyo 173-8605, Japan.
Adachi
Tetsuya
T
Masuda
Tomoko
T
Kojima
Yasuhiro
Y
Hara
Asae
A
Toda
Takako
T
Nagase
Hiroyuki
H
Ohta
Ken
K
D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D004804:Eosinophils / Q000276:immunology* / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D015848:Interleukin-5 / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology; D015230:Prostaglandin D2 / Q000494:pharmacology; D015854:Up-Regulation
D016207:Cytokines; D015848:Interleukin-5; D047908:Intracellular Signaling Peptides and Proteins; D010972:Platelet Activating Factor; D038362:Glycogen Synthase Kinase 3; D015230:Prostaglandin D2