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An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis.

Hettmann T, DiDonato J, Karin M, Leiden JM - J. Exp. Med. (1999)

Bottom Line: However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals.The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor.Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Pathology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
To examine the role of nuclear factor (NF)-kappaB in T cell development and activation in vivo, we produced transgenic mice that express a superinhibitory mutant form of inhibitor kappaB-alpha (IkappaB-alphaA32/36) under the control of the T cell-specific CD2 promoter and enhancer (mutant [m]IkappaB-alpha mice). Thymocyte development proceeded normally in the mIkappaB-alpha mice. However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals. The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4(+)CD8(+); DP) thymocytes from the mIkappaB-alpha mice were resistant to alpha-CD3-mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by gamma-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mIkappaB-alpha thymocytes maintained high level expression of bcl-xL after alpha-CD3 treatment. Taken together, these results demonstrated important roles for NF-kappaB in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-kappaB is required for the alpha-CD3-mediated apoptosis of DP thymocytes through a pathway that involves the regulation of the antiapoptotic gene, bcl-xL.

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IκB-α and NF-κB expression in the mIκB-α transgenic  mice. (A) Western blot analysis of IκB-α expression in thymocytes from  mIκB-α transgenic mice after treatment with TNF-α. Thymocytes from  wild-type (WT) and mIκB-α transgenic (Tg) mice were incubated with  TNF-α (15 ng/ml) for the indicated times and cytoplasmic extracts were  separated by electrophoresis in a denaturing polyacrylamide gel. Proteins  were transferred to a PDVF membrane and immunoblotted with either a  murine HA-specific antibody (α-HA) or a rabbit polyclonal antibody specific for IκB-α (α-MAD) (13). The positions of the endogenous IκB-α  and IκB-αA32/36 transgene-encoded proteins are shown to the left of the  autoradiogram. Size markers in kilodaltons are shown to the right of the  autoradiogram. (B) EMSA of NF-κB expression in transgenic thymocytes  after stimulation in vivo with α-CD3 mAb. Thymic nuclear extracts (2 μg)  from mice treated for 3 h with a single intraperitoneal injection of α-CD3  mAb or PBS (control) were analyzed by EMSA with a radiolabeled κB  oligonucleotide probe. For antibody supershift experiments, thymic nuclear extracts were preincubated with antibodies specific for NF-κB p50,  p65, c-Rel, or RelB. For cold competition experiments, binding reactions contained a 50-fold molar excess of unlabeled competitor oligonucleotide. The positions of bands corresponding to binding of NF-κB p50  homodimers and p65/p50 as well as c-Rel/p50 and RelB/p50 heterodimers are indicated, as are “supershifted” complexes containing p50,  p65, c-Rel, and RelB proteins (•).
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Figure 2: IκB-α and NF-κB expression in the mIκB-α transgenic mice. (A) Western blot analysis of IκB-α expression in thymocytes from mIκB-α transgenic mice after treatment with TNF-α. Thymocytes from wild-type (WT) and mIκB-α transgenic (Tg) mice were incubated with TNF-α (15 ng/ml) for the indicated times and cytoplasmic extracts were separated by electrophoresis in a denaturing polyacrylamide gel. Proteins were transferred to a PDVF membrane and immunoblotted with either a murine HA-specific antibody (α-HA) or a rabbit polyclonal antibody specific for IκB-α (α-MAD) (13). The positions of the endogenous IκB-α and IκB-αA32/36 transgene-encoded proteins are shown to the left of the autoradiogram. Size markers in kilodaltons are shown to the right of the autoradiogram. (B) EMSA of NF-κB expression in transgenic thymocytes after stimulation in vivo with α-CD3 mAb. Thymic nuclear extracts (2 μg) from mice treated for 3 h with a single intraperitoneal injection of α-CD3 mAb or PBS (control) were analyzed by EMSA with a radiolabeled κB oligonucleotide probe. For antibody supershift experiments, thymic nuclear extracts were preincubated with antibodies specific for NF-κB p50, p65, c-Rel, or RelB. For cold competition experiments, binding reactions contained a 50-fold molar excess of unlabeled competitor oligonucleotide. The positions of bands corresponding to binding of NF-κB p50 homodimers and p65/p50 as well as c-Rel/p50 and RelB/p50 heterodimers are indicated, as are “supershifted” complexes containing p50, p65, c-Rel, and RelB proteins (•).

