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EN
Adenosine 5'-triphosphate (ATP) and adenosine are the crucial endogenous signaling molecules in immunity and inflammation. In this study we identified the source of extracellular adenosine in human B lymphoblasts, and evaluate the ATP release and metabolism. We observed that the B cells continuously released substantial quantities of ATP (35 pmol/106 cells) when subjected to slow motion in the incubation medium. The adenosine level in the B cell incubation medium was very low, and increased (5-fold) upon inhibition of adenosine deaminase activity with 10 µM of 2-deoxycoformycin (DCF). Inclusion of an inhibitor of equilibrate nucleoside transport (nitrobenzylthioinosine) in the incubation medium in the presence of DCF resulted in the elevation of adenosine level by 9-fold. Inhibition of ecto-ATPase activity with 100 µM of ARL67156 was associated with a 2-fold increase of the extracellular ATP level and a 3-fold decrease of adenosine concentration in the cell culture media. Inclusion of ,ß-methyleneadenosine 5'-diphosphate, a selective inhibitor of ecto-5'-nucleotidase in the incubation medium resulted in a significant decrease (7-fold) the adenosine concentration. In conclusion, our results indicate that ATP released from the B cell is the primary source of peripheral adenosine, and that the activities of ecto enzymes and the efficiency of Ado uptake through the nucleoside transporters determine the Ado level on the B cell surface.
EN
Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects induction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential (ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27Kip1. The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide (H2O2) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H2O2 activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27Kip1 in WEHI-231 B lymphoma cells.
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