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Nicotinic receptor β2 determines NK cell-dependent metastasis in a murine model of metastatic lung cancer.

Hao J, Shi FD, Abdelwahab M, Shi SX, Simard A, Whiteaker P, Lukas R, Zhou Q - PLoS ONE (2013)

Bottom Line: Further, nicotinic suppression of NF-κB-induced transcriptional activity in NK cells is dependent on nAChR β2.This nAChR subtype also plays a large role in the NK cell-mediated control of melanoma lung metastasis, in a murine lung metastasis model exposed to nicotine.Our findings suggest nAChR β2 as a prominent pathway for nicotine induced impairment of NK cell functions which contributes to the occurrence of smoking-related pathologies.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China. jhao0216@gmail.com

ABSTRACT
Cigarette smoke exposure markedly compromises the ability of the immune system to protect against invading pathogens and tumorigenesis. Nicotine is a psychoactive component of tobacco products that acts as does the natural neurotransmitter, acetylcholine, on nicotinic receptors (nAChRs). Here we demonstrate that natural killer (NK) cells strongly express nAChR β2. Nicotine exposure impairs the ability of NK cells to kill target cells and release cytokines, a process that is largely abrogated by nAChR β2 deficiency. Further, nicotinic suppression of NF-κB-induced transcriptional activity in NK cells is dependent on nAChR β2. This nAChR subtype also plays a large role in the NK cell-mediated control of melanoma lung metastasis, in a murine lung metastasis model exposed to nicotine. Our findings suggest nAChR β2 as a prominent pathway for nicotine induced impairment of NK cell functions which contributes to the occurrence of smoking-related pathologies.

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Nicotinic acetylcholine receptor (nAChR) expression on NK cells.Spleen, or lymph node cell suspensions were obtained RAG2−/− mice, and FACS was performed as described in the Materials and Methods section. A. Representative dot plots of cell populations before and after sorting are shown in the left- and right-hand panels, respectively. The purity of NK cells reached >98% purity after sorting. B. mRNA was purified from the sorted NK cells, and cDNA was synthesized by reverse transcription. PCR was then performed using primers specific for each mouse nAChR subunit. A positive control (+), using whole mouse brain cDNA, and a negative control (-), without cDNA, were included in each reaction. For each nAChR subunit, bands from the positive control and at least one sample from each cell type were sequenced and confirmed as the correct PCR product. C. nAChR expression assessed by radioligand saturation binding. Data depicted are representative from 2 to 3 separate experiments with similar results.
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pone-0057495-g001: Nicotinic acetylcholine receptor (nAChR) expression on NK cells.Spleen, or lymph node cell suspensions were obtained RAG2−/− mice, and FACS was performed as described in the Materials and Methods section. A. Representative dot plots of cell populations before and after sorting are shown in the left- and right-hand panels, respectively. The purity of NK cells reached >98% purity after sorting. B. mRNA was purified from the sorted NK cells, and cDNA was synthesized by reverse transcription. PCR was then performed using primers specific for each mouse nAChR subunit. A positive control (+), using whole mouse brain cDNA, and a negative control (-), without cDNA, were included in each reaction. For each nAChR subunit, bands from the positive control and at least one sample from each cell type were sequenced and confirmed as the correct PCR product. C. nAChR expression assessed by radioligand saturation binding. Data depicted are representative from 2 to 3 separate experiments with similar results.

Mentions: To determine nAChR mRNA expression on NK cells, we sorted NK cells from pooled splenocytes of RAG2−/− C57BL/6 mice that are devoid of T cells, NKT cells and B cells. mRNA was extracted from highly purified NK cells (Figure 1A) and nAChR expression profile was determined by PCR. Our results indicate that NK cells express nAChR α4, α5, α6, β2 and β3 but not α3, α7, α9; nor β4. Furthermore expression of β2 was particularly strong (Figure 1B). We then investigated whether nAChR proteins capable of engaging in nicotinic radioligand binding are assembled in NK cells by using an 125I-labeled epibatidine (I-Epi) binding assay (200 pM I-Epi +/− 100 mM carbamylcholine), as described elsewhere [14], [19], [20], [21]. NK cells (∼1 million) were purified by FACS, and mouse brain cortex homogenate was used as a positive control. Specific I-Epi binding was 99 fmol/mg protein in NK cells, about equal to specific binding levels in mouse whole brain (Figure 1C). These data demonstrate that NK cells express significant numbers of assembled nAChRs (as well as nAChR subunit mRNAs as shown previously) at levels that can be quantified with this assay. Since I-Epi binds to both β2- and β4-containing nAChRs [14], competitive binding with the nAChR β2-selective compound A85380 was used to discriminate between these two receptor subtypes. We found that A85380 largely abrogated I-Epi binding, demonstrating that the vast majority of nAChRs present on NK cells contain the β2 subunit (Figure 1C). Collectively, our results reveal that NK cells express an array of nAChR mRNAs and that most of these subunits assemble into β2-containing nAChRs. The strong expression of nAChR β2 in NK cells, both at the mRNA and protein levels suggest that β2-containing nAChRs are candidates for mediation of nicotine’s effects on NK cell activity.


