Immune Netw.  2011 Dec;11(6):390-398. 10.4110/in.2011.11.6.390.

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

• KMID: 2150725
• DOI: http://doi.org/10.4110/in.2011.11.6.390

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.

Keyword

CD80; CD86; EBV; CD54; FAK

MeSH Terms

Antibodies, Monoclonal
Apoptosis
B-Lymphocytes
Cell Adhesion
Cell Line
Focal Adhesion Protein-Tyrosine Kinases
Herpesvirus 4, Human
Proteins
Antibodies, Monoclonal
Focal Adhesion Protein-Tyrosine Kinases
Proteins

Figure

• 

  Figure 1 CD86 and CD80 expression during EBV-transformation process of B cells. Resting B cells purified from human blood were immortalized with a medium containing EBV particles. Harvesting cells at each indicated period, cells were stained with FITC-conjugated mouse anti-human CD80 (first lane) or CD86 (third lane) and PE-conjugated mouse anti-human CD19 (second and fourth lane) and analyzed by confocal microscope (×400 magnification) as described in Materials and Methods.

• 

  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.

• 

  Figure 3 Reduced expression of CD54 after CD80 and CD86 stimulation. (A) EBV-transformed B cells were incubated with anti-CD80, anti-CD86, or isotype control antibodies (MOPC) for 3 hours. The cells were observed and visualized using inverted microscope (×100 magnification, Leica 351, Wetzlar, Germany). (B) After 3 hours stimulation of CD80 or CD86 (5 ug/ml), flow cytometric analysis was performed to examine surface CD54 expression. Histogram shows the expression levels of CD54 by CD80/86 engagement (bold line), isotype-matched control group (MOPC, spotted line), and negative control (solid line). (C) EBV-transformed B cells (5×105 cells/well, 200 ul) were triggered on anti-CD80/86 antibody (5 ug/ml) plates for 1 hour. The cells were washed three times with PBS. RNA was extracted and cDNA was produced using RT premix as described in Materials and Methods. RT-PCR for CD54 was performed. The PCR products obtained were electrophoresed on 1% agarose gel.

• 

  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.

• 

  Figure 5 Activation of RhoA, Ras, FAK, and JNK after CD80 and CD86 stimulation. (A) EBV-transformed B cells (5×105 cells/well, 200 ul) were triggered on anti-CD80/86 antibody (5 ug/ml) plates for 1 hour. RNA was extracted and cDNA was synthesized in Materials and Methods. RT-PCR for RhoA, Ras, and beta-actin as an normalization control was performed. (B, C) EBV-transformed B cells were incubated anti-CD80 or anti-CD86 antibody (5 ug/ml) for 3 hours. Cells was harvested and lysed in lysis buffer. Immunoblot for indicated molecules in the whole lysate was performed as decribed in Materials and Methods.

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