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NIK-dependent RelB activation defines a unique signaling pathway for the development of V alpha 14i NKT cells.

Elewaut D, Shaikh RB, Hammond KJ, De Winter H, Leishman AJ, Sidobre S, Turovskaya O, Prigozy TI, Ma L, Banks TA, Lo D, Ware CF, Cheroutre H, Kronenberg M - J. Exp. Med. (2003)

Bottom Line: RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells.In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors.These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA.

ABSTRACT
A defect in RelB, a member of the Rel/nuclear factor (NF)-kappa B family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-kappa B-inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/- x aly/+ compound heterozygous mice. After stimulation with alpha-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/- or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

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LTβR mediated activation of RelB through NIK. (A) ICAM-1 induction by LTβR stimulation. Fibroblasts from the kidneys of RelB+/+ or RelB−/− mice were stimulated with an agonistic α-LTβR mAb (2 μg/ml) for 24 h. Cell surface levels of ICAM-1 were determined by flow cytometry. Histograms representing ICAM-1 levels before (thin line) and after stimulation (bold line) are shown. One representative example of three independent experiments is shown. (B) NF-κB/Rel binding activities in wild-type and aly/aly mice after LTβR ligation. MEFs from C57BL/6 and aly/aly mice were stimulated with an α-LTβR mAb (2 μg/ml) for 8 h. Nuclear extracts were prepared from unstimulated and LTβR triggered cells. Extracts were incubated with a palindromic κB-binding site as described in Materials and Methods. The results from addition of specific anti-sera against RelA (p65) and RelB are indicated at the bottom. One representative experiment of three is shown.
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fig5: LTβR mediated activation of RelB through NIK. (A) ICAM-1 induction by LTβR stimulation. Fibroblasts from the kidneys of RelB+/+ or RelB−/− mice were stimulated with an agonistic α-LTβR mAb (2 μg/ml) for 24 h. Cell surface levels of ICAM-1 were determined by flow cytometry. Histograms representing ICAM-1 levels before (thin line) and after stimulation (bold line) are shown. One representative example of three independent experiments is shown. (B) NF-κB/Rel binding activities in wild-type and aly/aly mice after LTβR ligation. MEFs from C57BL/6 and aly/aly mice were stimulated with an α-LTβR mAb (2 μg/ml) for 8 h. Nuclear extracts were prepared from unstimulated and LTβR triggered cells. Extracts were incubated with a palindromic κB-binding site as described in Materials and Methods. The results from addition of specific anti-sera against RelA (p65) and RelB are indicated at the bottom. One representative experiment of three is shown.

Mentions: In addition to the Vα14i NKT cell defect, NIK and RelB mutant mice share defects in secondary lymphoid organ development (22). These observations suggested that RelB may act downstream of NIK to govern the development of Vα14i NKT cells as well as secondary lymphoid organs, although NIK also can activate other NF-κB family transcription factors (39). We therefore performed experiments in vitro to provide evidence for NIK activation of RelB. Adhesion molecules are induced on primary fibroblasts after LTβR stimulation with an agonistic antibody (28). This induction is defective, however, in NIK mutant aly/aly fibroblasts (28). To determine if RelB might act downstream of NIK, we analyzed the induction of adhesion molecules on primary fibroblasts from wild-type and RelB mutant mice after LTβR stimulation. Interestingly, similar to aly/aly mice, up-regulation of both ICAM-1 (Fig. 5 A) and VCAM-1 (unpublished data) were absent in RelB−/− fibroblasts after stimulation with an agonistic anti-LTβR mAb. Upon LTβR triggering, however, RelB−/− fibroblasts displayed a similar degree of IκBα degradation as RelB+/+ cells (unpublished data). To directly assess RelB activation, NF-κB DNA-binding activity was determined by EMSA using a consensus NF-κB DNA binding sequence as a probe. Stimulation of mouse embryonic fibroblasts (MEFs) from wild-type or aly/aly mice with an agonistic LTβR antibody resulted in a profound activation of NF-κB (Fig. 5 B), which lasted for more than 8 h. To determine the identity of the NF-κB containing complexes, supershift assays were performed. These assays revealed that RelB and p65 containing complexes were present in wild-type MEFs after LTβR signaling (Fig. 5 B). By contrast, in aly/aly MEFs, LTβR stimulation did not result in RelB activation. Taken together, these data unambiguously demonstrate that NIK is essential for RelB activation after LTβR stimulation in fibroblasts, but not for the activation of complexes containing RelA (p65).