Nicotinic receptor β2 determines NK cell-dependent metastasis in a murine model of metastatic lung cancer.

Hao J, Shi FD, Abdelwahab M, Shi SX, Simard A, Whiteaker P, Lukas R, Zhou Q - PLoS ONE (2013)

Nicotinic acetylcholine receptor (nAChR) expression on NK cells.Spleen, or lymph node cell suspensions were obtained RAG2−/− mice, and FACS was performed as described in the Materials and Methods section. A. Representative dot plots of cell populations before and after sorting are shown in the left- and right-hand panels, respectively. The purity of NK cells reached >98% purity after sorting. B. mRNA was purified from the sorted NK cells, and cDNA was synthesized by reverse transcription. PCR was then performed using primers specific for each mouse nAChR subunit. A positive control (+), using whole mouse brain cDNA, and a negative control (-), without cDNA, were included in each reaction. For each nAChR subunit, bands from the positive control and at least one sample from each cell type were sequenced and confirmed as the correct PCR product. C. nAChR expression assessed by radioligand saturation binding. Data depicted are representative from 2 to 3 separate experiments with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585320&req=5

pone-0057495-g001: Nicotinic acetylcholine receptor (nAChR) expression on NK cells.Spleen, or lymph node cell suspensions were obtained RAG2−/− mice, and FACS was performed as described in the Materials and Methods section. A. Representative dot plots of cell populations before and after sorting are shown in the left- and right-hand panels, respectively. The purity of NK cells reached >98% purity after sorting. B. mRNA was purified from the sorted NK cells, and cDNA was synthesized by reverse transcription. PCR was then performed using primers specific for each mouse nAChR subunit. A positive control (+), using whole mouse brain cDNA, and a negative control (-), without cDNA, were included in each reaction. For each nAChR subunit, bands from the positive control and at least one sample from each cell type were sequenced and confirmed as the correct PCR product. C. nAChR expression assessed by radioligand saturation binding. Data depicted are representative from 2 to 3 separate experiments with similar results.
Mentions: To determine nAChR mRNA expression on NK cells, we sorted NK cells from pooled splenocytes of RAG2−/− C57BL/6 mice that are devoid of T cells, NKT cells and B cells. mRNA was extracted from highly purified NK cells (Figure 1A) and nAChR expression profile was determined by PCR. Our results indicate that NK cells express nAChR α4, α5, α6, β2 and β3 but not α3, α7, α9; nor β4. Furthermore expression of β2 was particularly strong (Figure 1B). We then investigated whether nAChR proteins capable of engaging in nicotinic radioligand binding are assembled in NK cells by using an 125I-labeled epibatidine (I-Epi) binding assay (200 pM I-Epi +/− 100 mM carbamylcholine), as described elsewhere [14], [19], [20], [21]. NK cells (∼1 million) were purified by FACS, and mouse brain cortex homogenate was used as a positive control. Specific I-Epi binding was 99 fmol/mg protein in NK cells, about equal to specific binding levels in mouse whole brain (Figure 1C). These data demonstrate that NK cells express significant numbers of assembled nAChRs (as well as nAChR subunit mRNAs as shown previously) at levels that can be quantified with this assay. Since I-Epi binds to both β2- and β4-containing nAChRs [14], competitive binding with the nAChR β2-selective compound A85380 was used to discriminate between these two receptor subtypes. We found that A85380 largely abrogated I-Epi binding, demonstrating that the vast majority of nAChRs present on NK cells contain the β2 subunit (Figure 1C). Collectively, our results reveal that NK cells express an array of nAChR mRNAs and that most of these subunits assemble into β2-containing nAChRs. The strong expression of nAChR β2 in NK cells, both at the mRNA and protein levels suggest that β2-containing nAChRs are candidates for mediation of nicotine’s effects on NK cell activity.

Bottom Line: Further, nicotinic suppression of NF-κB-induced transcriptional activity in NK cells is dependent on nAChR β2.This nAChR subtype also plays a large role in the NK cell-mediated control of melanoma lung metastasis, in a murine lung metastasis model exposed to nicotine.Our findings suggest nAChR β2 as a prominent pathway for nicotine induced impairment of NK cell functions which contributes to the occurrence of smoking-related pathologies.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China. jhao0216@gmail.com

ABSTRACT
Cigarette smoke exposure markedly compromises the ability of the immune system to protect against invading pathogens and tumorigenesis. Nicotine is a psychoactive component of tobacco products that acts as does the natural neurotransmitter, acetylcholine, on nicotinic receptors (nAChRs). Here we demonstrate that natural killer (NK) cells strongly express nAChR β2. Nicotine exposure impairs the ability of NK cells to kill target cells and release cytokines, a process that is largely abrogated by nAChR β2 deficiency. Further, nicotinic suppression of NF-κB-induced transcriptional activity in NK cells is dependent on nAChR β2. This nAChR subtype also plays a large role in the NK cell-mediated control of melanoma lung metastasis, in a murine lung metastasis model exposed to nicotine. Our findings suggest nAChR β2 as a prominent pathway for nicotine induced impairment of NK cell functions which contributes to the occurrence of smoking-related pathologies.

Show MeSH
Related in: MedlinePlus