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Impairment of natural killer cytotoxic activity and interferon gamma production in CCAAT/enhancer binding protein gamma-deficient mice.

Kaisho T, Tsutsui H, Tanaka T, Tsujimura T, Takeda K, Kawai T, Yoshida N, Nakanishi K, Akira S - J. Exp. Med. (1999)

Bottom Line: However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells.NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPgamma(2/-) newborn spleen cells.In conclusion, our study reveals that C/EBPgamma is a critical transcription factor involved in the functional maturation of NK cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo 663-8501, Japan.

ABSTRACT
We have investigated in vivo roles of CCAAT/enhancer binding protein gamma (C/EBPgamma) by gene targeting. C/EBPgamma-deficient (C/EBPgamma(2/-)) mice showed a high mortality rate within 48 h after birth. To analyze the roles of C/EBPgamma in lymphoid lineage cells, bone marrow chimeras were established. C/EBPgamma(2/-) chimeras showed normal T and B cell development. However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells. In addition, the ability of C/EBPgamma(-/-) chimera splenocytes to produce interferon (IFN)-gamma in response to IL-12 and/or IL-18 was markedly impaired. NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPgamma(2/-) newborn spleen cells. However, they also showed lower cytotoxic activity and IFN-gamma production when stimulated with IL-12 plus IL-18 than control NK cells, as observed in C/EBPgamma(2/-) chimera splenocytes. In conclusion, our study reveals that C/EBPgamma is a critical transcription factor involved in the functional maturation of NK cells.

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Generation of C/EBPγ2/− mice and expression of C/EBPγ mRNA. (A) Schematic representation of the C/EBPγ targeting vector, the C/EBPγ genomic locus, and the targeted C/EBPγ allele. The targeting vector contains the herpes simplex virus thymidine kinase (HSV-TK) gene 5′ upstream of the long arm homology region and the neomycin resistance gene (NEO) in the middle of the second exon. (B) RT-PCR analysis of newborn spleen and liver RNAs from C/EBPγ1/+ and C/EBPγ2/− mice. Total RNAs were isolated and analyzed for C/EBPγ and β-actin expression by RT-PCR. M, ΦX174/HaeIII digest marker. (C) RT-PCR analysis for C/EBPγ in lymphoid lineage cells. Total RNAs were isolated from splenic CD3−DX5+ (NK), B220+ (B), and CD3+ (T) cells and analyzed for C/EBPγ and β-actin expression by RT-PCR.
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Figure 1: Generation of C/EBPγ2/− mice and expression of C/EBPγ mRNA. (A) Schematic representation of the C/EBPγ targeting vector, the C/EBPγ genomic locus, and the targeted C/EBPγ allele. The targeting vector contains the herpes simplex virus thymidine kinase (HSV-TK) gene 5′ upstream of the long arm homology region and the neomycin resistance gene (NEO) in the middle of the second exon. (B) RT-PCR analysis of newborn spleen and liver RNAs from C/EBPγ1/+ and C/EBPγ2/− mice. Total RNAs were isolated and analyzed for C/EBPγ and β-actin expression by RT-PCR. M, ΦX174/HaeIII digest marker. (C) RT-PCR analysis for C/EBPγ in lymphoid lineage cells. Total RNAs were isolated from splenic CD3−DX5+ (NK), B220+ (B), and CD3+ (T) cells and analyzed for C/EBPγ and β-actin expression by RT-PCR.

Mentions: The C/EBPγ genomic locus was disrupted by inserting the neomycin resistance gene into the second exon (Fig. 1 A). This insertion resulted in disruption of the basic and leucine zipper domains, which are essential for DNA binding and dimer formation. Homologous recombinants were obtained through a double selection with G418 and gancyclovir. Targeted clones were injected into B6 blastocysts to generate chimeric mice, which were bred to achieve germline transmission. By mating heterozygous mutants, homozygous mutants were obtained at a frequency of the expected Mendelian ratio (22.6% of littermates). C/EBPγ expression was detected in wild-type newborn spleens and livers, but not in those of homozygous mutants as expected (Fig. 1 B). General appearance at birth was indistinguishable among C/EBPγ1/+, C/EBPγ1/−, and C/EBPγ2/− mice. However, only 11% of homozygous mutants could survive >60 h, although they were healthy until 12 h after they were born (Table ). These results indicate that C/EBPγ is involved in early neonatal survival but not embryonic survival. By histological examination, C/EBPγ2/− mice at 24 h showed emphysematous changes in their lungs (data not shown). However, it is not clear at the time of this writing whether the lung lesions alone can account for the high mortality of homozygous mutants at the early neonatal stage.


