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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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An NF-κB–mediated pathway downregulates the expression of bcl-xL after α-CD3 administration in vivo. (A) Expression of Bcl-2–related genes in thymocytes after administration of α-CD3  mAb. Wild-type (WT) or mIκB-α transgenic mice (IκB-αA32/36) were injected intraperitoneally with  200 μl of PBS containing 0 (−) or 40 μg of α-CD3 mAb and thymocyte RNA was prepared 24 or 48 h  after treatment. Expression of Bcl-2–related mRNAs was quantitated by RNAse protection assay. L32  is a control RNA that is present at equivalent levels in each of the samples assayed. Note the specific reduction in bcl-xL mRNA levels in the WT thymocytes that was prevented in the mIκB-α thymocytes.  (B) Western blot analysis of Bcl-xL expression in wild-type (WT) or mIκB-α (IκB-αA32/36) thymocytes  from mice injected intraperitoneally with 40 μg of α-CD3 mAb for the times shown. Equivalent levels  of expression of a slow mobility nonspecific control band demonstrate equal loading of the gel. (C and D) Expression of Bcl-xL in viable thymocytes from  wild-type (WT) or mIκB-α (IκB-αA32/36) transgenic mice 24 h after intraperitoneal injection with 40 μg of α-CD3 mAb (dotted lines) or an isotype-matched control antibody (Control Ab; solid lines). Viable thymocytes (as determined by forward and side scatter gating) were analyzed by flow cytometry with Cy-Chrome–α-CD4 and PE–α-CD8 (see inserts for FACS® profiles). Thymocytes were fixed, permeabilized, and stained with an FITC– α-Bcl-xL mAb. Levels of intracellular Bcl-xL in specific subpopulations of thymocytes were analyzed by gating on DP (C) and CD4+ SP cells (D). Note  the reduction in intracellular Bcl-xL levels seen in the wild-type DP thymocytes after α-CD3 treatment, which was observed neither in the mIκB-α DP  thymocytes nor in the wild-type or mIκB-α SP thymocytes from these same animals.
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Figure 9: An NF-κB–mediated pathway downregulates the expression of bcl-xL after α-CD3 administration in vivo. (A) Expression of Bcl-2–related genes in thymocytes after administration of α-CD3 mAb. Wild-type (WT) or mIκB-α transgenic mice (IκB-αA32/36) were injected intraperitoneally with 200 μl of PBS containing 0 (−) or 40 μg of α-CD3 mAb and thymocyte RNA was prepared 24 or 48 h after treatment. Expression of Bcl-2–related mRNAs was quantitated by RNAse protection assay. L32 is a control RNA that is present at equivalent levels in each of the samples assayed. Note the specific reduction in bcl-xL mRNA levels in the WT thymocytes that was prevented in the mIκB-α thymocytes. (B) Western blot analysis of Bcl-xL expression in wild-type (WT) or mIκB-α (IκB-αA32/36) thymocytes from mice injected intraperitoneally with 40 μg of α-CD3 mAb for the times shown. Equivalent levels of expression of a slow mobility nonspecific control band demonstrate equal loading of the gel. (C and D) Expression of Bcl-xL in viable thymocytes from wild-type (WT) or mIκB-α (IκB-αA32/36) transgenic mice 24 h after intraperitoneal injection with 40 μg of α-CD3 mAb (dotted lines) or an isotype-matched control antibody (Control Ab; solid lines). Viable thymocytes (as determined by forward and side scatter gating) were analyzed by flow cytometry with Cy-Chrome–α-CD4 and PE–α-CD8 (see inserts for FACS® profiles). Thymocytes were fixed, permeabilized, and stained with an FITC– α-Bcl-xL mAb. Levels of intracellular Bcl-xL in specific subpopulations of thymocytes were analyzed by gating on DP (C) and CD4+ SP cells (D). Note the reduction in intracellular Bcl-xL levels seen in the wild-type DP thymocytes after α-CD3 treatment, which was observed neither in the mIκB-α DP thymocytes nor in the wild-type or mIκB-α SP thymocytes from these same animals.

