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Cross-linking of CD80 and CD86 Diminishes Expression of CD54 on EBV-transformed B Cells through Inactivation of RhoA and Ras.

Park GB, Kim YS, Song H, Kim S, Park DM, Lee WJ, Hur DY - Immune Netw (2011)

Bottom Line: We also examined that their stimulation induced ROS accumulation and reduced CD54 expression.CD80 stimulus inhibited CD54 expression through mainly RhoA inactivation, while CD86 down-regulated Ras and JNK phosphorylation.These results suggest that co-stimulatory CD80 and CD86 molecules, expressed EBV-transformed B cells, may play a role in apoptosis and cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Korea.

ABSTRACT

Background: Epstein Barr virus (EBV) infected B cells are transformed into lymphoblastoid cell lines. Some researchers suggested some a few similarities between this process and carcinogenesis. We observed the expression of CD80 and CD86, co-stimulatory molecules on EBV-transformed B cells and changes of CD54 expression after stimulation of CD80 and CD86.

Methods: CD80 and CD86 were stimulated using anti-CD80 and anti-CD86 monoclonal antibodies. To assess apoptosis and surface protein expression, flow cytometric analysis was performed. Intracellular signal molecules were evaluated by RT-PCR and immunoblot. Morphology and localization of proteins were examined using inverted or confocal microscope.

Results: Cross-linking of CD80 and CD86 induced apoptosis and interfered with proliferation of EBV-transformed B cells, and dispersion of clumped cells. We also examined that their stimulation induced ROS accumulation and reduced CD54 expression. Interestingly, we observed that CD80 and CD86 diminished the expression of CD54 in different methods. Both CD80 and CD86 down-regulated activation of focal adhesion kinase. CD80 stimulus inhibited CD54 expression through mainly RhoA inactivation, while CD86 down-regulated Ras and JNK phosphorylation.

Conclusion: These results suggest that co-stimulatory CD80 and CD86 molecules, expressed EBV-transformed B cells, may play a role in apoptosis and cell adhesion.

No MeSH data available.


Related in: MedlinePlus

ROS generation after CD80 and CD86 stimulation. EBV-transformed B cells were pretreated with 10 uM DCFH-DA (Molecular probes) for 30 minutes. Cells (5×105 cells/well, 200 ul) were triggered on anti-CD80/86 antibody (5 ug/ml) plates for 4 hour. Cells were harvested, and then ROS levels determined using a flow cytometer. Histogram shows ROS level generated by CD80/86 stimulation (dot line) and isotype control group (MOPC, solid line). NAC pretreatment (10 mM, 1 hour) preceded all procedures.
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Figure 4: ROS generation after CD80 and CD86 stimulation. EBV-transformed B cells were pretreated with 10 uM DCFH-DA (Molecular probes) for 30 minutes. Cells (5×105 cells/well, 200 ul) were triggered on anti-CD80/86 antibody (5 ug/ml) plates for 4 hour. Cells were harvested, and then ROS levels determined using a flow cytometer. Histogram shows ROS level generated by CD80/86 stimulation (dot line) and isotype control group (MOPC, solid line). NAC pretreatment (10 mM, 1 hour) preceded all procedures.

Mentions: ROS (reactive oxygen species) level was investigated after CD80 and CD86 stimulation because ROS generation is associated with cellular stress. DCFH-DA was pretreated for 30 minutes before incubation with anti-CD80 and CD86 antibodies. After 4 hours stimulation, ROS was detected using flow cytometry. Ligation of CD80 or CD86 increased ROS level on EBV-transformed B cells (Fig. 4 upper panel). Both CD80 and CD86 stimulation did not increase ROS generation additively more than single stimulation. Pretreatment of NAC as a ROS accumulation inhibitor completely blocked ROS generation by CD80 and CD86 stimulation (Fig. 4 lower panel).


Cross-linking of CD80 and CD86 Diminishes Expression of CD54 on EBV-transformed B Cells through Inactivation of RhoA and Ras.

Park GB, Kim YS, Song H, Kim S, Park DM, Lee WJ, Hur DY - Immune Netw (2011)

ROS generation after CD80 and CD86 stimulation. EBV-transformed B cells were pretreated with 10 uM DCFH-DA (Molecular probes) for 30 minutes. Cells (5×105 cells/well, 200 ul) were triggered on anti-CD80/86 antibody (5 ug/ml) plates for 4 hour. Cells were harvested, and then ROS levels determined using a flow cytometer. Histogram shows ROS level generated by CD80/86 stimulation (dot line) and isotype control group (MOPC, solid line). NAC pretreatment (10 mM, 1 hour) preceded all procedures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275709&req=5

Figure 4: ROS generation after CD80 and CD86 stimulation. EBV-transformed B cells were pretreated with 10 uM DCFH-DA (Molecular probes) for 30 minutes. Cells (5×105 cells/well, 200 ul) were triggered on anti-CD80/86 antibody (5 ug/ml) plates for 4 hour. Cells were harvested, and then ROS levels determined using a flow cytometer. Histogram shows ROS level generated by CD80/86 stimulation (dot line) and isotype control group (MOPC, solid line). NAC pretreatment (10 mM, 1 hour) preceded all procedures.
Mentions: ROS (reactive oxygen species) level was investigated after CD80 and CD86 stimulation because ROS generation is associated with cellular stress. DCFH-DA was pretreated for 30 minutes before incubation with anti-CD80 and CD86 antibodies. After 4 hours stimulation, ROS was detected using flow cytometry. Ligation of CD80 or CD86 increased ROS level on EBV-transformed B cells (Fig. 4 upper panel). Both CD80 and CD86 stimulation did not increase ROS generation additively more than single stimulation. Pretreatment of NAC as a ROS accumulation inhibitor completely blocked ROS generation by CD80 and CD86 stimulation (Fig. 4 lower panel).

Bottom Line: We also examined that their stimulation induced ROS accumulation and reduced CD54 expression.CD80 stimulus inhibited CD54 expression through mainly RhoA inactivation, while CD86 down-regulated Ras and JNK phosphorylation.These results suggest that co-stimulatory CD80 and CD86 molecules, expressed EBV-transformed B cells, may play a role in apoptosis and cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Korea.

ABSTRACT

Background: Epstein Barr virus (EBV) infected B cells are transformed into lymphoblastoid cell lines. Some researchers suggested some a few similarities between this process and carcinogenesis. We observed the expression of CD80 and CD86, co-stimulatory molecules on EBV-transformed B cells and changes of CD54 expression after stimulation of CD80 and CD86.

Methods: CD80 and CD86 were stimulated using anti-CD80 and anti-CD86 monoclonal antibodies. To assess apoptosis and surface protein expression, flow cytometric analysis was performed. Intracellular signal molecules were evaluated by RT-PCR and immunoblot. Morphology and localization of proteins were examined using inverted or confocal microscope.

Results: Cross-linking of CD80 and CD86 induced apoptosis and interfered with proliferation of EBV-transformed B cells, and dispersion of clumped cells. We also examined that their stimulation induced ROS accumulation and reduced CD54 expression. Interestingly, we observed that CD80 and CD86 diminished the expression of CD54 in different methods. Both CD80 and CD86 down-regulated activation of focal adhesion kinase. CD80 stimulus inhibited CD54 expression through mainly RhoA inactivation, while CD86 down-regulated Ras and JNK phosphorylation.

Conclusion: These results suggest that co-stimulatory CD80 and CD86 molecules, expressed EBV-transformed B cells, may play a role in apoptosis and cell adhesion.

No MeSH data available.


Related in: MedlinePlus