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Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction.

Scharton-Kersten T, Contursi C, Masumi A, Sher A, Ozato K - J. Exp. Med. (1997)

Bottom Line: Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-gamma and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP-/- animals.This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-gamma.Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-gamma-dependent host resistance.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-2753, USA.

ABSTRACT
Mice lacking the transcription factor interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family of transcription proteins, were infected with the intracellular protozoan, Toxoplasma gondii. ICSBP-deficient mice exhibited unchecked parasite replication in vivo and rapidly succumbed within 14 d after inoculation with an avirulent Toxoplasma strain. In contrast, few intracellular parasites were observed in wild-type littermates and these animals survived for at least 60 d after infection. Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-gamma and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP-/- animals. In related experiments, macrophages from uninfected ICSBP-/- mice were shown to display a selective impairment in the mRNA expression of IL-12 p40 but not IL-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, or TNF-alpha in response to live parasites, parasite antigen, lipopolysaccharide, or Staphylococcus aureus. This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-gamma. We hypothesize that the impaired synthesis of IL-12 p40 in ICSBP-/- animals is the primary lesion responsible for the loss in resistance to T. gondii because IFN-gamma-induced parasite killing was unimpaired in vitro and, more importantly, administration of exogenous IL-12 in vivo significantly prolonged survival of the infected mice. Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-gamma-dependent host resistance.

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Survey of cytokine expression (TNF-α, IL-12 p40, IL-10, and  IL-6) by RT-PCR analysis in thioglycollate elicited macrophage populations after stimulation with IFN-γ and/or SAC, STAg or LPS. (A) Two to  four million peritoneal macrophages were cultured with (+) or without  (−) pretreatment with IFN-γ at 200 U/ml for 2 h followed by the addition  of STAg (5 μg/ml), SAC (1:1,000) or LPS (200 ng/ml) for 6 h. RNA was  extracted as described in Materials and Methods and samples were subjected  to quantitative RT-PCR as in Fig. 4. In lane 7, anti–IL-4 and anti–IL-10  mAbs (10 μg/ml each) were included in the cultures. ND indicates that the  cDNA sample could not be evaluated. (B) Relative levels of mRNA induction were calculated by first normalizing the densitometry units for each cytokine to the densitometry units of the housekeeping gene (HPRT) for  each sample. Second, a ratio of the values determined for ICSBP−/− and  ICSBP+/+ samples was calculated. Values of 100% indicate equal expression  between the two groups, those <100% signify ICSBP−/− <ICSBP+/+ and  those >100% that ICSBP−/− >ICSBP+/+. (−) indicate that a value could  not be determined because below the level of detection (see Media −IFN)  or because the HPRT value for at least 1 sample was not available (see  STAg–IFNγ). The experiment shown is representative of three performed.
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Figure 6: Survey of cytokine expression (TNF-α, IL-12 p40, IL-10, and IL-6) by RT-PCR analysis in thioglycollate elicited macrophage populations after stimulation with IFN-γ and/or SAC, STAg or LPS. (A) Two to four million peritoneal macrophages were cultured with (+) or without (−) pretreatment with IFN-γ at 200 U/ml for 2 h followed by the addition of STAg (5 μg/ml), SAC (1:1,000) or LPS (200 ng/ml) for 6 h. RNA was extracted as described in Materials and Methods and samples were subjected to quantitative RT-PCR as in Fig. 4. In lane 7, anti–IL-4 and anti–IL-10 mAbs (10 μg/ml each) were included in the cultures. ND indicates that the cDNA sample could not be evaluated. (B) Relative levels of mRNA induction were calculated by first normalizing the densitometry units for each cytokine to the densitometry units of the housekeeping gene (HPRT) for each sample. Second, a ratio of the values determined for ICSBP−/− and ICSBP+/+ samples was calculated. Values of 100% indicate equal expression between the two groups, those <100% signify ICSBP−/− <ICSBP+/+ and those >100% that ICSBP−/− >ICSBP+/+. (−) indicate that a value could not be determined because below the level of detection (see Media −IFN) or because the HPRT value for at least 1 sample was not available (see STAg–IFNγ). The experiment shown is representative of three performed.

Mentions: To further address the selectivity of the ICSBP macrophage deficiency the expression of three additional cytokines, TNFα, IL-6, and IL-10, was analyzed by RT-PCR (Fig. 6). IL-12 p40 mRNA induction was negligible in ICSBP−/− macrophages in response to SAC, LPS, or STAg even after priming with IFN-γ. Addition of anti–IL-4 and anti–IL-10 antibodies failed to reveal IL-12 p40 mRNA expression in SAC stimulated ICSBP macrophages suggesting that the defect observed is not due to IL-4−/− IL-10– mediated suppression. In contrast to the impaired IL-12 p40 mRNA induction, ICSBP−/− macrophages expressed TNFα mRNA in response to IFN-γ, STAg and LPS at levels comparable with ICSBP+/+ cells. Nevertheless, TNFα mRNAs induced by SAC alone, but not SAC + IFN-γ, were reduced in macrophages from ICSBP−/− mice. Macrophages from both strains of mice also synthesized comparable levels of IL-6 transcripts in response to all of the agents tested. Interestingly, ICSBP−/− macrophages constitutively expressed IL-10 mRNA and higher levels of this cytokine transcript were induced in ICSBP−/− as compared with ICSBP+/+ macrophages in response to most of the stimuli. Quantitative analysis of the cytokine transcript data in Fig. 6 A is provided in Fig. 6 B.


Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction.

