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Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29
Life Sciences 72 (2003) 2091 – 2094 www.elsevier.com/locate/lifescie
Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29 Andrea E. Summers, Clifford J. Whelan *, Mike E. Parsons Department of Biosciences, CP Snow Building, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, AL10 9AB, UK
Abstract In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFa-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFa (50 ng ml 1) in the absence and presence of nicotine (10 11 – 10 6 M). HT29 cells contained mRNA for h1, a4, a5, and a7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 F 0.51 ng ml 1 (n = 5). Nicotine significantly inhibited TNFa-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10 7 M (2.39 F 0.78 ng ml 1, n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are a7. D 2003 Elsevier Science Inc. All rights reserved.
Introduction Smoking has been found to reduce development and severity of ulcerative colitis (UC) [1]. Nicotine has been shown to be effective in the treatment of UC, but the mechanism underlying this action is still unclear [1]. The synthesis of the neutrophil chemoattractant interleukin-8 (IL-8), a potent factor for recruitment and activation of inflammatory cells, has been shown to be generated in large amounts in the colonic mucosa of patients with UC [2]. Studies on the effect of transdermal nicotine on patients with the active form of UC showed a significant decrease of IL-8 mRNA expression as determined by colonic * Corresponding author. Tel.: +44-1707-284-000; fax: +44-1707-285-046. E-mail address: [email protected] (C.J. Whelan). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00089-4
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biopsies [3]. Moreover, IL-8 was found to be significantly downregulated in colonic tissue sections after 24 hrs nicotine exposure in vitro [4]. These data thus provide part of a possible explanation for the beneficial effects of nicotine observed in UC. However, a number of infiltrating inflammatory cells, such as monocytes and macrophages are potent generators of IL-8, and it was thus the aim of the present experiments to investigate the extent to which colonic epithelial cells contribute to IL-8 release and the inhibitory effect of nicotine on the release of this cytokine in active UC.
Methods The human colonic epithelial cell line HT29 was cultured as previously described [5]. For stimulation experiments, cells were seeded at 5 105 cells ml 1 in 24 well culture plates (BD, Falcon, UK) and grown to confluence. The wells were washed twice with PBS (pH 7.4), 1 ml of foetal calf serum free medium and ( )-nicotine (Sigma, UK) to a final concentration of 1 10 11 –1 10 6 M was added per well. The plates were incubated for 2 h after which the cells were activated with TNFa (50 ng ml 1) (PeproTech, UK) and incubated for a further 6 h. The cell free supernatant was removed and assayed by ELISA for IL-8 content. IL-8 ELISA was carried out as per the manufacturer’s instructions (Pharmingen, UK). Total RNA was extracted from HT29 cells by kit as per manufacturer’s protocol (QIAGEN, UK). RT-PCR primers were designed for the human a1–7, a10 and h1–4 nAChR subunit from mRNA sequences published in NCBI, with the Generunner program. Primers were synthesised by Invitrogen, (UK). RT-PCR was carried out from 1Ag total RNA with reagents from the Access RT-PCR System kit (Promega, UK), following the manufacturer’s instructions. The RT-PCR products were examined by electrophoresis on 1% agarose gels and visualised by ethidium bromide. The size of the products was estimated from the migration of a DNA marker run concurrently (GIBCO, UK).
