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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

The expression of IDOEGFP in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP at MOI of 100 for 60 h. (A) After 60 h of infection, the infection was monitored by EGFP expression under fluorescent microscopy. Original magnification 300×; (B) The efficiency of infection was determined by flow cytometry. After 60 h of infection, the cells were harvested and the numbers of EGFP positive cells were estimated by flow cytometry. Data are shown as mean ± SD, representative of three independent experiments; (C) Western blot analysis of IDOEGFP expression. After 60 h of infection, the noninfected (control) and infected cells were harvested and cell lysates from about 3 × 105 cells were fractionated by SDS-PAGE. IDOEGFP protein was detected using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000. EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MOI = multiplicity of infection; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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f1-ijms-13-10863: The expression of IDOEGFP in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP at MOI of 100 for 60 h. (A) After 60 h of infection, the infection was monitored by EGFP expression under fluorescent microscopy. Original magnification 300×; (B) The efficiency of infection was determined by flow cytometry. After 60 h of infection, the cells were harvested and the numbers of EGFP positive cells were estimated by flow cytometry. Data are shown as mean ± SD, representative of three independent experiments; (C) Western blot analysis of IDOEGFP expression. After 60 h of infection, the noninfected (control) and infected cells were harvested and cell lysates from about 3 × 105 cells were fractionated by SDS-PAGE. IDOEGFP protein was detected using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000. EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MOI = multiplicity of infection; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mentions: MT-2 cells were susceptible to the adenoviral infection. GFP expression could be visualized by fluorescent microscopy within 12 h of the addition of recombinant adenovirus (data not shown). The efficiency of infection was determined by fluorescent microscopy and flow cytometry (Figure 1A,B). As illustrated in Figure 1, more than 80% of MT-2 cells were infected by pAd-EGFP or pAd-IDOEGFP after 60 h of infection. To determine the IDOEGFP protein expression, MT-2 cells were infected with pAd-IDOEGFP at MOI of 100 for 60 h, then harvested and lysed with RIPA buffer and checked for IDOEGFP expression by Western blot using anti-GFP monoclonal antibody. As expected, EGFP (26-kDa) and IDOEGFP (68-kDa) protein bands were observed in cells infected with pAd-EGFP and pAd-IDOEGFP respectively, but not in non-infected cells (Figure 1C). To further confirm the expression of IDOEGFP fusion protein, the MT-2 cells infected with pAd-IDOEGFP were lysed and analyzed by Western blot with IDO polyclonal antibody and the results showed that a IDOEGFP protein band with a 68-kDa molecular weight was observed in these infected cells, but not in pAd-EGFP infected cells or non-infected cells (data not shown).


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)

The expression of IDOEGFP in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP at MOI of 100 for 60 h. (A) After 60 h of infection, the infection was monitored by EGFP expression under fluorescent microscopy. Original magnification 300×; (B) The efficiency of infection was determined by flow cytometry. After 60 h of infection, the cells were harvested and the numbers of EGFP positive cells were estimated by flow cytometry. Data are shown as mean ± SD, representative of three independent experiments; (C) Western blot analysis of IDOEGFP expression. After 60 h of infection, the noninfected (control) and infected cells were harvested and cell lysates from about 3 × 105 cells were fractionated by SDS-PAGE. IDOEGFP protein was detected using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000. EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MOI = multiplicity of infection; 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

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Show All Figures
getmorefigures.php?uid=PMC3472717&req=5

f1-ijms-13-10863: The expression of IDOEGFP in MT-2 cells. MT-2 cells were infected with either pAd-EGFP or pAd-IDOEGFP at MOI of 100 for 60 h. (A) After 60 h of infection, the infection was monitored by EGFP expression under fluorescent microscopy. Original magnification 300×; (B) The efficiency of infection was determined by flow cytometry. After 60 h of infection, the cells were harvested and the numbers of EGFP positive cells were estimated by flow cytometry. Data are shown as mean ± SD, representative of three independent experiments; (C) Western blot analysis of IDOEGFP expression. After 60 h of infection, the noninfected (control) and infected cells were harvested and cell lysates from about 3 × 105 cells were fractionated by SDS-PAGE. IDOEGFP protein was detected using purified mouse monoclonal anti-GFP antibody at a concentration of 1:1000. EGFP = enhanced green fluorescent protein; IDO = indoleamine 2,3-dioxygenase; MOI = multiplicity of infection; pAd-EGFP = recombinant adenovirus containing EGFP gene; pAd-IDOEGFP = recombinant adenovirus containing IDOEGFP gene; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Mentions: MT-2 cells were susceptible to the adenoviral infection. GFP expression could be visualized by fluorescent microscopy within 12 h of the addition of recombinant adenovirus (data not shown). The efficiency of infection was determined by fluorescent microscopy and flow cytometry (Figure 1A,B). As illustrated in Figure 1, more than 80% of MT-2 cells were infected by pAd-EGFP or pAd-IDOEGFP after 60 h of infection. To determine the IDOEGFP protein expression, MT-2 cells were infected with pAd-IDOEGFP at MOI of 100 for 60 h, then harvested and lysed with RIPA buffer and checked for IDOEGFP expression by Western blot using anti-GFP monoclonal antibody. As expected, EGFP (26-kDa) and IDOEGFP (68-kDa) protein bands were observed in cells infected with pAd-EGFP and pAd-IDOEGFP respectively, but not in non-infected cells (Figure 1C). To further confirm the expression of IDOEGFP fusion protein, the MT-2 cells infected with pAd-IDOEGFP were lysed and analyzed by Western blot with IDO polyclonal antibody and the results showed that a IDOEGFP protein band with a 68-kDa molecular weight was observed in these infected cells, but not in pAd-EGFP infected cells or non-infected cells (data not shown).

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