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Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival.

Xiang J, Gu X, Qian S, Chen Z - J. Biomed. Biotechnol. (2010)

Bottom Line: This approach could specifically and effectively knock down CD80 and CD86 expression.We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group.Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China.

ABSTRACT
Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

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DCs were transduced with nothing (No treated), CD80 lenti, CD86 lenti, NC lenti, or both CD80 lenti and CD86 lenti. (a) After 6 days of culture, the cells were then incubated with LPS (1 μg/mL) for 18 hours. Expression of CD80 and CD86 was determined by flow cytometry. The data show the effect of recombinant lentivirus on inhibition of target molecule expression. Values expressed as mean ± 1 SD from three independent experiments; *P < .05 compared to NC lenti/No treated; **P < .05 compared to CD80 lenti; ***P < .05 compared to CD86 lenti. (b) C3H BM-derived DCs were cultured over 6 days. DCs were stimulated or unstimulated with LPS (1 μg/mL) for 18 hours. Upregulation of CD80 and CD86 in response to LPS was also suppressed by CD80 lenti and CD86 lenti. The mean fluorescence intensity (MFI) of GFP+ cells expressing the marker of interest is indicated.
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fig3: DCs were transduced with nothing (No treated), CD80 lenti, CD86 lenti, NC lenti, or both CD80 lenti and CD86 lenti. (a) After 6 days of culture, the cells were then incubated with LPS (1 μg/mL) for 18 hours. Expression of CD80 and CD86 was determined by flow cytometry. The data show the effect of recombinant lentivirus on inhibition of target molecule expression. Values expressed as mean ± 1 SD from three independent experiments; *P < .05 compared to NC lenti/No treated; **P < .05 compared to CD80 lenti; ***P < .05 compared to CD86 lenti. (b) C3H BM-derived DCs were cultured over 6 days. DCs were stimulated or unstimulated with LPS (1 μg/mL) for 18 hours. Upregulation of CD80 and CD86 in response to LPS was also suppressed by CD80 lenti and CD86 lenti. The mean fluorescence intensity (MFI) of GFP+ cells expressing the marker of interest is indicated.

Mentions: To determine the lentiviral transduction efficiency in DCs, GFP expression was examined by microscopy and flow cytometry at different MOIs on day 4 after transduction. Lentivirus demonstrated a high (79.8% ± 5.0%) transduction efficiency in DCs at MOI of 20 (Figure 2). The percentage of GFP-expressing cells remained minor changes when MOI is >20. Therefore, in the following experiments all viral titers were at MOI of 20. To blockade both CD80 and CD86 expression, DCs were transduced with both CD80 lenti and CD86 lenti. DCs were collected after LPS stimulation. As measured by flow cytometry, we found that CD80 lenti and CD86 lenti together could enhance suppressive effect on CD80 and CD86 expression (Figure 3(a)). After the activation of LPS, untreated DCs underwent marked maturation progress. But as to those transduced DCs LPS failed to bring great changes of the CD80 and CD86 expression. On the other hand, CD80 lenti and CD86 lenti had no effect on expression of unrelated proteins (such as MHC-II and CD40) (Figure 3(b)). Our study indicates that recombinant lentivirus can provide highly efficient and specific CD80 and CD86 knockdown in DCs.


Endoplasmic reticulum stress-mediated apoptosis involved in indirect recognition pathway blockade induces long-term heart allograft survival.

Xiang J, Gu X, Qian S, Chen Z - J. Biomed. Biotechnol. (2010)

DCs were transduced with nothing (No treated), CD80 lenti, CD86 lenti, NC lenti, or both CD80 lenti and CD86 lenti. (a) After 6 days of culture, the cells were then incubated with LPS (1 μg/mL) for 18 hours. Expression of CD80 and CD86 was determined by flow cytometry. The data show the effect of recombinant lentivirus on inhibition of target molecule expression. Values expressed as mean ± 1 SD from three independent experiments; *P < .05 compared to NC lenti/No treated; **P < .05 compared to CD80 lenti; ***P < .05 compared to CD86 lenti. (b) C3H BM-derived DCs were cultured over 6 days. DCs were stimulated or unstimulated with LPS (1 μg/mL) for 18 hours. Upregulation of CD80 and CD86 in response to LPS was also suppressed by CD80 lenti and CD86 lenti. The mean fluorescence intensity (MFI) of GFP+ cells expressing the marker of interest is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871569&req=5

fig3: DCs were transduced with nothing (No treated), CD80 lenti, CD86 lenti, NC lenti, or both CD80 lenti and CD86 lenti. (a) After 6 days of culture, the cells were then incubated with LPS (1 μg/mL) for 18 hours. Expression of CD80 and CD86 was determined by flow cytometry. The data show the effect of recombinant lentivirus on inhibition of target molecule expression. Values expressed as mean ± 1 SD from three independent experiments; *P < .05 compared to NC lenti/No treated; **P < .05 compared to CD80 lenti; ***P < .05 compared to CD86 lenti. (b) C3H BM-derived DCs were cultured over 6 days. DCs were stimulated or unstimulated with LPS (1 μg/mL) for 18 hours. Upregulation of CD80 and CD86 in response to LPS was also suppressed by CD80 lenti and CD86 lenti. The mean fluorescence intensity (MFI) of GFP+ cells expressing the marker of interest is indicated.
Mentions: To determine the lentiviral transduction efficiency in DCs, GFP expression was examined by microscopy and flow cytometry at different MOIs on day 4 after transduction. Lentivirus demonstrated a high (79.8% ± 5.0%) transduction efficiency in DCs at MOI of 20 (Figure 2). The percentage of GFP-expressing cells remained minor changes when MOI is >20. Therefore, in the following experiments all viral titers were at MOI of 20. To blockade both CD80 and CD86 expression, DCs were transduced with both CD80 lenti and CD86 lenti. DCs were collected after LPS stimulation. As measured by flow cytometry, we found that CD80 lenti and CD86 lenti together could enhance suppressive effect on CD80 and CD86 expression (Figure 3(a)). After the activation of LPS, untreated DCs underwent marked maturation progress. But as to those transduced DCs LPS failed to bring great changes of the CD80 and CD86 expression. On the other hand, CD80 lenti and CD86 lenti had no effect on expression of unrelated proteins (such as MHC-II and CD40) (Figure 3(b)). Our study indicates that recombinant lentivirus can provide highly efficient and specific CD80 and CD86 knockdown in DCs.

Bottom Line: This approach could specifically and effectively knock down CD80 and CD86 expression.We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group.Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China.

ABSTRACT
Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

Show MeSH
Related in: MedlinePlus