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Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha.

Neumann H, Schmidt H, Cavalié A, Jenne D, Wekerle H - J. Exp. Med. (1997)

Bottom Line: The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate.In contrast to IFN-gamma, treatment with TNF-alpha did neither upregulate TAP1/TAP2 nor beta2-microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons.Consequently, no MHC class I molecules were detectable on the membranes of TNF-alpha-treated neurons.

View Article: PubMed Central - PubMed

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

ABSTRACT
This study examined the effect of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the induction of MHC class I-related genes in functionally mature brain neurons derived from cultures of dissociated rat hippocampal tissue. Patch clamp electrophysiology combined with single cell RT-PCR demonstrated that approximately 50% of the untreated neurons contained mRNA for MHC class I heavy chains, while, with few exceptions, the cells failed to transcribe beta2-microglobulin and TAP1/TAP2 gene transcripts. No constitutive expression of MHC class I protein was detectable by confocal laser microscopy on the surface of neurons. All neurons transcribed the alpha-chain of the interferon-type II receptor (binding IFN-gamma) along with the p55 receptor for TNF-alpha. Sustained exposure to IFN-gamma resulted in transcription of beta2-microglobulin and TAP1/TAP2 genes and MHC class I surface expression in a minor part of the neurons, but did not alter their electrophysiological activities as assessed by whole cell electrophysiology. Suppression of neuronal electric activity by the sodium channel blocker tetrodotoxin drastically increased to almost 100% IFN-gamma-mediated induction of MHC class I chains, of both TAP transporters, and of membrane expression of MHC class I protein. The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate. In contrast to IFN-gamma, treatment with TNF-alpha did neither upregulate TAP1/TAP2 nor beta2-microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons. Consequently, no MHC class I molecules were detectable on the membranes of TNF-alpha-treated neurons.

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Spontaneous bioelectric activity of hippocampal neurons  with or without IFN-γ treatment.  (A) Spontaneous firing of action  potentials persisting over at least 10  min by an untreated neuron (upper  trace). Additional suprathreshold  current pulses elicited single action  potentials (lower trace, left). Depolarising ramps demonstrated sodium  and potassium currents, downward  and upward deflections, respectively (lower trace, right). (B) Hippocampal neuron treated with  IFN-γ (100 U/ml) for 3 d.
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Figure 2: Spontaneous bioelectric activity of hippocampal neurons with or without IFN-γ treatment. (A) Spontaneous firing of action potentials persisting over at least 10 min by an untreated neuron (upper trace). Additional suprathreshold current pulses elicited single action potentials (lower trace, left). Depolarising ramps demonstrated sodium and potassium currents, downward and upward deflections, respectively (lower trace, right). (B) Hippocampal neuron treated with IFN-γ (100 U/ml) for 3 d.

Mentions: Cells dissociated from fetal rat hippocampal tissue (E18) were allowed to differentiate for up to 2 wk. By day 12 in culture the neuronal cells formed a network of dense processes placed on top of an astrocyte monolayer (Fig. 1, A–C). Individual neurons were selected morphologically and identified by whole cell patch–clamp recording of spontaneous or evoked fast action potential firing (Fig. 2). Immediately after electrophysiological analysis, samples of cytoplasm were drawn from the recorded cell through the micropipette. MessengerRNA of each single cell was reversely transcribed into cDNA and analyzed after PCR amplification with specific primers. Efficiency and specificity of the patch-clamp RTPCR method was verified by co-amplifying housekeeping and lineage-specific gene transcripts. These included GAPDH as housekeeping gene, along with the cytoskeleton proteins MAP2 specific for neurons, and GFAP for astrocytes, the most important non-neuronal cells present in the cultures. Fig. 1 D displays the correct transcription of the lineage specific gene markers in a series of selected neurons and astrocytes, respectively. MAP2, but not GFAP, was exclusively detected in neuron-derived material, while all samples from neighboring astrocytes yielded solely cDNA for GFAP.


Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha.

