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NF-kappa B RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses.

Doi TS, Takahashi T, Taguchi O, Azuma T, Obata Y - J. Exp. Med. (1997)

Bottom Line: However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells.Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3 + anti-CD28, LPS, anti-IgM, and PMA + calcium ionophore.The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan.

ABSTRACT
To investigate the function of NF-kappa B RelA (p65), we generated mice deficient in this NF-kappa B family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA-/- fetal liver transplants, similar to the relA+/+ and +/- cases. T cells were found to mature to Thy-1+/TCR alpha beta +/CD3+/CD4+ or CD8+, while B cells had the ability to differentiate to IgM+/B220+ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3 + anti-CD28, LPS, anti-IgM, and PMA + calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.

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Liver degeneration and the absence of relA transcripts and  RelA protein in relA−/− embryos. (A) Histological features of livers of  ED14.5 relA+/+ and relA−/− embryos. In the liver of a relA+/+ embryo, hepatocytes with large cell size and light nuclear staining are mixed  with the hematopoietic cells with dark nuclear staining. In the liver of  relA−/− embryos, hepatocytes are disintegrated, while the hematopoietic cells are apparently normal. Magnification is 240-fold. (B) RNA blot  analysis of ED13.5 relA+/+, +/−, and −/− embryos. 10 μg of total  RNA were loaded per lane and analyzed with the relA, c-rel, relB, and p50  cDNA probes. relA transcripts were present in the relA+/+ and +/−  embryos, but not in the relA−/− embryos. The relA+/+, +/− and −/−  embryos expressed similar amounts of c-rel and p50 transcripts. No relB  transcripts were detected in ED13.5 embryos (data not shown). (C) RelA  protein in ED13.5 relA+/+ and relA−/− embryos. With rabbit antiRelA antibody and the ABC method, RelA protein is ubiquitously detected in the relA+/+ embryo, but is completely absent in the relA−/−  embryo. Magnification is eightfold.
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Figure 2: Liver degeneration and the absence of relA transcripts and RelA protein in relA−/− embryos. (A) Histological features of livers of ED14.5 relA+/+ and relA−/− embryos. In the liver of a relA+/+ embryo, hepatocytes with large cell size and light nuclear staining are mixed with the hematopoietic cells with dark nuclear staining. In the liver of relA−/− embryos, hepatocytes are disintegrated, while the hematopoietic cells are apparently normal. Magnification is 240-fold. (B) RNA blot analysis of ED13.5 relA+/+, +/−, and −/− embryos. 10 μg of total RNA were loaded per lane and analyzed with the relA, c-rel, relB, and p50 cDNA probes. relA transcripts were present in the relA+/+ and +/− embryos, but not in the relA−/− embryos. The relA+/+, +/− and −/− embryos expressed similar amounts of c-rel and p50 transcripts. No relB transcripts were detected in ED13.5 embryos (data not shown). (C) RelA protein in ED13.5 relA+/+ and relA−/− embryos. With rabbit antiRelA antibody and the ABC method, RelA protein is ubiquitously detected in the relA+/+ embryo, but is completely absent in the relA−/− embryo. Magnification is eightfold.

Mentions: Mice heterozygous for a disrupted relA were normal and fertile, but no homozygous relA-deficient mice were born from heterozygote mating. Sequential DNA blot analysis and histological examination of embryos from timed matings of heterozygous mice were conducted. Until embryonic day (ED) 13.5, all embryos were apparently normal and homozygous mutants (−/−) were present in an expected ratio. On ED14.5, the relA−/− embryos were still present but some showed signs of abnormalities in their liver (Fig. 2 A). On ED15.5, a portion of the embryos became necrotic and were typed homozygous mutant (−/−), while normal embryos were all wild type (+/+) or heterozygous (+/−). RNA blot analysis of relA+/+ embryos from ED8.5 until birth as well as CCE ES cells showed the presence of relA transcripts, while no relA transcripts could be detected in relA−/− embryos at any stage (Fig. 2 B). Positive immunostaining with anti-RelA antibody correlated with the presence of RNA transcripts, showing that RelA proteins were present in almost all tissues of ED13.5 relA+/+ embryos, while no staining of relA−/− embryos (Fig. 2 C). Although a different vector construct was used in our study, the generated RelA-deficient mice showed the same phenotype as those of Beg et al. (11), indicating that RelA is essential for embryonic development of the mouse. In contrast to RelA-deficient mice, mice lacking other NF-κB proteins are known to develop normally at least until birth (12–14). The difference may simply reflect the fact that RelA is expressed ubiquitously from an early stage of development while the others are expressed in restricted tissues from a much later stage (34, 35). Identification of RelA responsive genes in developing embryos, especially in the liver, should open new avenues for elucidation of the function of NF-κB in embryonic development.


NF-kappa B RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses.