Mentions: Endogenous IκB-α is phosphorylated and degraded after treatment of T cells with the NF-κB inducer, TNF-α (47). To analyze the relative levels of endogenous and transgene-encoded IκB-α and to study the differential regulation of the two proteins in response to TNF-α, we performed Western blot analyses using whole cell extracts from mIκB-α and control thymocytes that had been treated for different times with TNF-α (Fig. 2 A). Basal levels of the IκB-αA32/36 protein as detected with both the α-HA and α-IκB-α (α-MAD) antibodies slightly exceeded levels of the endogenous IκB-α protein in the mIκB-α thymocytes. In both wild-type and mIκB-α thymocytes, endogenous IκB-α was almost completely degraded after 15 min of TNF-α treatment and remained undetectable until ∼30 min after treatment. As previously reported (9, 15), endogenous IκB-α was reexpressed between 30 and 60 min after TNF-α treatment (data not shown). This reexpression reflects NF-κB–mediated activation of the IκB-α promoter. In marked contrast, levels of the transgene-encoded IκB-αA32/36 were unchanged by TNF-α treatment at all time points (Fig. 2 A). Activation of mIκB-α thymocytes with PMA plus ionomycin or TCR cross-linking resulted in a similar degradation of endogenous IκB-α, whereas the levels of IκB-αA32/36 remain unaffected (data not shown).


An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis.

Hettmann T, DiDonato J, Karin M, Leiden JM - J. Exp. Med. (1999)