Results Using the RT-PCR technique we have demonstrated the presence of the a4, a5 and a7 as well as h1 subunit mRNA of the nAChR in HT29 cells. However, none of the bands corresponding to the a1–3, 10 and h2–4 amplification products could be detected, indicating that these cells lack these subtypes. As a positive control for the quality of RNA and cDNA synthesis, amplification of the housekeeping gene mRNA h-actin was performed and the expected product of 285 bp was detected. PCR performed on each sample of RNA that had not been reverse transcribed to cDNA were used as negative controls and showed no amplification products. In all three experiments the mRNAs encoding the a1–3, 10 and h2– 4 nAChR subunits were not found to be present in HT29 cells, although the PCR primers specific for the a1–3, 10 and h2–4 subunit mRNA were shown to amplify fragments of the expected sizes (190, 306, 296, 327 bp and 309, 418 and 220 bp respectively) from commercially available total human brain mRNA (ORIGENE, UK). Product for the amplification of the a6 subunit was absent from both HT29 cells and total human brain mRNA. Activation of HT29 cells increased IL-8 release from undetectable amounts to a peak value of 3.92 F 0.51 ng ml 1 (n = 5). Addition of ( )-nicotine significantly inhibited IL-8 release in a concentration related manner with significant inhibition occurring at 10 9 to 10 7 M (2.67 F 0.63, 2.58 F 0.52 and 2.39 F 0.78 ng ml 1 respectively), which represents a maximum inhibition of 39% (Fig. 1) The
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Fig. 1. Effect of ( )-nicotine on the IL-8 release from HT29 cells activated for 6h with TNFa (50 ng ml 1) in the absence and presence of nicotine (10 11 – 10 6 M). Nicotine caused a concentration related significant inhibition of IL-8 release from TNFa (50 ng ml 1) activated HT29 cells after 6 h. Cytokine release is presented as mean F 1SD and represent 5 individual experiments carried out in duplicate. Data were analysed with one way ANOVA (* p < 0.05).
addition of ( )-nicotine to unstimulated HT29 cells had no effect on IL-8 release. The viability of HT29 cells was not affected by the addition of ( )-nicotine (10 11 –10 6 M) after 6 hrs, as measured by the ability of cells to reduce 3-[4,5-2-yl]-2,5-diphenyltetrazolium bromide (data not shown).
Discussion The main purpose of the present study was to assess the effect of nicotine on the human colonic epithelial cell line HT29 and to determine if any effect could be mediated via a nAChR. Nicotine has been found to modulate secretory components in this cell line [6], indicating the possible presence of functional nAChRs in the membrane of these cells. Our results show that mRNAs encoding for the a4, a5 and a7 as well as the h1 subunit nAChRs are expressed in the HT29 cell line. However, in order to form a functional receptor, both a4 and a5 subunits require the presence of a structural h subunit other than h1, which is known to only combine with a1 and g, y or q to form the pentameric muscle nAChR. Thus of those subunit mRNAs present in the HT29 cells, only a7 is able to form a functional receptor by homodimerisation. Due to the absence of an amplification product for the a6 subunit from both HT29 cells and the human brain mRNA control, the involvement of a6 cannot be shown. The concentration related inhibition by ( )-nicotine of IL-8 release from HT29 cells observed in these experiments, together with the modulation by ( )-nicotine of secretory components shown in the same cell line [6], suggests that there are functional nAChRs in the membrane of these cells. The molecular biology described above indicates that this receptor may be the a-bungarotoxin sensitive, homomeric a7 nAChR. However, further functional experiments will be required to demonstrate the presence of a fully functional a7 nAChR in the membrane of HT29 cells. The results on this colonic epithelial cell line appear to confirm the potential involvement in vivo of the colonic epithelium in the beneficial effects of nicotine in clinically active UC.
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References [1] Sandborn WJ. Nicotine therapy for ulcerative colitis: a review of rationale, mechanisms, pharmacology and clinical results. American Journal of Gastroenterology 1999;94:1161 – 71. [2] Daig R, Andus T, Aschenbrenner E, Falk W, Scholmerich J, Gross V. Increased interleukin 8 expression in the colon mucosa of patients with inflammatory bowel disease. Gut 1996;38:216 – 22. [3] Louvet B, Buisine MP, Desreumaux P, Tremaine WJ, Aubert JP, Porchet N, Capron M, Cortot A, Colombel JF, Sandborn WJ. Transdermal nicotine decreases mucosal IL-8 expression but has no effect on mucin gene expression in ulcerative colitis. Inflammatory Bowel Diseases 1999;5:174 – 81. [4] Bhatti MA, Hodgson HJF. Peripheral blood pro-inflammatory cytokine profile in active inflammatory bowel disease (IBD) differ between smokers and non-smokers. Gut 1997;40:F294. [5] Ihenetu K., Mollemen A., Parsons M.E., Whelan C.J. Inhibition of interleukin-8 release in the human colonic epithelial cell line HT-29 by cannabinoids. European Journal of Pharmacology 2003;458:207 – 15. [6] Gregory RL, Gfell LE. Effect of nicotine on secretory component synthesis by secretory epithelial cells. Clinical and Diagnostic Laboratory Immunology 1996;3:578 – 83.
Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29 Andrea E. Summers, Clifford J. Whelan *, Mike E. Parsons Department of Biosciences, CP Snow Building, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, AL10 9AB, UK
Abstract In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFa-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFa (50 ng ml 1) in the absence and presence of nicotine (10 11 – 10 6 M). HT29 cells contained mRNA for h1, a4, a5, and a7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 F 0.51 ng ml 1 (n = 5). Nicotine significantly inhibited TNFa-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10 7 M (2.39 F 0.78 ng ml 1, n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are a7. D 2003 Elsevier Science Inc. All rights reserved.
Introduction Smoking has been found to reduce development and severity of ulcerative colitis (UC) [1]. Nicotine has been shown to be effective in the treatment of UC, but the mechanism underlying this action is still unclear [1]. The synthesis of the neutrophil chemoattractant interleukin-8 (IL-8), a potent factor for recruitment and activation of inflammatory cells, has been shown to be generated in large amounts in the colonic mucosa of patients with UC [2]. Studies on the effect of transdermal nicotine on patients with the active form of UC showed a significant decrease of IL-8 mRNA expression as determined by colonic * Corresponding author. Tel.: +44-1707-284-000; fax: +44-1707-285-046. E-mail address: [email protected] (C.J. Whelan). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00089-4
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biopsies [3]. Moreover, IL-8 was found to be significantly downregulated in colonic tissue sections after 24 hrs nicotine exposure in vitro [4]. These data thus provide part of a possible explanation for the beneficial effects of nicotine observed in UC. However, a number of infiltrating inflammatory cells, such as monocytes and macrophages are potent generators of IL-8, and it was thus the aim of the present experiments to investigate the extent to which colonic epithelial cells contribute to IL-8 release and the inhibitory effect of nicotine on the release of this cytokine in active UC.
Methods The human colonic epithelial cell line HT29 was cultured as previously described [5]. For stimulation experiments, cells were seeded at 5 105 cells ml 1 in 24 well culture plates (BD, Falcon, UK) and grown to confluence. The wells were washed twice with PBS (pH 7.4), 1 ml of foetal calf serum free medium and ( )-nicotine (Sigma, UK) to a final concentration of 1 10 11 –1 10 6 M was added per well. The plates were incubated for 2 h after which the cells were activated with TNFa (50 ng ml 1) (PeproTech, UK) and incubated for a further 6 h. The cell free supernatant was removed and assayed by ELISA for IL-8 content. IL-8 ELISA was carried out as per the manufacturer’s instructions (Pharmingen, UK). Total RNA was extracted from HT29 cells by kit as per manufacturer’s protocol (QIAGEN, UK). RT-PCR primers were designed for the human a1–7, a10 and h1–4 nAChR subunit from mRNA sequences published in NCBI, with the Generunner program. Primers were synthesised by Invitrogen, (UK). RT-PCR was carried out from 1Ag total RNA with reagents from the Access RT-PCR System kit (Promega, UK), following the manufacturer’s instructions. The RT-PCR products were examined by electrophoresis on 1% agarose gels and visualised by ethidium bromide. The size of the products was estimated from the migration of a DNA marker run concurrently (GIBCO, UK).
Results Using the RT-PCR technique we have demonstrated the presence of the a4, a5 and a7 as well as h1 subunit mRNA of the nAChR in HT29 cells. However, none of the bands corresponding to the a1–3, 10 and h2–4 amplification products could be detected, indicating that these cells lack these subtypes. As a positive control for the quality of RNA and cDNA synthesis, amplification of the housekeeping gene mRNA h-actin was performed and the expected product of 285 bp was detected. PCR performed on each sample of RNA that had not been reverse transcribed to cDNA were used as negative controls and showed no amplification products. In all three experiments the mRNAs encoding the a1–3, 10 and h2– 4 nAChR subunits were not found to be present in HT29 cells, although the PCR primers specific for the a1–3, 10 and h2–4 subunit mRNA were shown to amplify fragments of the expected sizes (190, 306, 296, 327 bp and 309, 418 and 220 bp respectively) from commercially available total human brain mRNA (ORIGENE, UK). Product for the amplification of the a6 subunit was absent from both HT29 cells and total human brain mRNA. Activation of HT29 cells increased IL-8 release from undetectable amounts to a peak value of 3.92 F 0.51 ng ml 1 (n = 5). Addition of ( )-nicotine significantly inhibited IL-8 release in a concentration related manner with significant inhibition occurring at 10 9 to 10 7 M (2.67 F 0.63, 2.58 F 0.52 and 2.39 F 0.78 ng ml 1 respectively), which represents a maximum inhibition of 39% (Fig. 1) The
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Fig. 1. Effect of ( )-nicotine on the IL-8 release from HT29 cells activated for 6h with TNFa (50 ng ml 1) in the absence and presence of nicotine (10 11 – 10 6 M). Nicotine caused a concentration related significant inhibition of IL-8 release from TNFa (50 ng ml 1) activated HT29 cells after 6 h. Cytokine release is presented as mean F 1SD and represent 5 individual experiments carried out in duplicate. Data were analysed with one way ANOVA (* p < 0.05).