NIK-dependent RelB activation defines a unique signaling pathway for the development of V alpha 14i NKT cells.

Elewaut D, Shaikh RB, Hammond KJ, De Winter H, Leishman AJ, Sidobre S, Turovskaya O, Prigozy TI, Ma L, Banks TA, Lo D, Ware CF, Cheroutre H, Kronenberg M - J. Exp. Med. (2003)

LTβR mediated activation of RelB through NIK. (A) ICAM-1 induction by LTβR stimulation. Fibroblasts from the kidneys of RelB+/+ or RelB−/− mice were stimulated with an agonistic α-LTβR mAb (2 μg/ml) for 24 h. Cell surface levels of ICAM-1 were determined by flow cytometry. Histograms representing ICAM-1 levels before (thin line) and after stimulation (bold line) are shown. One representative example of three independent experiments is shown. (B) NF-κB/Rel binding activities in wild-type and aly/aly mice after LTβR ligation. MEFs from C57BL/6 and aly/aly mice were stimulated with an α-LTβR mAb (2 μg/ml) for 8 h. Nuclear extracts were prepared from unstimulated and LTβR triggered cells. Extracts were incubated with a palindromic κB-binding site as described in Materials and Methods. The results from addition of specific anti-sera against RelA (p65) and RelB are indicated at the bottom. One representative experiment of three is shown.
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Related In: Results  -  Collection

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fig5: LTβR mediated activation of RelB through NIK. (A) ICAM-1 induction by LTβR stimulation. Fibroblasts from the kidneys of RelB+/+ or RelB−/− mice were stimulated with an agonistic α-LTβR mAb (2 μg/ml) for 24 h. Cell surface levels of ICAM-1 were determined by flow cytometry. Histograms representing ICAM-1 levels before (thin line) and after stimulation (bold line) are shown. One representative example of three independent experiments is shown. (B) NF-κB/Rel binding activities in wild-type and aly/aly mice after LTβR ligation. MEFs from C57BL/6 and aly/aly mice were stimulated with an α-LTβR mAb (2 μg/ml) for 8 h. Nuclear extracts were prepared from unstimulated and LTβR triggered cells. Extracts were incubated with a palindromic κB-binding site as described in Materials and Methods. The results from addition of specific anti-sera against RelA (p65) and RelB are indicated at the bottom. One representative experiment of three is shown.
Mentions: In addition to the Vα14i NKT cell defect, NIK and RelB mutant mice share defects in secondary lymphoid organ development (22). These observations suggested that RelB may act downstream of NIK to govern the development of Vα14i NKT cells as well as secondary lymphoid organs, although NIK also can activate other NF-κB family transcription factors (39). We therefore performed experiments in vitro to provide evidence for NIK activation of RelB. Adhesion molecules are induced on primary fibroblasts after LTβR stimulation with an agonistic antibody (28). This induction is defective, however, in NIK mutant aly/aly fibroblasts (28). To determine if RelB might act downstream of NIK, we analyzed the induction of adhesion molecules on primary fibroblasts from wild-type and RelB mutant mice after LTβR stimulation. Interestingly, similar to aly/aly mice, up-regulation of both ICAM-1 (Fig. 5 A) and VCAM-1 (unpublished data) were absent in RelB−/− fibroblasts after stimulation with an agonistic anti-LTβR mAb. Upon LTβR triggering, however, RelB−/− fibroblasts displayed a similar degree of IκBα degradation as RelB+/+ cells (unpublished data). To directly assess RelB activation, NF-κB DNA-binding activity was determined by EMSA using a consensus NF-κB DNA binding sequence as a probe. Stimulation of mouse embryonic fibroblasts (MEFs) from wild-type or aly/aly mice with an agonistic LTβR antibody resulted in a profound activation of NF-κB (Fig. 5 B), which lasted for more than 8 h. To determine the identity of the NF-κB containing complexes, supershift assays were performed. These assays revealed that RelB and p65 containing complexes were present in wild-type MEFs after LTβR signaling (Fig. 5 B). By contrast, in aly/aly MEFs, LTβR stimulation did not result in RelB activation. Taken together, these data unambiguously demonstrate that NIK is essential for RelB activation after LTβR stimulation in fibroblasts, but not for the activation of complexes containing RelA (p65).

Bottom Line: RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells.In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors.These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA.

ABSTRACT
A defect in RelB, a member of the Rel/nuclear factor (NF)-kappa B family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-kappa B-inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/- x aly/+ compound heterozygous mice. After stimulation with alpha-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/- or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

Show MeSH