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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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Restriction site analysis of amplified PCR-fragments and  MHC class I heavy chain induction by TNF-α. (A) PCR-fragments amplified from IFN-γ plus TTX treated individual neurons, digested by restriction enzymes selected from the published sequence. PCR-fragments  for GAPDH (428 bp) cut into 324 bp and 104 bp, for β2-microglobulin  (332 bp) into 206 bp and 126 bp, for MHC class I heavy chain (198 bp)  into 160 bp and 38 bp. PCR-fragments for TAP1 (300 bp) cut into 194  bp and 106 bp. PCR-fragments for TAP2 (264 bp) cut into 192 bp and  72 bp. All analyzed PCR-fragments were in agreement with the published sequences. (B) Gene transcripts for GAPDH, β2-microglobulin and  MHC class I heavy chain coamplified from individual neurons (lanes 1 to  10) treated for 72 h with TNF-α. Lane N and lane M show negative control of PCR-amplification and molecular weight marker ΦX174/Hae III,  respectively.
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Figure 7: Restriction site analysis of amplified PCR-fragments and MHC class I heavy chain induction by TNF-α. (A) PCR-fragments amplified from IFN-γ plus TTX treated individual neurons, digested by restriction enzymes selected from the published sequence. PCR-fragments for GAPDH (428 bp) cut into 324 bp and 104 bp, for β2-microglobulin (332 bp) into 206 bp and 126 bp, for MHC class I heavy chain (198 bp) into 160 bp and 38 bp. PCR-fragments for TAP1 (300 bp) cut into 194 bp and 106 bp. PCR-fragments for TAP2 (264 bp) cut into 192 bp and 72 bp. All analyzed PCR-fragments were in agreement with the published sequences. (B) Gene transcripts for GAPDH, β2-microglobulin and MHC class I heavy chain coamplified from individual neurons (lanes 1 to 10) treated for 72 h with TNF-α. Lane N and lane M show negative control of PCR-amplification and molecular weight marker ΦX174/Hae III, respectively.

Mentions: IFN-γ-induced transcription of class I genes in neurons was, however, dramatically increased by the blockade of sodium channels by tetrodotoxin (TTX). After 72 h treatment with TTX plus IFN-γ all neurons expressed MHC class I heavy chain, β2-microglobulin (19/19), and almost all expressed TAP1 (18/19) and TAP2 (17/19) gene transcripts (Fig. 6 B, Table 2). Restriction fragment analysis and sequencing confirmed the identity of the amplified PCR fragments (Fig. 7 A).


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)

Restriction site analysis of amplified PCR-fragments and  MHC class I heavy chain induction by TNF-α. (A) PCR-fragments amplified from IFN-γ plus TTX treated individual neurons, digested by restriction enzymes selected from the published sequence. PCR-fragments  for GAPDH (428 bp) cut into 324 bp and 104 bp, for β2-microglobulin  (332 bp) into 206 bp and 126 bp, for MHC class I heavy chain (198 bp)  into 160 bp and 38 bp. PCR-fragments for TAP1 (300 bp) cut into 194  bp and 106 bp. PCR-fragments for TAP2 (264 bp) cut into 192 bp and  72 bp. All analyzed PCR-fragments were in agreement with the published sequences. (B) Gene transcripts for GAPDH, β2-microglobulin and  MHC class I heavy chain coamplified from individual neurons (lanes 1 to  10) treated for 72 h with TNF-α. Lane N and lane M show negative control of PCR-amplification and molecular weight marker ΦX174/Hae III,  respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Restriction site analysis of amplified PCR-fragments and MHC class I heavy chain induction by TNF-α. (A) PCR-fragments amplified from IFN-γ plus TTX treated individual neurons, digested by restriction enzymes selected from the published sequence. PCR-fragments for GAPDH (428 bp) cut into 324 bp and 104 bp, for β2-microglobulin (332 bp) into 206 bp and 126 bp, for MHC class I heavy chain (198 bp) into 160 bp and 38 bp. PCR-fragments for TAP1 (300 bp) cut into 194 bp and 106 bp. PCR-fragments for TAP2 (264 bp) cut into 192 bp and 72 bp. All analyzed PCR-fragments were in agreement with the published sequences. (B) Gene transcripts for GAPDH, β2-microglobulin and MHC class I heavy chain coamplified from individual neurons (lanes 1 to 10) treated for 72 h with TNF-α. Lane N and lane M show negative control of PCR-amplification and molecular weight marker ΦX174/Hae III, respectively.
Mentions: IFN-γ-induced transcription of class I genes in neurons was, however, dramatically increased by the blockade of sodium channels by tetrodotoxin (TTX). After 72 h treatment with TTX plus IFN-γ all neurons expressed MHC class I heavy chain, β2-microglobulin (19/19), and almost all expressed TAP1 (18/19) and TAP2 (17/19) gene transcripts (Fig. 6 B, Table 2). Restriction fragment analysis and sequencing confirmed the identity of the amplified PCR fragments (Fig. 7 A).

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