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Interferon gamma gene expression in sensory neurons: evidence for autocrine gene regulation.

Neumann H, Schmidt H, Wilharm E, Behrens L, Wekerle H - J. Exp. Med. (1997)

Bottom Line: Locally produced IFN-gamma acts back on its cellular source.Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-gamma-dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures.Our findings indicate a role of IFN-gamma in autocrine regulation of sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroimmunology, Max-Planck-Institute for Psychiatry, D-82152 Martinsried, Germany.

ABSTRACT
We explored expression and possible function of interferon-gamma (IFN-gamma) in cultured fetal (E15) rat dorsal root ganglion neurons combining whole cell patch-clamp electrophysiology with single cell reverse transcriptase polymerase chain reaction and confocal laser immunocytochemistry. Morphologically, we located IFN-gamma protein in the cytoplasm of the neurons in culture as well as in situ during peri- and postnatal development. Transcripts for classic IFN-gamma and for its receptor were determined in probes of cytoplasm sampled from individual cultured neurons, which had been identified by patch clamp electrophysiology. In addition, the cultured neurons expressed both chains of the IFN-gamma receptor. Locally produced IFN-gamma acts back on its cellular source. Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-gamma-dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures. However, both processes were substantially blocked in the presence of antibodies neutralizing IFN-gamma. Our findings indicate a role of IFN-gamma in autocrine regulation of sensory neurons.

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Related in: MedlinePlus

Nuclear localization of STAT1 and cell membrane expression of MHC class I  molecules. (A) Nuclear localization of STAT1 in sensory neurons. Embryonic sensory neurons were cultured for 2 d and  analyzed by confocal laser scanning microscopy for neurofilament and STAT1. (B) Cytoplasmic localization of STAT1 in  neurons of hippocampal cultures.  (C) Cytoplasmic localization of  STAT1 in sensory neurons (cultured for 2 d) in the presence of  neutralizing antibodies directed  against IFN-γ. (D) MHC class I  molecules detected on the cell  membrane of untreated sensory  neurons. Sensory neurons (cultured for 5 d) were identified by  antibodies recognizing neurofilament and were immunolabeled  with antibodies directed against  MHC class I on the cell surface.  Scale bar A, B, C, and D: 10 μm.
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Figure 8: Nuclear localization of STAT1 and cell membrane expression of MHC class I molecules. (A) Nuclear localization of STAT1 in sensory neurons. Embryonic sensory neurons were cultured for 2 d and analyzed by confocal laser scanning microscopy for neurofilament and STAT1. (B) Cytoplasmic localization of STAT1 in neurons of hippocampal cultures. (C) Cytoplasmic localization of STAT1 in sensory neurons (cultured for 2 d) in the presence of neutralizing antibodies directed against IFN-γ. (D) MHC class I molecules detected on the cell membrane of untreated sensory neurons. Sensory neurons (cultured for 5 d) were identified by antibodies recognizing neurofilament and were immunolabeled with antibodies directed against MHC class I on the cell surface. Scale bar A, B, C, and D: 10 μm.

Mentions: Western blot analysis of total cell lysate of DRG cultures demonstrated phosphorylated STAT1 (data not shown), but this method does not identify the cell type in the mixed DRG culture that has the active form of STAT1. However, confocal laser scanning microscopy clearly located STAT1 in the nuclei of almost all cultured neurofilament-positive DRG neurons (96 ± 4%, Figs. 8 and 9), and also in the glia cells. In contrast, in differentiated hippocampal neuronal cultures, where no IFN-γ synthesis is demonstrable, all STAT1 immunoreactivity was confined to the cytoplasm (Fig. 8). IFN-γ–neutralizing antibodies to DRG cultures profoundly interfered with nuclear translocation of STAT1. After neutralization of IFN-γ, most neurons displayed STAT1 within their cytoplasm, with nuclear location seen only in a minority of all cells (26 ± 11%, Figs. 8 and 9).