IκB-α and NF-κB expression in the mIκB-α transgenic  mice. (A) Western blot analysis of IκB-α expression in thymocytes from  mIκB-α transgenic mice after treatment with TNF-α. Thymocytes from  wild-type (WT) and mIκB-α transgenic (Tg) mice were incubated with  TNF-α (15 ng/ml) for the indicated times and cytoplasmic extracts were  separated by electrophoresis in a denaturing polyacrylamide gel. Proteins  were transferred to a PDVF membrane and immunoblotted with either a  murine HA-specific antibody (α-HA) or a rabbit polyclonal antibody specific for IκB-α (α-MAD) (13). The positions of the endogenous IκB-α  and IκB-αA32/36 transgene-encoded proteins are shown to the left of the  autoradiogram. Size markers in kilodaltons are shown to the right of the  autoradiogram. (B) EMSA of NF-κB expression in transgenic thymocytes  after stimulation in vivo with α-CD3 mAb. Thymic nuclear extracts (2 μg)  from mice treated for 3 h with a single intraperitoneal injection of α-CD3  mAb or PBS (control) were analyzed by EMSA with a radiolabeled κB  oligonucleotide probe. For antibody supershift experiments, thymic nuclear extracts were preincubated with antibodies specific for NF-κB p50,  p65, c-Rel, or RelB. For cold competition experiments, binding reactions contained a 50-fold molar excess of unlabeled competitor oligonucleotide. The positions of bands corresponding to binding of NF-κB p50  homodimers and p65/p50 as well as c-Rel/p50 and RelB/p50 heterodimers are indicated, as are “supershifted” complexes containing p50,  p65, c-Rel, and RelB proteins (•).
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Figure 2: IκB-α and NF-κB expression in the mIκB-α transgenic mice. (A) Western blot analysis of IκB-α expression in thymocytes from mIκB-α transgenic mice after treatment with TNF-α. Thymocytes from wild-type (WT) and mIκB-α transgenic (Tg) mice were incubated with TNF-α (15 ng/ml) for the indicated times and cytoplasmic extracts were separated by electrophoresis in a denaturing polyacrylamide gel. Proteins were transferred to a PDVF membrane and immunoblotted with either a murine HA-specific antibody (α-HA) or a rabbit polyclonal antibody specific for IκB-α (α-MAD) (13). The positions of the endogenous IκB-α and IκB-αA32/36 transgene-encoded proteins are shown to the left of the autoradiogram. Size markers in kilodaltons are shown to the right of the autoradiogram. (B) EMSA of NF-κB expression in transgenic thymocytes after stimulation in vivo with α-CD3 mAb. Thymic nuclear extracts (2 μg) from mice treated for 3 h with a single intraperitoneal injection of α-CD3 mAb or PBS (control) were analyzed by EMSA with a radiolabeled κB oligonucleotide probe. For antibody supershift experiments, thymic nuclear extracts were preincubated with antibodies specific for NF-κB p50, p65, c-Rel, or RelB. For cold competition experiments, binding reactions contained a 50-fold molar excess of unlabeled competitor oligonucleotide. The positions of bands corresponding to binding of NF-κB p50 homodimers and p65/p50 as well as c-Rel/p50 and RelB/p50 heterodimers are indicated, as are “supershifted” complexes containing p50, p65, c-Rel, and RelB proteins (•).
Mentions: Endogenous IκB-α is phosphorylated and degraded after treatment of T cells with the NF-κB inducer, TNF-α (47). To analyze the relative levels of endogenous and transgene-encoded IκB-α and to study the differential regulation of the two proteins in response to TNF-α, we performed Western blot analyses using whole cell extracts from mIκB-α and control thymocytes that had been treated for different times with TNF-α (Fig. 2 A). Basal levels of the IκB-αA32/36 protein as detected with both the α-HA and α-IκB-α (α-MAD) antibodies slightly exceeded levels of the endogenous IκB-α protein in the mIκB-α thymocytes. In both wild-type and mIκB-α thymocytes, endogenous IκB-α was almost completely degraded after 15 min of TNF-α treatment and remained undetectable until ∼30 min after treatment. As previously reported (9, 15), endogenous IκB-α was reexpressed between 30 and 60 min after TNF-α treatment (data not shown). This reexpression reflects NF-κB–mediated activation of the IκB-α promoter. In marked contrast, levels of the transgene-encoded IκB-αA32/36 were unchanged by TNF-α treatment at all time points (Fig. 2 A). Activation of mIκB-α thymocytes with PMA plus ionomycin or TCR cross-linking resulted in a similar degradation of endogenous IκB-α, whereas the levels of IκB-αA32/36 remain unaffected (data not shown).

Bottom Line: However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals.The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor.Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL.

View Article: PubMed Central - PubMed

Affiliation: Departments of Medicine and Pathology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
To examine the role of nuclear factor (NF)-kappaB in T cell development and activation in vivo, we produced transgenic mice that express a superinhibitory mutant form of inhibitor kappaB-alpha (IkappaB-alphaA32/36) under the control of the T cell-specific CD2 promoter and enhancer (mutant [m]IkappaB-alpha mice). Thymocyte development proceeded normally in the mIkappaB-alpha mice. However, the numbers of peripheral CD8(+) T cells were significantly reduced in these animals. The mIkappaB-alpha thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4(+)CD8(+); DP) thymocytes from the mIkappaB-alpha mice were resistant to alpha-CD3-mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by gamma-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of alpha-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mIkappaB-alpha thymocytes maintained high level expression of bcl-xL after alpha-CD3 treatment. Taken together, these results demonstrated important roles for NF-kappaB in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-kappaB is required for the alpha-CD3-mediated apoptosis of DP thymocytes through a pathway that involves the regulation of the antiapoptotic gene, bcl-xL.

Show MeSH
Related in: MedlinePlus