Limits...
Indoleamine 2,3-dioxygenase (IDO) downregulates the cell surface expression of the CD4 molecule.

Huang G, Zeng Y, Liang P, Zhou C, Zhao S, Huang X, Wu L, He X - Int J Mol Sci (2012)

Bottom Line: The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP.Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells.Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Medicine Center, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China; E-Mails: zhcr1234@126.com (C.Z.); zhaosy68@126.com (S.Z.) ; Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China; E-Mails: tzengyy@jnu.edu.cn (Y.Z.); huangxiuyan@gmail.com (X.H.); thexh@jnu.edu.cn (X.H.).

ABSTRACT
Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

No MeSH data available.


Related in: MedlinePlus

Addition of tryptophan and IDO inhibitor partially restored IDO induced downregulation of CD4 expression. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP and added with either 200 μM tryptophan or 800 μM 1-MT at the time of infection. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A,E) and the cell surface CD4 expression (B,C,F,G) in untreated, pAd-EGFP or pAd-IDOEGFP infected C8166 cells added with tryptophan or 1-MT or without. Pane A and pane B illustrate the results from two-color (PE and GFP) channel analysis, whereas pane C depicts the quantitative analysis of CD4 expressing levels in pAd-EGFP infected C8166 cells added with or without tryptophan or 1-MT. Pane E and pane F illustrate the results from two-color (PE and GFP) channel analysis, whereas pane G depicts the quantitative analysis of CD4 expressing levels in pAd-IDOEGFP infected C8166 cells added with or without tryptophan or 1-MT. Data are shown as mean ± SD, representative of three similar experiments. (D,H) The expression of cell surface CD4 was carried out by Western blot analysis. The plasmatic membrane proteins prepared from C8166 cells infected with either pAd-EGFP or pAd-IDOEGFP and added with tryptophan or 1-MT or without for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto PVDF membrane. CD4 was detected using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL in pAd-EGFP (D, upper line) or pAd-IDOEGFP (H, upper line) infected C8166 cells. In parallel, the cell lysate (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. EGFP or IDOEGFP protein was detected by using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000 and β-actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300 in pAd-EGFP (pane D, middle and low lines) or pAd-IDOEGFP (H, middle and low lines) infected C8166 cells. ★p < 0.05, compared with pAd-IDOEGFP. Abbreviations: EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MFI = mean fluorescence intensity; 1-MT = 1-methyl-dl-tryptophan; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3472717&req=5

