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Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

Wheeler DW, Thompson AJ, Corletto F, Reckless J, Loke JC, Lapaque N, Grant AJ, Mastroeni P, Grainger DJ, Padgett CL, O'Brien JA, Miller NG, Trowsdale J, Lummis SC, Menon DK, Beech JS - PLoS ONE (2011)

Bottom Line: Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear.We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die.The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

View Article: PubMed Central - PubMed

Affiliation: Division of Anaesthesia, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom.

ABSTRACT

Background: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.

Principal findings: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

Significance: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

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mRNA and protein expression of GABAA receptor subunits in monocytic cells.a) Typical RT-PCR of total RNA isolated from monocyte (M) and THP-1 cell (T) lysates detecting GABAA receptor subunits. Amplimers corresponding to the expected sizes were detected for α4, γ1 and δ subunits of the GABAA receptor in THP-1 cells, and β2 subunits in monocytes. RNA isolated from whole human brain (B) was used as a positive control. b) GABAA receptor β2 subunit expression in non–permeabilised human monocytes. Image of a human monocyte stained with Hoescht 33342 (left hand panel) to show the nucleus, and with a GABAA receptor β2-specific polyclonal antiserum (centre) revealing cell surface β2 subunits. The right panel shows the merged image. Positive controls were human cerebral cortex and negative controls were neutrophils (data not shown). Scale bar  = 5 µm. Data are typical of at least 6 independent experiments. Inset  =  typical immunoblot of a monocyte sample (left hand side) and control (neutrophil, right hand side) probed with the β2-specific antiserum and showing expected MWt for a β2 subunit.
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pone-0017152-g001: mRNA and protein expression of GABAA receptor subunits in monocytic cells.a) Typical RT-PCR of total RNA isolated from monocyte (M) and THP-1 cell (T) lysates detecting GABAA receptor subunits. Amplimers corresponding to the expected sizes were detected for α4, γ1 and δ subunits of the GABAA receptor in THP-1 cells, and β2 subunits in monocytes. RNA isolated from whole human brain (B) was used as a positive control. b) GABAA receptor β2 subunit expression in non–permeabilised human monocytes. Image of a human monocyte stained with Hoescht 33342 (left hand panel) to show the nucleus, and with a GABAA receptor β2-specific polyclonal antiserum (centre) revealing cell surface β2 subunits. The right panel shows the merged image. Positive controls were human cerebral cortex and negative controls were neutrophils (data not shown). Scale bar  = 5 µm. Data are typical of at least 6 independent experiments. Inset  =  typical immunoblot of a monocyte sample (left hand side) and control (neutrophil, right hand side) probed with the β2-specific antiserum and showing expected MWt for a β2 subunit.

Mentions: RT-PCR of RNA extracted from a human myleomonocytic cell line (THP-1 cells) and freshly prepared human monocytes showed that GABAA receptor subunits are expressed in these cells. In THP-1 cells, amplimers of the correct sizes and sequences were obtained for α4, β2, γ1 and δ GABAA receptor subunits (figure 1a). In fresh monocytes, only β2 subunits could be isolated (table 1). Expression of the β2 subunit protein was confirmed by immunoblotting and immunohistochemistry (figure 1b). These receptors are functional: whole cell patch clamp of THP-1 cells showed that both GABA and the GABAA-specific agonist muscimol elicited currents that were blocked by the GABAA antagonists picrotoxin and bicuculline at 1 mM and 100 µM respectively (figure 2a). The channels predominantly conducted chloride: in whole cell patch clamp experiments the reversal potential of the GABA-induced responses was 15.1±0.7 mV (all data  =  mean ± SEM, n≥5), close to the theoretical value of 16.1 mV for a chloride channel, and under bi-ionic conditions was predominantly chloride permeable (figures 2b and 2c). Due to the small amplitudes of the whole-cell responses and the fragility of the cells, it was not possible to obtain reliable concentration-response curves. However, the use of a membrane potential-sensitive dye revealed concentration-dependent decreases in fluorescence to the GABA-selective agonist muscimol (EC50  = 2.5 µM, pEC50  = 5.61±0.22, figures 2d and e), which were inhibited by the GABAA receptor antagonists bicuculline and picrotoxin with IC50s of 135 µM (pIC50  = 3.87±0.29) and 29.6 µM (pIC50  = 4.53±0.29) respectively. We also observed no potentiation of these effects in the presence of the benzodiazepine diazepam, and no responses to bicuculline and picrotoxin up to concentrations of 1 mM (data not shown). No specific radioligand binding could be observed with [3H]-GABA, [3H]-muscimol or [3H]-flunitrazepam (data not shown), which was consistent with the low levels of protein expression and small currents recorded in THP-1 cells.


Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

Wheeler DW, Thompson AJ, Corletto F, Reckless J, Loke JC, Lapaque N, Grant AJ, Mastroeni P, Grainger DJ, Padgett CL, O'Brien JA, Miller NG, Trowsdale J, Lummis SC, Menon DK, Beech JS - PLoS ONE (2011)

mRNA and protein expression of GABAA receptor subunits in monocytic cells.a) Typical RT-PCR of total RNA isolated from monocyte (M) and THP-1 cell (T) lysates detecting GABAA receptor subunits. Amplimers corresponding to the expected sizes were detected for α4, γ1 and δ subunits of the GABAA receptor in THP-1 cells, and β2 subunits in monocytes. RNA isolated from whole human brain (B) was used as a positive control. b) GABAA receptor β2 subunit expression in non–permeabilised human monocytes. Image of a human monocyte stained with Hoescht 33342 (left hand panel) to show the nucleus, and with a GABAA receptor β2-specific polyclonal antiserum (centre) revealing cell surface β2 subunits. The right panel shows the merged image. Positive controls were human cerebral cortex and negative controls were neutrophils (data not shown). Scale bar  = 5 µm. Data are typical of at least 6 independent experiments. Inset  =  typical immunoblot of a monocyte sample (left hand side) and control (neutrophil, right hand side) probed with the β2-specific antiserum and showing expected MWt for a β2 subunit.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044756&req=5

pone-0017152-g001: mRNA and protein expression of GABAA receptor subunits in monocytic cells.a) Typical RT-PCR of total RNA isolated from monocyte (M) and THP-1 cell (T) lysates detecting GABAA receptor subunits. Amplimers corresponding to the expected sizes were detected for α4, γ1 and δ subunits of the GABAA receptor in THP-1 cells, and β2 subunits in monocytes. RNA isolated from whole human brain (B) was used as a positive control. b) GABAA receptor β2 subunit expression in non–permeabilised human monocytes. Image of a human monocyte stained with Hoescht 33342 (left hand panel) to show the nucleus, and with a GABAA receptor β2-specific polyclonal antiserum (centre) revealing cell surface β2 subunits. The right panel shows the merged image. Positive controls were human cerebral cortex and negative controls were neutrophils (data not shown). Scale bar  = 5 µm. Data are typical of at least 6 independent experiments. Inset  =  typical immunoblot of a monocyte sample (left hand side) and control (neutrophil, right hand side) probed with the β2-specific antiserum and showing expected MWt for a β2 subunit.
Mentions: RT-PCR of RNA extracted from a human myleomonocytic cell line (THP-1 cells) and freshly prepared human monocytes showed that GABAA receptor subunits are expressed in these cells. In THP-1 cells, amplimers of the correct sizes and sequences were obtained for α4, β2, γ1 and δ GABAA receptor subunits (figure 1a). In fresh monocytes, only β2 subunits could be isolated (table 1). Expression of the β2 subunit protein was confirmed by immunoblotting and immunohistochemistry (figure 1b). These receptors are functional: whole cell patch clamp of THP-1 cells showed that both GABA and the GABAA-specific agonist muscimol elicited currents that were blocked by the GABAA antagonists picrotoxin and bicuculline at 1 mM and 100 µM respectively (figure 2a). The channels predominantly conducted chloride: in whole cell patch clamp experiments the reversal potential of the GABA-induced responses was 15.1±0.7 mV (all data  =  mean ± SEM, n≥5), close to the theoretical value of 16.1 mV for a chloride channel, and under bi-ionic conditions was predominantly chloride permeable (figures 2b and 2c). Due to the small amplitudes of the whole-cell responses and the fragility of the cells, it was not possible to obtain reliable concentration-response curves. However, the use of a membrane potential-sensitive dye revealed concentration-dependent decreases in fluorescence to the GABA-selective agonist muscimol (EC50  = 2.5 µM, pEC50  = 5.61±0.22, figures 2d and e), which were inhibited by the GABAA receptor antagonists bicuculline and picrotoxin with IC50s of 135 µM (pIC50  = 3.87±0.29) and 29.6 µM (pIC50  = 4.53±0.29) respectively. We also observed no potentiation of these effects in the presence of the benzodiazepine diazepam, and no responses to bicuculline and picrotoxin up to concentrations of 1 mM (data not shown). No specific radioligand binding could be observed with [3H]-GABA, [3H]-muscimol or [3H]-flunitrazepam (data not shown), which was consistent with the low levels of protein expression and small currents recorded in THP-1 cells.

Bottom Line: Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear.We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die.The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

View Article: PubMed Central - PubMed

Affiliation: Division of Anaesthesia, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom.

ABSTRACT

Background: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.

Principal findings: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

Significance: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

Show MeSH
Related in: MedlinePlus