CB1 and CB2 cannabinoid receptors differentially regulate the production of reactive oxygen species by macrophages.
OBJECTIVE
We investigated the mechanism by which cannabinoid receptors-1 (CB1) and -2 (CB2) modulate inflammatory activities of macrophages.


RESULTS
Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor-alpha and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production.


CONCLUSIONS
CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.
Han
Ki Hoon
KH
Division of Cardiology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Songpa-gu, Seoul, Republic of Korea. steadyhan@amc.seoul.kr
Lim
Sunny
S
Ryu
Jewon
J
Lee
Cheol-Whan
CW
Kim
Yuna
Y
Kang
Ju-Hee
JH
Kang
Soon-Suk
SS
Ahn
Yeong Ki
YK
Park
Chan-Sik
CS
Kim
Jae Joong
JJ
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D023421:Models, Animal; D017382:Reactive Oxygen Species / Q000378:metabolism*; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D018932:Chemokine CCL2; D016207:Cytokines; D017382:Reactive Oxygen Species; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D020827:rap1 GTP-Binding Proteins