f3-ijms-13-10863: Addition of tryptophan and IDO inhibitor partially restored IDO induced downregulation of CD4 expression. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP and added with either 200 μM tryptophan or 800 μM 1-MT at the time of infection. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A,E) and the cell surface CD4 expression (B,C,F,G) in untreated, pAd-EGFP or pAd-IDOEGFP infected C8166 cells added with tryptophan or 1-MT or without. Pane A and pane B illustrate the results from two-color (PE and GFP) channel analysis, whereas pane C depicts the quantitative analysis of CD4 expressing levels in pAd-EGFP infected C8166 cells added with or without tryptophan or 1-MT. Pane E and pane F illustrate the results from two-color (PE and GFP) channel analysis, whereas pane G depicts the quantitative analysis of CD4 expressing levels in pAd-IDOEGFP infected C8166 cells added with or without tryptophan or 1-MT. Data are shown as mean ± SD, representative of three similar experiments. (D,H) The expression of cell surface CD4 was carried out by Western blot analysis. The plasmatic membrane proteins prepared from C8166 cells infected with either pAd-EGFP or pAd-IDOEGFP and added with tryptophan or 1-MT or without for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto PVDF membrane. CD4 was detected using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL in pAd-EGFP (D, upper line) or pAd-IDOEGFP (H, upper line) infected C8166 cells. In parallel, the cell lysate (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. EGFP or IDOEGFP protein was detected by using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000 and β-actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300 in pAd-EGFP (pane D, middle and low lines) or pAd-IDOEGFP (H, middle and low lines) infected C8166 cells. ★p < 0.05, compared with pAd-IDOEGFP. Abbreviations: EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MFI = mean fluorescence intensity; 1-MT = 1-methyl-dl-tryptophan; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mentions: To test the possible mechanism through which IDO mediates the downregulation of CD4 expression on the cell surface, l-tryptophan or IDO inhibitor 1-MT was added to C8166 cells including non-infected, pAd-EGFP and pAd-IDOEGFP infected cells at the time of infection, and at 60 h post infection, the cell surface CD4 molecule was evaluated by flow cytometry and Western blot. Addition of l-tryptophan at a concentration of 200 μM or 1-MT at a concentration of 800 μM to RPMI 1640 medium (containing 10% FBS) had no effect on the expression of CD4 in infected C8166 cells (data not shown) or pAd-EGFP infected C8166 cells (Figure 3B–D); however, the addition of tryptophan markedly increased the expression of CD4 molecule and partially restored the level of CD4 expression in pAd-IDOEGFP infected C8166 cells (MFI of 92.4 ± 8.1 vs. 70.4 ± 10.1, p < 0.05, compared with pAd-IDOEGFP infected C8166 cells, Figure 3F–H). Meanwhile, the addition of 1-MT was also able to partially restore CD4 expression in pAd-IDOEGFP infected C8166 cells (MFI of 93.3 ± 7.6 vs. 70.4 ± 10.1, p < 0.05, compared with pAd-IDOEGFP infected C8166 cells, Figure 3F–H). This partial restoration of CD4 expression in IDO infected C8166 cells by addition of tryptophan or IDO inhibitor strongly suggested that the depletion of tryptophan is involved in IDO induced downregulation of CD4 expression.


Indoleamine 2,3-dioxygenase (IDO) downregulates the cell surface expression of the CD4 molecule.

Huang G, Zeng Y, Liang P, Zhou C, Zhao S, Huang X, Wu L, He X - Int J Mol Sci (2012)

Addition of tryptophan and IDO inhibitor partially restored IDO induced downregulation of CD4 expression. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP and added with either 200 μM tryptophan or 800 μM 1-MT at the time of infection. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A,E) and the cell surface CD4 expression (B,C,F,G) in untreated, pAd-EGFP or pAd-IDOEGFP infected C8166 cells added with tryptophan or 1-MT or without. Pane A and pane B illustrate the results from two-color (PE and GFP) channel analysis, whereas pane C depicts the quantitative analysis of CD4 expressing levels in pAd-EGFP infected C8166 cells added with or without tryptophan or 1-MT. Pane E and pane F illustrate the results from two-color (PE and GFP) channel analysis, whereas pane G depicts the quantitative analysis of CD4 expressing levels in pAd-IDOEGFP infected C8166 cells added with or without tryptophan or 1-MT. Data are shown as mean ± SD, representative of three similar experiments. (D,H) The expression of cell surface CD4 was carried out by Western blot analysis. The plasmatic membrane proteins prepared from C8166 cells infected with either pAd-EGFP or pAd-IDOEGFP and added with tryptophan or 1-MT or without for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto PVDF membrane. CD4 was detected using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL in pAd-EGFP (D, upper line) or pAd-IDOEGFP (H, upper line) infected C8166 cells. In parallel, the cell lysate (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. EGFP or IDOEGFP protein was detected by using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000 and β-actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300 in pAd-EGFP (pane D, middle and low lines) or pAd-IDOEGFP (H, middle and low lines) infected C8166 cells. ★p < 0.05, compared with pAd-IDOEGFP. Abbreviations: EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MFI = mean fluorescence intensity; 1-MT = 1-methyl-dl-tryptophan; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472717&req=5

