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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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Defective IL-12 p40 protein and mRNA response in  thioglycollate-elicited macrophage-enriched populations from ICSBP−/−  and ICSBP+/+ mice. (A) IL-12 p40 protein synthesis in cultures of −/−  (black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in  peritoneal macrophages stimulated in vitro. PECs were collected from  five mice that had been injected with thioglycollate 5 d earlier. Pooled  adherent cells (macrophage enriched) were cultured in triplicate in the  presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell),  STAg (5 μg/ml), or media alone. Where indicated, cells were incubated  with IFN-γ (200 U/ml) and/or anti–IL-4/10 mAbs (10 μg/ml each) for  2 h before the addition of stimuli and throughout the culture period.  Cells were collected at 6 h for RNase protection assay and supernatants at  48 h for protein measurement by ELISA. Three μg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1α,  IL-1β, IL-1Ra, and GAPDH. The experiment shown is representative of  three performed.
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Figure 5: Defective IL-12 p40 protein and mRNA response in thioglycollate-elicited macrophage-enriched populations from ICSBP−/− and ICSBP+/+ mice. (A) IL-12 p40 protein synthesis in cultures of −/− (black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in peritoneal macrophages stimulated in vitro. PECs were collected from five mice that had been injected with thioglycollate 5 d earlier. Pooled adherent cells (macrophage enriched) were cultured in triplicate in the presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell), STAg (5 μg/ml), or media alone. Where indicated, cells were incubated with IFN-γ (200 U/ml) and/or anti–IL-4/10 mAbs (10 μg/ml each) for 2 h before the addition of stimuli and throughout the culture period. Cells were collected at 6 h for RNase protection assay and supernatants at 48 h for protein measurement by ELISA. Three μg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1α, IL-1β, IL-1Ra, and GAPDH. The experiment shown is representative of three performed.

Mentions: Having demonstrated that mice deficient in ICSBP express negligible amounts of IL-12 p40 after exposure to T. gondii in vivo, we next asked whether cells from these animals are also defective in their IL-12 response to other stimuli both in the absence and presence of an IFN-γ priming signal. The ability of adherent, thioglycollate-elicited peritoneal macrophages from uninfected mice to produce IL-12 p40 protein after stimulation in vitro was assessed. In both +/+ and −/− cultures, >85% of the adherent population were macrophages as determined by their expression of the Mac-1 but not GR-1 cell surface markers (data not shown). Exposure of wt cells to live tachyzoites, STAg, LPS, or heat-killed SAC resulted in increased IL-12 p40 levels. In contrast, while cells from ICSBP−/− mice also responded to these stimuli, the relative increase in IL-12 p40 production was markedly less (Fig. 5 A, left). In the four individual experiments performed, the most dramatic reduction in IL-12 synthesis resulting from the ICSBP mutation was observed in the response to live tachyzoites or STAg (always >80%), followed by LPS (>70%) and then SAC (>50%). This hierarchy was consistently observed in each experiment.


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)

Defective IL-12 p40 protein and mRNA response in  thioglycollate-elicited macrophage-enriched populations from ICSBP−/−  and ICSBP+/+ mice. (A) IL-12 p40 protein synthesis in cultures of −/−  (black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in  peritoneal macrophages stimulated in vitro. PECs were collected from  five mice that had been injected with thioglycollate 5 d earlier. Pooled  adherent cells (macrophage enriched) were cultured in triplicate in the  presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell),  STAg (5 μg/ml), or media alone. Where indicated, cells were incubated  with IFN-γ (200 U/ml) and/or anti–IL-4/10 mAbs (10 μg/ml each) for  2 h before the addition of stimuli and throughout the culture period.  Cells were collected at 6 h for RNase protection assay and supernatants at  48 h for protein measurement by ELISA. Three μg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1α,  IL-1β, IL-1Ra, and GAPDH. The experiment shown is representative of  three performed.
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Figure 5: Defective IL-12 p40 protein and mRNA response in thioglycollate-elicited macrophage-enriched populations from ICSBP−/− and ICSBP+/+ mice. (A) IL-12 p40 protein synthesis in cultures of −/− (black bars) and +/+ (white bars) cells. (B) IL-12 p40 mRNA induction in peritoneal macrophages stimulated in vitro. PECs were collected from five mice that had been injected with thioglycollate 5 d earlier. Pooled adherent cells (macrophage enriched) were cultured in triplicate in the presence of SAC (1:1,000), LPS (200 ng/ml), RH (0.2 tachyzoites/cell), STAg (5 μg/ml), or media alone. Where indicated, cells were incubated with IFN-γ (200 U/ml) and/or anti–IL-4/10 mAbs (10 μg/ml each) for 2 h before the addition of stimuli and throughout the culture period. Cells were collected at 6 h for RNase protection assay and supernatants at 48 h for protein measurement by ELISA. Three μg of RNA were subjected to RNase protection assay to detect transcripts for IL-12 p40, IL-1α, IL-1β, IL-1Ra, and GAPDH. The experiment shown is representative of three performed.
Mentions: Having demonstrated that mice deficient in ICSBP express negligible amounts of IL-12 p40 after exposure to T. gondii in vivo, we next asked whether cells from these animals are also defective in their IL-12 response to other stimuli both in the absence and presence of an IFN-γ priming signal. The ability of adherent, thioglycollate-elicited peritoneal macrophages from uninfected mice to produce IL-12 p40 protein after stimulation in vitro was assessed. In both +/+ and −/− cultures, >85% of the adherent population were macrophages as determined by their expression of the Mac-1 but not GR-1 cell surface markers (data not shown). Exposure of wt cells to live tachyzoites, STAg, LPS, or heat-killed SAC resulted in increased IL-12 p40 levels. In contrast, while cells from ICSBP−/− mice also responded to these stimuli, the relative increase in IL-12 p40 production was markedly less (Fig. 5 A, left). In the four individual experiments performed, the most dramatic reduction in IL-12 synthesis resulting from the ICSBP mutation was observed in the response to live tachyzoites or STAg (always >80%), followed by LPS (>70%) and then SAC (>50%). This hierarchy was consistently observed in each experiment.

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