addition of ( )-nicotine to unstimulated HT29 cells had no effect on IL-8 release. The viability of HT29 cells was not affected by the addition of ( )-nicotine (10 11 –10 6 M) after 6 hrs, as measured by the ability of cells to reduce 3-[4,5-2-yl]-2,5-diphenyltetrazolium bromide (data not shown).
Discussion The main purpose of the present study was to assess the effect of nicotine on the human colonic epithelial cell line HT29 and to determine if any effect could be mediated via a nAChR. Nicotine has been found to modulate secretory components in this cell line [6], indicating the possible presence of functional nAChRs in the membrane of these cells. Our results show that mRNAs encoding for the a4, a5 and a7 as well as the h1 subunit nAChRs are expressed in the HT29 cell line. However, in order to form a functional receptor, both a4 and a5 subunits require the presence of a structural h subunit other than h1, which is known to only combine with a1 and g, y or q to form the pentameric muscle nAChR. Thus of those subunit mRNAs present in the HT29 cells, only a7 is able to form a functional receptor by homodimerisation. Due to the absence of an amplification product for the a6 subunit from both HT29 cells and the human brain mRNA control, the involvement of a6 cannot be shown. The concentration related inhibition by ( )-nicotine of IL-8 release from HT29 cells observed in these experiments, together with the modulation by ( )-nicotine of secretory components shown in the same cell line [6], suggests that there are functional nAChRs in the membrane of these cells. The molecular biology described above indicates that this receptor may be the a-bungarotoxin sensitive, homomeric a7 nAChR. However, further functional experiments will be required to demonstrate the presence of a fully functional a7 nAChR in the membrane of HT29 cells. The results on this colonic epithelial cell line appear to confirm the potential involvement in vivo of the colonic epithelium in the beneficial effects of nicotine in clinically active UC.
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A.E. Summers et al. / Life Sciences 72 (2003) 2091–2094
References [1] Sandborn WJ. Nicotine therapy for ulcerative colitis: a review of rationale, mechanisms, pharmacology and clinical results. American Journal of Gastroenterology 1999;94:1161 – 71. [2] Daig R, Andus T, Aschenbrenner E, Falk W, Scholmerich J, Gross V. Increased interleukin 8 expression in the colon mucosa of patients with inflammatory bowel disease. Gut 1996;38:216 – 22. [3] Louvet B, Buisine MP, Desreumaux P, Tremaine WJ, Aubert JP, Porchet N, Capron M, Cortot A, Colombel JF, Sandborn WJ. Transdermal nicotine decreases mucosal IL-8 expression but has no effect on mucin gene expression in ulcerative colitis. Inflammatory Bowel Diseases 1999;5:174 – 81. [4] Bhatti MA, Hodgson HJF. Peripheral blood pro-inflammatory cytokine profile in active inflammatory bowel disease (IBD) differ between smokers and non-smokers. Gut 1997;40:F294. [5] Ihenetu K., Mollemen A., Parsons M.E., Whelan C.J. Inhibition of interleukin-8 release in the human colonic epithelial cell line HT-29 by cannabinoids. European Journal of Pharmacology 2003;458:207 – 15. [6] Gregory RL, Gfell LE. Effect of nicotine on secretory component synthesis by secretory epithelial cells. Clinical and Diagnostic Laboratory Immunology 1996;3:578 – 83.