Mentions: The Bcl-2 family of proteins is comprised of multiple members that function either as death agonists (Bax, Bak, Bcl-XS, and Bad) or death antagonists (Bcl-2, Bcl-xL, Bcl-xγ, Mcl-1, A1, and Bcl-w) (65). Two antiapoptotic members of this family, Bcl-xL and Bcl-2, are expressed in a reciprocal pattern during thymocyte development. Bcl-2 is expressed at high levels in immature DN thymocytes. Its expression is downregulated in DP cells and it is then reexpressed as these DP cells mature to SP thymocytes and peripheral T cells (66, 67). Conversely, Bcl-xL is expressed at low or undetectable levels in immature DN cells. Its expression is significantly upregulated in DP thymocytes, and it is then downregulated as these cells progress to the SP stage of thymocyte ontogeny (68, 69). Interestingly, DP thymocytes from transgenic mice that overexpress Bcl-2 or Bcl-xL are protected from multiple proapoptotic stimuli, including α-CD3 treatment in vivo (69, 70). Given these findings, it was logical to postulate that altered expression of Bcl-2-family genes in the mIκB-α thymocytes might account for their decreased susceptibility to proapoptotic stimuli. Accordingly, we used an RNAse protection assay to directly monitor the expression of Bcl-2–related genes in thymocytes from wild-type and mIκB-α transgenic mice. As shown in Fig. 9 A, both basal and α-CD3–treated levels of bfl-1, bak, bax, bcl-2, and bad mRNAs were equivalent in wild-type and mIκB-α thymocytes. Similarly, we failed to detect differential expression of mRNAs encoding Fas, Fas-L, TRAF, TRADD, Fadd, RIP, and FLICE in either unstimulated or α-CD3–treated wild-type and mIκB-α thymocytes (data not shown). Basal levels of bcl-xL were equivalent in wild-type and mIκB-α thymocytes. However, after α-CD3 treatment, the expression of bcl-xL was significantly decreased in the wild-type cells but was maintained at pretreatment levels in the mIκB-α thymocytes (Fig. 9 A).


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)

An NF-κB–mediated pathway downregulates the expression of bcl-xL after α-CD3 administration in vivo. (A) Expression of Bcl-2–related genes in thymocytes after administration of α-CD3  mAb. Wild-type (WT) or mIκB-α transgenic mice (IκB-αA32/36) were injected intraperitoneally with  200 μl of PBS containing 0 (−) or 40 μg of α-CD3 mAb and thymocyte RNA was prepared 24 or 48 h  after treatment. Expression of Bcl-2–related mRNAs was quantitated by RNAse protection assay. L32  is a control RNA that is present at equivalent levels in each of the samples assayed. Note the specific reduction in bcl-xL mRNA levels in the WT thymocytes that was prevented in the mIκB-α thymocytes.  (B) Western blot analysis of Bcl-xL expression in wild-type (WT) or mIκB-α (IκB-αA32/36) thymocytes  from mice injected intraperitoneally with 40 μg of α-CD3 mAb for the times shown. Equivalent levels  of expression of a slow mobility nonspecific control band demonstrate equal loading of the gel. (C and D) Expression of Bcl-xL in viable thymocytes from  wild-type (WT) or mIκB-α (IκB-αA32/36) transgenic mice 24 h after intraperitoneal injection with 40 μg of α-CD3 mAb (dotted lines) or an isotype-matched control antibody (Control Ab; solid lines). Viable thymocytes (as determined by forward and side scatter gating) were analyzed by flow cytometry with Cy-Chrome–α-CD4 and PE–α-CD8 (see inserts for FACS® profiles). Thymocytes were fixed, permeabilized, and stained with an FITC– α-Bcl-xL mAb. Levels of intracellular Bcl-xL in specific subpopulations of thymocytes were analyzed by gating on DP (C) and CD4+ SP cells (D). Note  the reduction in intracellular Bcl-xL levels seen in the wild-type DP thymocytes after α-CD3 treatment, which was observed neither in the mIκB-α DP  thymocytes nor in the wild-type or mIκB-α SP thymocytes from these same animals.