Interferon gamma gene expression in sensory neurons: evidence for autocrine gene regulation.

Neumann H, Schmidt H, Wilharm E, Behrens L, Wekerle H - J. Exp. Med. (1997)

Nuclear localization of STAT1 and cell membrane expression of MHC class I  molecules. (A) Nuclear localization of STAT1 in sensory neurons. Embryonic sensory neurons were cultured for 2 d and  analyzed by confocal laser scanning microscopy for neurofilament and STAT1. (B) Cytoplasmic localization of STAT1 in  neurons of hippocampal cultures.  (C) Cytoplasmic localization of  STAT1 in sensory neurons (cultured for 2 d) in the presence of  neutralizing antibodies directed  against IFN-γ. (D) MHC class I  molecules detected on the cell  membrane of untreated sensory  neurons. Sensory neurons (cultured for 5 d) were identified by  antibodies recognizing neurofilament and were immunolabeled  with antibodies directed against  MHC class I on the cell surface.  Scale bar A, B, C, and D: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199162&req=5

Figure 8: Nuclear localization of STAT1 and cell membrane expression of MHC class I molecules. (A) Nuclear localization of STAT1 in sensory neurons. Embryonic sensory neurons were cultured for 2 d and analyzed by confocal laser scanning microscopy for neurofilament and STAT1. (B) Cytoplasmic localization of STAT1 in neurons of hippocampal cultures. (C) Cytoplasmic localization of STAT1 in sensory neurons (cultured for 2 d) in the presence of neutralizing antibodies directed against IFN-γ. (D) MHC class I molecules detected on the cell membrane of untreated sensory neurons. Sensory neurons (cultured for 5 d) were identified by antibodies recognizing neurofilament and were immunolabeled with antibodies directed against MHC class I on the cell surface. Scale bar A, B, C, and D: 10 μm.
Mentions: Western blot analysis of total cell lysate of DRG cultures demonstrated phosphorylated STAT1 (data not shown), but this method does not identify the cell type in the mixed DRG culture that has the active form of STAT1. However, confocal laser scanning microscopy clearly located STAT1 in the nuclei of almost all cultured neurofilament-positive DRG neurons (96 ± 4%, Figs. 8 and 9), and also in the glia cells. In contrast, in differentiated hippocampal neuronal cultures, where no IFN-γ synthesis is demonstrable, all STAT1 immunoreactivity was confined to the cytoplasm (Fig. 8). IFN-γ–neutralizing antibodies to DRG cultures profoundly interfered with nuclear translocation of STAT1. After neutralization of IFN-γ, most neurons displayed STAT1 within their cytoplasm, with nuclear location seen only in a minority of all cells (26 ± 11%, Figs. 8 and 9).

Bottom Line: Locally produced IFN-gamma acts back on its cellular source.Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-gamma-dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures.Our findings indicate a role of IFN-gamma in autocrine regulation of sensory neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroimmunology, Max-Planck-Institute for Psychiatry, D-82152 Martinsried, Germany.

ABSTRACT
We explored expression and possible function of interferon-gamma (IFN-gamma) in cultured fetal (E15) rat dorsal root ganglion neurons combining whole cell patch-clamp electrophysiology with single cell reverse transcriptase polymerase chain reaction and confocal laser immunocytochemistry. Morphologically, we located IFN-gamma protein in the cytoplasm of the neurons in culture as well as in situ during peri- and postnatal development. Transcripts for classic IFN-gamma and for its receptor were determined in probes of cytoplasm sampled from individual cultured neurons, which had been identified by patch clamp electrophysiology. In addition, the cultured neurons expressed both chains of the IFN-gamma receptor. Locally produced IFN-gamma acts back on its cellular source. Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-gamma-dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures. However, both processes were substantially blocked in the presence of antibodies neutralizing IFN-gamma. Our findings indicate a role of IFN-gamma in autocrine regulation of sensory neurons.

Show MeSH
Related in: MedlinePlus