ABSTRACT
Objective@#To study the distribution of natural killer (NK) cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis (RA) patients and the mechanism of CD38 agonist.@*Methods@#The levels of NK cells, CD38 positive subpopulation and NK cell surface chemokine receptors (CXCR3, CCR5), as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid. The isolated cells were then cultured in vitro and divided into 3 groups, namely blank control group, IB4 stimulation group (Anti-CD38 mAb), IL-2 and IB4 co-stimulation group. And the concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA). Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients, and transfection efficiency was detected by reverse transcription-polymerase chain reaction (RT-PCR). The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1 (PLC-γ1) protein in the peripheral blood lymphocytes of RA was detected by Western blot.@*Results@#The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects (P<0.05). The two chemokine receptors (CXCR3, CCR5) were mainly expressed in CD38+ NK cell subsets. The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes (P<0.05). The secretion of IL-6 and TNF-α in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group (P<0.05). The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA (P<0.05).@*Conclusions@#The synovial NK cells are more lethal than the peripheral NK cells in the RA patients. CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C (PKC) pathway. Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins. CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.
ABSTRACT
Objective To study the distribution of natural killer (NK) cells and the CD38 positive subpopulations in different tissues in rheumatoid arthritis (RA) patients and the mechanism of CD38 agonist.Methods The levels of NK cells,CD38 positive subpopulation and NK cell surface chemokine receptors (CXCR3,CCRS),as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid.The isolated cells were then cultured in vitro and divided into 3 groups,namely blank control group,IB4 stimulation group (Anti-CD38 mAb),IL-2 and IB4 co-stimulation group.And the concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA).Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients,and transfection efficiency was detected by reverse transcription-polymerase chain reaction (RT-PCR).The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C-gamma 1 (PLC-γ1) protein in the peripheral blood lymphocytes of RA was detected by Western blot.Results The expression of CCR5 and CXCR3 in NK cells in peripercal NK cells of RA was significantly higher than that in normal healthy subjects (P ＜ 0.05).The two chemokine receptors (CXCR3,CCRS) were mainly expressed in CD38 + NK cell subsets.The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes (P ＜0.05).The secretion of IL-6 and TNF-α in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group (P ＜0.05).The levels of PLC-γ1 protein in the peripheral blood lymphocytes of RA patients was significantly decreased than that in the blank control group after the treatment with CD38-siRNA (P ＜ 0.05).Conclusions The synovial NK cells are more lethal than the peripheral NK cells in the RA patients.CD38 might mediate the phosphorylation of intracellular proteins via the Protain kinase C (PKC) pathway.Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins.CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.
ABSTRACT
ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cyclic ADP- ribose (cADPR), from beta-NAD+. A prototype of mammalian ADPR-cyclases is a lymphocyte antigen CD38. Accumulating evidence indicates that ADPR-cyclases other than CD38 are expressed in various cells and organs. In this study, we discovered a small molecule inhibitor of kidney ADPR-cyclase. This compound inhibited kidney ADPR-cyclase activity but not CD38, spleen, heart or brain ADPR-cyclase activity in vitro. Characterization of the compound in a cell-based system revealed that an extracellular calcium-sensing receptor (CaSR)- mediated cADPR production and a later long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse mesangial cells were inhibited by the pre-treatment with this compound. In contrast, the compound did not block CD3/TCR-induced cADPR production and the increase of [Ca2+]i in Jurkat T cells, which express CD38 exclusively. The long-lasting Ca2+ signal generated by both receptors was inhibited by pre-treatment with an antagonistic cADPR derivative, 8-Br-cADPR, indicating that the Ca2+ signal is mediated by the ADPR-cyclse metabolite, cADPR. Moreover, among structurally similar compounds tested, the compound inhibited most potently the cADPR production and Ca2+ signal induced by CaSR. These findings provide evidence for existence of a distinct ADPR-cyclase in the kidney and basis for the development of tissue specific inhibitors.