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Figure 9: An NF-κB–mediated pathway downregulates the expression of bcl-xL after α-CD3 administration in vivo. (A) Expression of Bcl-2–related genes in thymocytes after administration of α-CD3 mAb. Wild-type (WT) or mIκB-α transgenic mice (IκB-αA32/36) were injected intraperitoneally with 200 μl of PBS containing 0 (−) or 40 μg of α-CD3 mAb and thymocyte RNA was prepared 24 or 48 h after treatment. Expression of Bcl-2–related mRNAs was quantitated by RNAse protection assay. L32 is a control RNA that is present at equivalent levels in each of the samples assayed. Note the specific reduction in bcl-xL mRNA levels in the WT thymocytes that was prevented in the mIκB-α thymocytes. (B) Western blot analysis of Bcl-xL expression in wild-type (WT) or mIκB-α (IκB-αA32/36) thymocytes from mice injected intraperitoneally with 40 μg of α-CD3 mAb for the times shown. Equivalent levels of expression of a slow mobility nonspecific control band demonstrate equal loading of the gel. (C and D) Expression of Bcl-xL in viable thymocytes from wild-type (WT) or mIκB-α (IκB-αA32/36) transgenic mice 24 h after intraperitoneal injection with 40 μg of α-CD3 mAb (dotted lines) or an isotype-matched control antibody (Control Ab; solid lines). Viable thymocytes (as determined by forward and side scatter gating) were analyzed by flow cytometry with Cy-Chrome–α-CD4 and PE–α-CD8 (see inserts for FACS® profiles). Thymocytes were fixed, permeabilized, and stained with an FITC– α-Bcl-xL mAb. Levels of intracellular Bcl-xL in specific subpopulations of thymocytes were analyzed by gating on DP (C) and CD4+ SP cells (D). Note the reduction in intracellular Bcl-xL levels seen in the wild-type DP thymocytes after α-CD3 treatment, which was observed neither in the mIκB-α DP thymocytes nor in the wild-type or mIκB-α SP thymocytes from these same animals.
Mentions: The Bcl-2 family of proteins is comprised of multiple members that function either as death agonists (Bax, Bak, Bcl-XS, and Bad) or death antagonists (Bcl-2, Bcl-xL, Bcl-xγ, Mcl-1, A1, and Bcl-w) (65). Two antiapoptotic members of this family, Bcl-xL and Bcl-2, are expressed in a reciprocal pattern during thymocyte development. Bcl-2 is expressed at high levels in immature DN thymocytes. Its expression is downregulated in DP cells and it is then reexpressed as these DP cells mature to SP thymocytes and peripheral T cells (66, 67). Conversely, Bcl-xL is expressed at low or undetectable levels in immature DN cells. Its expression is significantly upregulated in DP thymocytes, and it is then downregulated as these cells progress to the SP stage of thymocyte ontogeny (68, 69). Interestingly, DP thymocytes from transgenic mice that overexpress Bcl-2 or Bcl-xL are protected from multiple proapoptotic stimuli, including α-CD3 treatment in vivo (69, 70). Given these findings, it was logical to postulate that altered expression of Bcl-2-family genes in the mIκB-α thymocytes might account for their decreased susceptibility to proapoptotic stimuli. Accordingly, we used an RNAse protection assay to directly monitor the expression of Bcl-2–related genes in thymocytes from wild-type and mIκB-α transgenic mice. As shown in Fig. 9 A, both basal and α-CD3–treated levels of bfl-1, bak, bax, bcl-2, and bad mRNAs were equivalent in wild-type and mIκB-α thymocytes. Similarly, we failed to detect differential expression of mRNAs encoding Fas, Fas-L, TRAF, TRADD, Fadd, RIP, and FLICE in either unstimulated or α-CD3–treated wild-type and mIκB-α thymocytes (data not shown). Basal levels of bcl-xL were equivalent in wild-type and mIκB-α thymocytes. However, after α-CD3 treatment, the expression of bcl-xL was significantly decreased in the wild-type cells but was maintained at pretreatment levels in the mIκB-α thymocytes (Fig. 9 A).

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