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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

Single cell RT-PCR of CD4+ T lymphoblasts and DRG satellite cells. Gene transcripts for GAPDH (A), GFAP (B), CD4 (C), and  IFN-γ (D) were analyzed by single-cell RT-PCR of activated T cells  (lanes 1–3) and of DRG satellite cells (lanes 4–6). N and M show negative  PCR control and molecular weight marker, respectively.
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Figure 4: Single cell RT-PCR of CD4+ T lymphoblasts and DRG satellite cells. Gene transcripts for GAPDH (A), GFAP (B), CD4 (C), and IFN-γ (D) were analyzed by single-cell RT-PCR of activated T cells (lanes 1–3) and of DRG satellite cells (lanes 4–6). N and M show negative PCR control and molecular weight marker, respectively.

Mentions: We then used single-cell RT-PCR analysis to identify the intraneuronal IFN-γ–like material. After electrophysiological characterization, we sampled cytoplasmic specimens from individual neurons through the patch-clamp micropipette, and assessed current gene transcription by RT-PCR. Oligonucleotides specific for IFN-γ were used to amplify gene transcripts for the cytokine. In addition, as in our previous studies, coamplification of mRNA for the house-keeping enzyme GAPDH and for cell lineage markers served as internal quality standards (12, 14). The validity of our method was corroborated by parallel analyses of activated CD4+ T lymphocytes with a Th1-like phenotype (13) and DRG-derived glia cells (Fig. 4). Each single T lymphoblast analyzed contained mRNA for the lineage marker gene CD4 along with IFN-γ. In contrast, satellite cells from DRG cultures, which expressed glia specific GFAP gene transcripts, and were negative for IFN-γ and CD4 gene transcripts (Fig. 4). In a subsequent cytokine study of 19 individual DRG neurons, mRNA for IFN-γ was identified in 13 cells cultured for 7 d (Fig. 5). Southern blot analysis and sequencing confirmed the identity of the amplified fragments with classic IFN-γ. IFN-γ gene expression in cultured DRG neurons developed steadily over time. Among neurons cultured for 2 h, only a minority (1/11) of the (electrophysiologically immature) cells expressed IFN-γ gene transcripts (Fig. 5). However, the percentage of IFN-γ–expressing neurons continuously increased through the first 24 h in culture. After 6 h, 3/13 neurons, and after 24 h, 7/11 DRG neurons, expressed IFN-γ gene transcripts. In contrast, differentiated neurons derived from hippocampus tissue transcribed IFN-γ at no time.


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)

Single cell RT-PCR of CD4+ T lymphoblasts and DRG satellite cells. Gene transcripts for GAPDH (A), GFAP (B), CD4 (C), and  IFN-γ (D) were analyzed by single-cell RT-PCR of activated T cells  (lanes 1–3) and of DRG satellite cells (lanes 4–6). N and M show negative  PCR control and molecular weight marker, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Single cell RT-PCR of CD4+ T lymphoblasts and DRG satellite cells. Gene transcripts for GAPDH (A), GFAP (B), CD4 (C), and IFN-γ (D) were analyzed by single-cell RT-PCR of activated T cells (lanes 1–3) and of DRG satellite cells (lanes 4–6). N and M show negative PCR control and molecular weight marker, respectively.
Mentions: We then used single-cell RT-PCR analysis to identify the intraneuronal IFN-γ–like material. After electrophysiological characterization, we sampled cytoplasmic specimens from individual neurons through the patch-clamp micropipette, and assessed current gene transcription by RT-PCR. Oligonucleotides specific for IFN-γ were used to amplify gene transcripts for the cytokine. In addition, as in our previous studies, coamplification of mRNA for the house-keeping enzyme GAPDH and for cell lineage markers served as internal quality standards (12, 14). The validity of our method was corroborated by parallel analyses of activated CD4+ T lymphocytes with a Th1-like phenotype (13) and DRG-derived glia cells (Fig. 4). Each single T lymphoblast analyzed contained mRNA for the lineage marker gene CD4 along with IFN-γ. In contrast, satellite cells from DRG cultures, which expressed glia specific GFAP gene transcripts, and were negative for IFN-γ and CD4 gene transcripts (Fig. 4). In a subsequent cytokine study of 19 individual DRG neurons, mRNA for IFN-γ was identified in 13 cells cultured for 7 d (Fig. 5). Southern blot analysis and sequencing confirmed the identity of the amplified fragments with classic IFN-γ. IFN-γ gene expression in cultured DRG neurons developed steadily over time. Among neurons cultured for 2 h, only a minority (1/11) of the (electrophysiologically immature) cells expressed IFN-γ gene transcripts (Fig. 5). However, the percentage of IFN-γ–expressing neurons continuously increased through the first 24 h in culture. After 6 h, 3/13 neurons, and after 24 h, 7/11 DRG neurons, expressed IFN-γ gene transcripts. In contrast, differentiated neurons derived from hippocampus tissue transcribed IFN-γ at no time.

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