f3-ijms-13-10863: Addition of tryptophan and IDO inhibitor partially restored IDO induced downregulation of CD4 expression. C8166 cells were infected with either pAd-EGFP or pAd-IDOEGFP and added with either 200 μM tryptophan or 800 μM 1-MT at the time of infection. At 60 h post infection, the cells were harvested and stained with PE-conjugated anti-CD4 monoclonal antibody. FACS analysis was used to determine the efficiency of infection (A,E) and the cell surface CD4 expression (B,C,F,G) in untreated, pAd-EGFP or pAd-IDOEGFP infected C8166 cells added with tryptophan or 1-MT or without. Pane A and pane B illustrate the results from two-color (PE and GFP) channel analysis, whereas pane C depicts the quantitative analysis of CD4 expressing levels in pAd-EGFP infected C8166 cells added with or without tryptophan or 1-MT. Pane E and pane F illustrate the results from two-color (PE and GFP) channel analysis, whereas pane G depicts the quantitative analysis of CD4 expressing levels in pAd-IDOEGFP infected C8166 cells added with or without tryptophan or 1-MT. Data are shown as mean ± SD, representative of three similar experiments. (D,H) The expression of cell surface CD4 was carried out by Western blot analysis. The plasmatic membrane proteins prepared from C8166 cells infected with either pAd-EGFP or pAd-IDOEGFP and added with tryptophan or 1-MT or without for 60 h, were fractionated by SDS electrophoresis on 8% acrylamide gel and electrotransferred onto PVDF membrane. CD4 was detected using mouse monoclonal anti-human CD4 antibody at a concentration of 1 μg/mL in pAd-EGFP (D, upper line) or pAd-IDOEGFP (H, upper line) infected C8166 cells. In parallel, the cell lysate (with RIPA buffer) from the same amount of these cells were fractionated by SDS-PAGE. EGFP or IDOEGFP protein was detected by using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000 and β-actin was detected using rabbit polyclonal anti-β-actin antibody at a concentration of 1:300 in pAd-EGFP (pane D, middle and low lines) or pAd-IDOEGFP (H, middle and low lines) infected C8166 cells. ★p < 0.05, compared with pAd-IDOEGFP. Abbreviations: EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MFI = mean fluorescence intensity; 1-MT = 1-methyl-dl-tryptophan; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Mentions: To test the possible mechanism through which IDO mediates the downregulation of CD4 expression on the cell surface, l-tryptophan or IDO inhibitor 1-MT was added to C8166 cells including non-infected, pAd-EGFP and pAd-IDOEGFP infected cells at the time of infection, and at 60 h post infection, the cell surface CD4 molecule was evaluated by flow cytometry and Western blot. Addition of l-tryptophan at a concentration of 200 μM or 1-MT at a concentration of 800 μM to RPMI 1640 medium (containing 10% FBS) had no effect on the expression of CD4 in infected C8166 cells (data not shown) or pAd-EGFP infected C8166 cells (Figure 3B–D); however, the addition of tryptophan markedly increased the expression of CD4 molecule and partially restored the level of CD4 expression in pAd-IDOEGFP infected C8166 cells (MFI of 92.4 ± 8.1 vs. 70.4 ± 10.1, p < 0.05, compared with pAd-IDOEGFP infected C8166 cells, Figure 3F–H). Meanwhile, the addition of 1-MT was also able to partially restore CD4 expression in pAd-IDOEGFP infected C8166 cells (MFI of 93.3 ± 7.6 vs. 70.4 ± 10.1, p < 0.05, compared with pAd-IDOEGFP infected C8166 cells, Figure 3F–H). This partial restoration of CD4 expression in IDO infected C8166 cells by addition of tryptophan or IDO inhibitor strongly suggested that the depletion of tryptophan is involved in IDO induced downregulation of CD4 expression.

Bottom Line: The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP.Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells.Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

View Article: PubMed Central - PubMed

Affiliation: Reproductive Medicine Center, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China; E-Mails: zhcr1234@126.com (C.Z.); zhaosy68@126.com (S.Z.) ; Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China; E-Mails: tzengyy@jnu.edu.cn (Y.Z.); huangxiuyan@gmail.com (X.H.); thexh@jnu.edu.cn (X.H.).

ABSTRACT
Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses.

No MeSH data available.


Related in: MedlinePlus