Neumann H, Schmidt H, Cavalié A, Jenne D, Wekerle H - J. Exp. Med. (1997)

Spontaneous bioelectric activity of hippocampal neurons  with or without IFN-γ treatment.  (A) Spontaneous firing of action  potentials persisting over at least 10  min by an untreated neuron (upper  trace). Additional suprathreshold  current pulses elicited single action  potentials (lower trace, left). Depolarising ramps demonstrated sodium  and potassium currents, downward  and upward deflections, respectively (lower trace, right). (B) Hippocampal neuron treated with  IFN-γ (100 U/ml) for 3 d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196130&req=5

Figure 2: Spontaneous bioelectric activity of hippocampal neurons with or without IFN-γ treatment. (A) Spontaneous firing of action potentials persisting over at least 10 min by an untreated neuron (upper trace). Additional suprathreshold current pulses elicited single action potentials (lower trace, left). Depolarising ramps demonstrated sodium and potassium currents, downward and upward deflections, respectively (lower trace, right). (B) Hippocampal neuron treated with IFN-γ (100 U/ml) for 3 d.
Mentions: Cells dissociated from fetal rat hippocampal tissue (E18) were allowed to differentiate for up to 2 wk. By day 12 in culture the neuronal cells formed a network of dense processes placed on top of an astrocyte monolayer (Fig. 1, A–C). Individual neurons were selected morphologically and identified by whole cell patch–clamp recording of spontaneous or evoked fast action potential firing (Fig. 2). Immediately after electrophysiological analysis, samples of cytoplasm were drawn from the recorded cell through the micropipette. MessengerRNA of each single cell was reversely transcribed into cDNA and analyzed after PCR amplification with specific primers. Efficiency and specificity of the patch-clamp RTPCR method was verified by co-amplifying housekeeping and lineage-specific gene transcripts. These included GAPDH as housekeeping gene, along with the cytoskeleton proteins MAP2 specific for neurons, and GFAP for astrocytes, the most important non-neuronal cells present in the cultures. Fig. 1 D displays the correct transcription of the lineage specific gene markers in a series of selected neurons and astrocytes, respectively. MAP2, but not GFAP, was exclusively detected in neuron-derived material, while all samples from neighboring astrocytes yielded solely cDNA for GFAP.

Bottom Line: The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate.In contrast to IFN-gamma, treatment with TNF-alpha did neither upregulate TAP1/TAP2 nor beta2-microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons.Consequently, no MHC class I molecules were detectable on the membranes of TNF-alpha-treated neurons.

View Article: PubMed Central - PubMed

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

ABSTRACT
This study examined the effect of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the induction of MHC class I-related genes in functionally mature brain neurons derived from cultures of dissociated rat hippocampal tissue. Patch clamp electrophysiology combined with single cell RT-PCR demonstrated that approximately 50% of the untreated neurons contained mRNA for MHC class I heavy chains, while, with few exceptions, the cells failed to transcribe beta2-microglobulin and TAP1/TAP2 gene transcripts. No constitutive expression of MHC class I protein was detectable by confocal laser microscopy on the surface of neurons. All neurons transcribed the alpha-chain of the interferon-type II receptor (binding IFN-gamma) along with the p55 receptor for TNF-alpha. Sustained exposure to IFN-gamma resulted in transcription of beta2-microglobulin and TAP1/TAP2 genes and MHC class I surface expression in a minor part of the neurons, but did not alter their electrophysiological activities as assessed by whole cell electrophysiology. Suppression of neuronal electric activity by the sodium channel blocker tetrodotoxin drastically increased to almost 100% IFN-gamma-mediated induction of MHC class I chains, of both TAP transporters, and of membrane expression of MHC class I protein. The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate. In contrast to IFN-gamma, treatment with TNF-alpha did neither upregulate TAP1/TAP2 nor beta2-microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons. Consequently, no MHC class I molecules were detectable on the membranes of TNF-alpha-treated neurons.

Show MeSH
Related in: MedlinePlus