Doi TS, Takahashi T, Taguchi O, Azuma T, Obata Y - J. Exp. Med. (1997)

Liver degeneration and the absence of relA transcripts and  RelA protein in relA−/− embryos. (A) Histological features of livers of  ED14.5 relA+/+ and relA−/− embryos. In the liver of a relA+/+ embryo, hepatocytes with large cell size and light nuclear staining are mixed  with the hematopoietic cells with dark nuclear staining. In the liver of  relA−/− embryos, hepatocytes are disintegrated, while the hematopoietic cells are apparently normal. Magnification is 240-fold. (B) RNA blot  analysis of ED13.5 relA+/+, +/−, and −/− embryos. 10 μg of total  RNA were loaded per lane and analyzed with the relA, c-rel, relB, and p50  cDNA probes. relA transcripts were present in the relA+/+ and +/−  embryos, but not in the relA−/− embryos. The relA+/+, +/− and −/−  embryos expressed similar amounts of c-rel and p50 transcripts. No relB  transcripts were detected in ED13.5 embryos (data not shown). (C) RelA  protein in ED13.5 relA+/+ and relA−/− embryos. With rabbit antiRelA antibody and the ABC method, RelA protein is ubiquitously detected in the relA+/+ embryo, but is completely absent in the relA−/−  embryo. Magnification is eightfold.
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Figure 2: Liver degeneration and the absence of relA transcripts and RelA protein in relA−/− embryos. (A) Histological features of livers of ED14.5 relA+/+ and relA−/− embryos. In the liver of a relA+/+ embryo, hepatocytes with large cell size and light nuclear staining are mixed with the hematopoietic cells with dark nuclear staining. In the liver of relA−/− embryos, hepatocytes are disintegrated, while the hematopoietic cells are apparently normal. Magnification is 240-fold. (B) RNA blot analysis of ED13.5 relA+/+, +/−, and −/− embryos. 10 μg of total RNA were loaded per lane and analyzed with the relA, c-rel, relB, and p50 cDNA probes. relA transcripts were present in the relA+/+ and +/− embryos, but not in the relA−/− embryos. The relA+/+, +/− and −/− embryos expressed similar amounts of c-rel and p50 transcripts. No relB transcripts were detected in ED13.5 embryos (data not shown). (C) RelA protein in ED13.5 relA+/+ and relA−/− embryos. With rabbit antiRelA antibody and the ABC method, RelA protein is ubiquitously detected in the relA+/+ embryo, but is completely absent in the relA−/− embryo. Magnification is eightfold.
Mentions: Mice heterozygous for a disrupted relA were normal and fertile, but no homozygous relA-deficient mice were born from heterozygote mating. Sequential DNA blot analysis and histological examination of embryos from timed matings of heterozygous mice were conducted. Until embryonic day (ED) 13.5, all embryos were apparently normal and homozygous mutants (−/−) were present in an expected ratio. On ED14.5, the relA−/− embryos were still present but some showed signs of abnormalities in their liver (Fig. 2 A). On ED15.5, a portion of the embryos became necrotic and were typed homozygous mutant (−/−), while normal embryos were all wild type (+/+) or heterozygous (+/−). RNA blot analysis of relA+/+ embryos from ED8.5 until birth as well as CCE ES cells showed the presence of relA transcripts, while no relA transcripts could be detected in relA−/− embryos at any stage (Fig. 2 B). Positive immunostaining with anti-RelA antibody correlated with the presence of RNA transcripts, showing that RelA proteins were present in almost all tissues of ED13.5 relA+/+ embryos, while no staining of relA−/− embryos (Fig. 2 C). Although a different vector construct was used in our study, the generated RelA-deficient mice showed the same phenotype as those of Beg et al. (11), indicating that RelA is essential for embryonic development of the mouse. In contrast to RelA-deficient mice, mice lacking other NF-κB proteins are known to develop normally at least until birth (12–14). The difference may simply reflect the fact that RelA is expressed ubiquitously from an early stage of development while the others are expressed in restricted tissues from a much later stage (34, 35). Identification of RelA responsive genes in developing embryos, especially in the liver, should open new avenues for elucidation of the function of NF-κB in embryonic development.

Bottom Line: However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells.Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3 + anti-CD28, LPS, anti-IgM, and PMA + calcium ionophore.The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan.

ABSTRACT
To investigate the function of NF-kappa B RelA (p65), we generated mice deficient in this NF-kappa B family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA-/- fetal liver transplants, similar to the relA+/+ and +/- cases. T cells were found to mature to Thy-1+/TCR alpha beta +/CD3+/CD4+ or CD8+, while B cells had the ability to differentiate to IgM+/B220+ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3 + anti-CD28, LPS, anti-IgM, and PMA + calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.

Show MeSH
Related in: MedlinePlus