Effect of CD40-CD40 ligand interaction on diacylglycerol-protein kinase C signal transduction pathway and intracellular calcium in cultured human monocytes.
OBJECTIVE
To investigate whether CD40-CD40 ligand interaction can activate diacylglycerol (DAG)-protein kinase C (PKC) signaling pathway and intracellular free calcium ([Ca2+]i) in cultured human peripheral blood monocytes (PBMC).


METHODS
The DAG levels in PBMC were studied with radio-enzymatic assay. Quantitative measurements of (32)P-phosphatidic acid were performed by thin-layer chromatography and autoradiography. The activity of PKC and [Ca2+]i induced by CD40 ligand (CD40L) in PBMC were measured by its ability to transfer phosphate from [gamma-32P]ATP to lysine-rich histone and flow cytometric analysis loading with the Ca2+ dye Fluo-3/Am, respectively.


RESULTS
The DAG levels in PBMC were increased by CD40L in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 20 s; the late phase reached the maximal level at 10 min and then decayed slowly. CD40L increased the PKC total activity in a dose-dependent manner with phase peaking at 12 min, then decreased slowly and maintained for at least 20 min. CD40L induced PKC activity translocation from the cytosol to membrane. Moreover, CD40L also induced biphasic [Ca2+]i responses including the rapid initial transient phase and the sustained phase. Removal of extracellular Ca2+ did not inhibit the rapid phase of CD40L-induced rise in [Ca2+]i, but abolished the sustained phase of [Ca2+]i response to CD40L. Anti-CD40 monoclone antibody 10 mg/L significantly suppressed CD40L-induced DAG-PKC signal transduction pathway activation and [Ca2+]i changes in PBMC.


CONCLUSIONS
CD40-CD40 ligand interaction induced a robust stimulation of the DAG-PKC pathway and calcium mobilization from intracellular pool in PBMC.
Yan
Jin-Chuan
JC
Department of Cardiology, Affiliated Zhong Da Hospital, Southeast University, Nanjing 210009, China. jinchuanyan@sina.com
Wu
Zong-Gui
ZG
Kong
Xian-Tao
XT
Zong
Ren-Qian
RQ
Zhang
Lin-Zhen
LZ
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D006801:Humans; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
C011439:1,2-diacylglycerol; D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D004075:Diglycerides; D023201:CD40 Ligand; D011493:Protein Kinase C; D002118:Calcium