IkappaBalpha/IkappaBepsilon deficiency reveals that a critical NF-kappaB dosage is required for lymphocyte survival - PubMed (original) (raw)

IkappaBalpha/IkappaBepsilon deficiency reveals that a critical NF-kappaB dosage is required for lymphocyte survival

Bertrand Goudeau et al. Proc Natl Acad Sci U S A. 2003.

Abstract

In most cells, the NF-kappaB transcription factor is sequestered in the cytoplasm by interaction with inhibitory proteins, the IkappaBs. Here, we show that combined IkappaBalpha/IkappaBepsilon deficiency in mice leads to neonatal death, elevated kappaB binding activity, overexpression of NF-kappaB target genes, and disruption of lymphocyte production. In IkappaBalpha/IkappaBepsilon-deficient fetuses, B220+IgM+ B cells and single-positive T cells die by apoptosis. In adults, IkappaBalpha-/-IkappaBepsilon-/- reconstituted chimeras exhibit a nearly complete absence of T and B cells that is not rescued by cotransfer with wild-type bone marrow. These findings demonstrate that IkappaBs tightly control NF-kappaB activity in vivo and that increased NF-kappaB activity intrinsically impairs lymphocyte survival. Because reduction or rise of NF-kappaB activity leads to similar dysfunction, they also reveal that only a narrow window of NF-kappaB activity is tolerated by lymphocytes.

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Figures

Fig. 1.

Fig. 1.

Constitutive up-regulation of NF-κB DNA binding activity and overexpression of NF-κB target genes in IκBα/IκBε-nullizygous mice. (A)IκBα/IκBε status in total thymic protein extracts (120 μg) from E18.5 fetuses analyzed by Western blot. Genotypes are indicated; ns, nonspecific band. (B) Nuclear extracts (0.5 μg) from thymus of E18.5 fetuses analyzed by electrophoretic mobility-shift assay for binding to a canonical κB site. Arrows indicate the complexes visualized: I and II for the different Rel/NF-κB dimers; CSL was used as an internal control. (C) Expression of H-2Kb+d and I-A antigens on CD8+ thymocytes was assayed by two-color flow cytometry. An identical number of CD8+ cells was found in all genotypes. Percentages of positive events in the corresponding quadrant thus reflect the levels of expression of MHC antigens.

Fig. 2.

Fig. 2.

Impaired B cell development in IκBα/IκBε-nullizygous fetuses. Single-cell suspensions from thymus (A), liver (B), and spleen (C) of E18.5 fetuses genotyped as indicated were stained for CD4, CD8, IgM, B220, CD43, GR1, or Mac1 and then analyzed by flow cytometry. Gates are shown by boxes, and percentages of gated cells within the plot are specified above or inside the quadrant. For single-color staining, percentages of positive events are indicated in each graph. (D) Analysis of Ig heavy (D-JH) and light (V-Jκ) chain rearrangement status in E18.5 FL lymphocytes genotyped as indicated. No DNA corresponds to the analysis done in the absence of DNA as a negative control. TCR gene amplification was done in parallel on the same DNA preparations as an internal control.

Fig. 3.

Fig. 3.

In IκBα/IκBε-nullizygous fetuses, programmed cell death is a feature of immature B cells and SP thymocytes. (A) Spleen lymphocytes from E18.5 fetuses genotyped as indicated were stained for B220 and IgM, assayed for apoptosis by DioC6 staining, and analyzed by flow cytometry. Percentages of apoptotic cells within the B220+IgM- and B220+IgM+ populations are indicated in the histograms. Results are representative of at least six separate experiments. (B) Thymocytes from 2.5-day FTOC of E18.5 thymi were stained for CD4 and CD8 and analyzed by flow cytometry (Top). Gates are shown by boxes, and percentages of gated cells within the plot are specified above or inside the boxes. Cells gated as SP CD4+ (Middle) or CD8+ (Bottom) were further assayed for apoptosis by DioC6 staining. Percentages indicate the apoptotic cells. Results are representative of at least four separate experiments.

Fig. 4.

Fig. 4.

Impaired B cell survival of IκBα/IκBε-nullizygous FL cultures in vitro. Flow cytometry analysis of E15.5 FL cells cultured for 7 days and stained for B220 and IgM. Gates are shown by boxes, and percentages of gated cells of total cells within the plot are specified at the right of the quadrant. Data shown are examples of three independent experiments.

Fig. 5.

Fig. 5.

Extended lymphopenia in IκBα/IκBε-nullizygous chimeras. Single-cell suspensions from bone marrow (A), spleen (B), and thymus (D) isolated from RAG2/γc mice nonreconstituted (n.r.) or reconstituted with FL cells genotyped as indicated were stained for IgM, B220, CD43, GR1, Mac1, CD3, CD19, CD4, CD8, or TCRβ and analyzed by flow cytometry. Gates are shown by boxes, and percentages of gated cells within the plot are specified above or inside the quadrant. (C) Analysis of Ig heavy (D-JH) and light (V-Jκ) chain rearrangement status in nonreconstituted (n.r., lane 1), wild-type (lanes 2 and 3), IκBε (lanes 4 and 5), IκBα (lanes 6 and 7), and IκBα/IκBε-nullizygous (lanes 8-10) chimeric splenocytes. TCR gene amplification was done in parallel on the same DNA preparations as an internal control. Note the absence of Ig heavy and light chain gene rearrangement in the nonreconstituted host despite normal TCR gene amplification. (E) T and B cell absolute numbers in the spleen of RAG2/γc chimeras.*, P < 0.00023; **, P < 4 × 10-7; ***, P < 4 × 10-17.

Fig. 6.

Fig. 6.

T and B lymphopoiesis defect of IκBα/IκBε-nullizygous RAG2/γc reconstituted chimeras is cell intrinsic. Immunostaining of splenocytes from FL wild-type- or IκBα/IκBε-nullizygous- and Ly-5.1-coinjected mice. Cells were stained for specific markers of donor (Ly-5.2) macrophages (Mac1), B lymphocytes (CD19), and T lymphocytes (TCRβ) and analyzed by fluorescence-activated cell sorting. The percentage of gated cells of total cells within the plot are specified inside the corresponding quadrant. Genotypes above plots refer to that of donor Ly-5.2 FL cells.

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