Impairment of natural killer cytotoxic activity and interferon gamma production in CCAAT/enhancer binding protein gamma-deficient mice.

Kaisho T, Tsutsui H, Tanaka T, Tsujimura T, Takeda K, Kawai T, Yoshida N, Nakanishi K, Akira S - J. Exp. Med. (1999)

Generation of C/EBPγ2/− mice and expression of C/EBPγ mRNA. (A) Schematic representation of the C/EBPγ targeting vector, the C/EBPγ genomic locus, and the targeted C/EBPγ allele. The targeting vector contains the herpes simplex virus thymidine kinase (HSV-TK) gene 5′ upstream of the long arm homology region and the neomycin resistance gene (NEO) in the middle of the second exon. (B) RT-PCR analysis of newborn spleen and liver RNAs from C/EBPγ1/+ and C/EBPγ2/− mice. Total RNAs were isolated and analyzed for C/EBPγ and β-actin expression by RT-PCR. M, ΦX174/HaeIII digest marker. (C) RT-PCR analysis for C/EBPγ in lymphoid lineage cells. Total RNAs were isolated from splenic CD3−DX5+ (NK), B220+ (B), and CD3+ (T) cells and analyzed for C/EBPγ and β-actin expression by RT-PCR.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195736&req=5

Figure 1: Generation of C/EBPγ2/− mice and expression of C/EBPγ mRNA. (A) Schematic representation of the C/EBPγ targeting vector, the C/EBPγ genomic locus, and the targeted C/EBPγ allele. The targeting vector contains the herpes simplex virus thymidine kinase (HSV-TK) gene 5′ upstream of the long arm homology region and the neomycin resistance gene (NEO) in the middle of the second exon. (B) RT-PCR analysis of newborn spleen and liver RNAs from C/EBPγ1/+ and C/EBPγ2/− mice. Total RNAs were isolated and analyzed for C/EBPγ and β-actin expression by RT-PCR. M, ΦX174/HaeIII digest marker. (C) RT-PCR analysis for C/EBPγ in lymphoid lineage cells. Total RNAs were isolated from splenic CD3−DX5+ (NK), B220+ (B), and CD3+ (T) cells and analyzed for C/EBPγ and β-actin expression by RT-PCR.
Mentions: The C/EBPγ genomic locus was disrupted by inserting the neomycin resistance gene into the second exon (Fig. 1 A). This insertion resulted in disruption of the basic and leucine zipper domains, which are essential for DNA binding and dimer formation. Homologous recombinants were obtained through a double selection with G418 and gancyclovir. Targeted clones were injected into B6 blastocysts to generate chimeric mice, which were bred to achieve germline transmission. By mating heterozygous mutants, homozygous mutants were obtained at a frequency of the expected Mendelian ratio (22.6% of littermates). C/EBPγ expression was detected in wild-type newborn spleens and livers, but not in those of homozygous mutants as expected (Fig. 1 B). General appearance at birth was indistinguishable among C/EBPγ1/+, C/EBPγ1/−, and C/EBPγ2/− mice. However, only 11% of homozygous mutants could survive >60 h, although they were healthy until 12 h after they were born (Table ). These results indicate that C/EBPγ is involved in early neonatal survival but not embryonic survival. By histological examination, C/EBPγ2/− mice at 24 h showed emphysematous changes in their lungs (data not shown). However, it is not clear at the time of this writing whether the lung lesions alone can account for the high mortality of homozygous mutants at the early neonatal stage.

Bottom Line: However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells.NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPgamma(2/-) newborn spleen cells.In conclusion, our study reveals that C/EBPgamma is a critical transcription factor involved in the functional maturation of NK cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo 663-8501, Japan.

ABSTRACT
We have investigated in vivo roles of CCAAT/enhancer binding protein gamma (C/EBPgamma) by gene targeting. C/EBPgamma-deficient (C/EBPgamma(2/-)) mice showed a high mortality rate within 48 h after birth. To analyze the roles of C/EBPgamma in lymphoid lineage cells, bone marrow chimeras were established. C/EBPgamma(2/-) chimeras showed normal T and B cell development. However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells. In addition, the ability of C/EBPgamma(-/-) chimera splenocytes to produce interferon (IFN)-gamma in response to IL-12 and/or IL-18 was markedly impaired. NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPgamma(2/-) newborn spleen cells. However, they also showed lower cytotoxic activity and IFN-gamma production when stimulated with IL-12 plus IL-18 than control NK cells, as observed in C/EBPgamma(2/-) chimera splenocytes. In conclusion, our study reveals that C/EBPgamma is a critical transcription factor involved in the functional maturation of NK cells.

Show MeSH
Related in: MedlinePlus