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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

Proliferation and apoptosis after CD80 and CD86 stimulation. (A) EBV-transformed B cells (4 weeks, 1×104 cells/well) were incubated with 0.01~10 ug/ml of anti-CD80, anti-CD80, or isotype control (MOPC) for 72 hours. AlamarBlue dye was then added (10% by volume) to each well and relative fluorescence was measured 7 hours later using a fluorometer as described in Materials and Methods. Reduction ratio was calculated by manufacturer's protocol. (B) EBV-transformed B cells were incubated with anti-CD80 and CD86 antibodies (0.312~5 ug/ml) or isotype control antibody (MOPC, 0.312~5 ug/ml) for 24 hours. The cells were harvested (5×105 cells/well, 200 ul) and then stained with a FITC-conjugated annexin V. Thin line represents isotype control and thick line represents CD80 or CD86 stimulation.
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Figure 2: Proliferation and apoptosis after CD80 and CD86 stimulation. (A) EBV-transformed B cells (4 weeks, 1×104 cells/well) were incubated with 0.01~10 ug/ml of anti-CD80, anti-CD80, or isotype control (MOPC) for 72 hours. AlamarBlue dye was then added (10% by volume) to each well and relative fluorescence was measured 7 hours later using a fluorometer as described in Materials and Methods. Reduction ratio was calculated by manufacturer's protocol. (B) EBV-transformed B cells were incubated with anti-CD80 and CD86 antibodies (0.312~5 ug/ml) or isotype control antibody (MOPC, 0.312~5 ug/ml) for 24 hours. The cells were harvested (5×105 cells/well, 200 ul) and then stained with a FITC-conjugated annexin V. Thin line represents isotype control and thick line represents CD80 or CD86 stimulation.

Mentions: Freshly isolated B cells did not express both CD80 and CD86 on surface (Fig. 1), but intracellular staining revealed that CD86 was abundant in B cell cytosol (data not shown). After 1 week of EBV infection, intracellular and surface expression of CD80 and surface expression of CD86 were increased. At 4 week which is known as a stable period of EBV-transformation, phenotype of most cells was CD19+CD80+CD86+ determining by flow cytometric analysis (data not shown). To examine cellular response after CD80 and CD86 stimulation, EBV-transformed B cells (4 weeks) were incubated with different concentrations of anti-CD80, anti-CD86, or isotype control (IgG1, MOPC21). Stimulation of CD80 or CD86 inhibited cell proliferation after 72 hours incubation with 0.1~10 ug/ml of antibodies (Fig. 2A). In addition, apoptosis was detected at incubation with 0.3125~5 µg/ml of antibodies for 24 hours (Fig. 2B).


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)

Proliferation and apoptosis after CD80 and CD86 stimulation. (A) EBV-transformed B cells (4 weeks, 1×104 cells/well) were incubated with 0.01~10 ug/ml of anti-CD80, anti-CD80, or isotype control (MOPC) for 72 hours. AlamarBlue dye was then added (10% by volume) to each well and relative fluorescence was measured 7 hours later using a fluorometer as described in Materials and Methods. Reduction ratio was calculated by manufacturer's protocol. (B) EBV-transformed B cells were incubated with anti-CD80 and CD86 antibodies (0.312~5 ug/ml) or isotype control antibody (MOPC, 0.312~5 ug/ml) for 24 hours. The cells were harvested (5×105 cells/well, 200 ul) and then stained with a FITC-conjugated annexin V. Thin line represents isotype control and thick line represents CD80 or CD86 stimulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Proliferation and apoptosis after CD80 and CD86 stimulation. (A) EBV-transformed B cells (4 weeks, 1×104 cells/well) were incubated with 0.01~10 ug/ml of anti-CD80, anti-CD80, or isotype control (MOPC) for 72 hours. AlamarBlue dye was then added (10% by volume) to each well and relative fluorescence was measured 7 hours later using a fluorometer as described in Materials and Methods. Reduction ratio was calculated by manufacturer's protocol. (B) EBV-transformed B cells were incubated with anti-CD80 and CD86 antibodies (0.312~5 ug/ml) or isotype control antibody (MOPC, 0.312~5 ug/ml) for 24 hours. The cells were harvested (5×105 cells/well, 200 ul) and then stained with a FITC-conjugated annexin V. Thin line represents isotype control and thick line represents CD80 or CD86 stimulation.
Mentions: Freshly isolated B cells did not express both CD80 and CD86 on surface (Fig. 1), but intracellular staining revealed that CD86 was abundant in B cell cytosol (data not shown). After 1 week of EBV infection, intracellular and surface expression of CD80 and surface expression of CD86 were increased. At 4 week which is known as a stable period of EBV-transformation, phenotype of most cells was CD19+CD80+CD86+ determining by flow cytometric analysis (data not shown). To examine cellular response after CD80 and CD86 stimulation, EBV-transformed B cells (4 weeks) were incubated with different concentrations of anti-CD80, anti-CD86, or isotype control (IgG1, MOPC21). Stimulation of CD80 or CD86 inhibited cell proliferation after 72 hours incubation with 0.1~10 ug/ml of antibodies (Fig. 2A). In addition, apoptosis was detected at incubation with 0.3125~5 µg/ml of antibodies for 24 hours (Fig. 2B).

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