Scharton-Kersten T, Contursi C, Masumi A, Sher A, Ozato K - J. Exp. Med. (1997)

Survey of cytokine expression (TNF-α, IL-12 p40, IL-10, and  IL-6) by RT-PCR analysis in thioglycollate elicited macrophage populations after stimulation with IFN-γ and/or SAC, STAg or LPS. (A) Two to  four million peritoneal macrophages were cultured with (+) or without  (−) pretreatment with IFN-γ at 200 U/ml for 2 h followed by the addition  of STAg (5 μg/ml), SAC (1:1,000) or LPS (200 ng/ml) for 6 h. RNA was  extracted as described in Materials and Methods and samples were subjected  to quantitative RT-PCR as in Fig. 4. In lane 7, anti–IL-4 and anti–IL-10  mAbs (10 μg/ml each) were included in the cultures. ND indicates that the  cDNA sample could not be evaluated. (B) Relative levels of mRNA induction were calculated by first normalizing the densitometry units for each cytokine to the densitometry units of the housekeeping gene (HPRT) for  each sample. Second, a ratio of the values determined for ICSBP−/− and  ICSBP+/+ samples was calculated. Values of 100% indicate equal expression  between the two groups, those <100% signify ICSBP−/− <ICSBP+/+ and  those >100% that ICSBP−/− >ICSBP+/+. (−) indicate that a value could  not be determined because below the level of detection (see Media −IFN)  or because the HPRT value for at least 1 sample was not available (see  STAg–IFNγ). The experiment shown is representative of three performed.
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Figure 6: Survey of cytokine expression (TNF-α, IL-12 p40, IL-10, and IL-6) by RT-PCR analysis in thioglycollate elicited macrophage populations after stimulation with IFN-γ and/or SAC, STAg or LPS. (A) Two to four million peritoneal macrophages were cultured with (+) or without (−) pretreatment with IFN-γ at 200 U/ml for 2 h followed by the addition of STAg (5 μg/ml), SAC (1:1,000) or LPS (200 ng/ml) for 6 h. RNA was extracted as described in Materials and Methods and samples were subjected to quantitative RT-PCR as in Fig. 4. In lane 7, anti–IL-4 and anti–IL-10 mAbs (10 μg/ml each) were included in the cultures. ND indicates that the cDNA sample could not be evaluated. (B) Relative levels of mRNA induction were calculated by first normalizing the densitometry units for each cytokine to the densitometry units of the housekeeping gene (HPRT) for each sample. Second, a ratio of the values determined for ICSBP−/− and ICSBP+/+ samples was calculated. Values of 100% indicate equal expression between the two groups, those <100% signify ICSBP−/− <ICSBP+/+ and those >100% that ICSBP−/− >ICSBP+/+. (−) indicate that a value could not be determined because below the level of detection (see Media −IFN) or because the HPRT value for at least 1 sample was not available (see STAg–IFNγ). The experiment shown is representative of three performed.
Mentions: To further address the selectivity of the ICSBP macrophage deficiency the expression of three additional cytokines, TNFα, IL-6, and IL-10, was analyzed by RT-PCR (Fig. 6). IL-12 p40 mRNA induction was negligible in ICSBP−/− macrophages in response to SAC, LPS, or STAg even after priming with IFN-γ. Addition of anti–IL-4 and anti–IL-10 antibodies failed to reveal IL-12 p40 mRNA expression in SAC stimulated ICSBP macrophages suggesting that the defect observed is not due to IL-4−/− IL-10– mediated suppression. In contrast to the impaired IL-12 p40 mRNA induction, ICSBP−/− macrophages expressed TNFα mRNA in response to IFN-γ, STAg and LPS at levels comparable with ICSBP+/+ cells. Nevertheless, TNFα mRNAs induced by SAC alone, but not SAC + IFN-γ, were reduced in macrophages from ICSBP−/− mice. Macrophages from both strains of mice also synthesized comparable levels of IL-6 transcripts in response to all of the agents tested. Interestingly, ICSBP−/− macrophages constitutively expressed IL-10 mRNA and higher levels of this cytokine transcript were induced in ICSBP−/− as compared with ICSBP+/+ macrophages in response to most of the stimuli. Quantitative analysis of the cytokine transcript data in Fig. 6 A is provided in Fig. 6 B.

Bottom Line: Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-gamma and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP-/- animals.This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-gamma.Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-gamma-dependent host resistance.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-2753, USA.

ABSTRACT
Mice lacking the transcription factor interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family of transcription proteins, were infected with the intracellular protozoan, Toxoplasma gondii. ICSBP-deficient mice exhibited unchecked parasite replication in vivo and rapidly succumbed within 14 d after inoculation with an avirulent Toxoplasma strain. In contrast, few intracellular parasites were observed in wild-type littermates and these animals survived for at least 60 d after infection. Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-gamma and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP-/- animals. In related experiments, macrophages from uninfected ICSBP-/- mice were shown to display a selective impairment in the mRNA expression of IL-12 p40 but not IL-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, or TNF-alpha in response to live parasites, parasite antigen, lipopolysaccharide, or Staphylococcus aureus. This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-gamma. We hypothesize that the impaired synthesis of IL-12 p40 in ICSBP-/- animals is the primary lesion responsible for the loss in resistance to T. gondii because IFN-gamma-induced parasite killing was unimpaired in vitro and, more importantly, administration of exogenous IL-12 in vivo significantly prolonged survival of the infected mice. Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-gamma-dependent host resistance.

Show MeSH
Related in: MedlinePlus