Rapid and quantitative detection of p38 kinase pathway in mouse blood monocyte.
The p38 alpha mitogen-activated protein kinase (MAPK) is essential in controlling the production of many proinflammatory cytokines, and its specific inhibitor can effectively block their production for treating human diseases. To effectively identify highly specific p38 alpha inhibitors in vivo, we developed an ex vivo mouse blood cell-based assay by flow cytometry to measure the intracellular p38 alpha kinase activation. We first attempted to identify the individual blood cell population in which the p38 alpha kinase pathway is highly expressed and activated. Based on CD11b, combined with Ly-6G cell surface expression, we identified two distinct subsets of non-neutrophilic myeloid cells, CD11b(Med)Ly-6G(-) and CD11b(Lo)Ly-6G(-), and characterized them as monocytes and natural killer (NK) cells, respectively. Then, we demonstrated that only monocytes, not NK cells, expressed a high level of p38 alpha kinase, which was rapidly activated by anisomycin stimulation as evidenced by the phosphorylation of both p38 and its substrate, MAPKAP-K2 (MK2). Finally, the p38 alpha kinase pathway activation in monocytes was fully inhibited by a highly selective p38 alpha kinase inhibitor dose-dependently in vitro and in vivo. In conclusion, we demonstrated an effective method for separating blood monocytes from other cells and for detecting the expression level and activation of the p38 alpha kinase pathway in monocytes, which provided a new approach for the rapid identification of specific p38 alpha inhibitors.
Zhao
Jingyong
J
Cancer Inflammation and Cell Survival, Lilly Research Laboratories, Eli Lilly, Indianapolis, IN 46285, USA.
Evans
Glenn
G
Li
Weiming
W
Green
Lisa
L
Chu
Shaoyou
S
Marder
Phil
P
Na
Songqing
S
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D018845:CD11 Antigens; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000097:blood; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000097:blood; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D000099190:MAP-Kinase-Activated Kinase 2
D018845:CD11 Antigens; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2