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Expression of alpha subunit genes of nicotinic acetylcholine receptors in human lymphocytes
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Neuroscience Letters 214 (1996) 171-174
NIUIIOSHHg LETTERS
Expression of alpha subunit genes of nicotinic acetylcholine receptors in human lymphocytes 1 C. Hiernke a'*, M. Stolp a, S. Reuss b, A. Wevers d, S. Reinhardt c, A. Maelicke c, S.
S c h l e g e l a,
H. S c h r 6 d e r d aDepartment of Psychiatry, University of Mainz, Untere Zahlbacher Strausse 8, D-55101 Mainz, Germany bDepartment of Anatomy, University of Mainz, Mainz, Germany CDepartmentof Physiological Chemistry, University of Mainz, Mainz, Germany dDepartment of Anatomy, University of K6ln, Ki~ln, Germany Received 13 May 1996; revised version received 17 July 1996; accepted 17 July 1996
Abstract Using immunohistochemistry and in situ hybridisation, we have studied whether c~-subunits of nicotinic acetylcholine receptors (nAChRs) are expressed in human lymphocytes. Cells were isolated by differential low speed gradient centrifugation from heparinised venous blood of 10 healthy volunteers. Receptor sites were visualised using the monoclonal antibody WF6 which specifically recognises c~-isoforms from several species including man. For visualisation of transcripts, digoxigenin-labeUed cRNA probes for a4- and t~3subunits were used. Immunostaining revealed specific binding of WF6 to isolated human lymphoid cells. The antibody was bound to most cells and concentraled preferentially in the perinuclear/surface region. The immunoreactivity resembled that observed after application of an antibody recognising CD4 surface proteins which was conducted for comparison. In situ-hybridisation revealed that the t~4-subunit genes of nAChRs was expressed in lymphocytes of all probands. The a3-subunit was found~ with lower intensity than t~4-transcripts, in eight of the 10 individuals. Control incubations with corresponding sense cRNAs were negative. It is concluded that human lymphocytes are able to express c~-subunit genes of nAChRs.
Keywords: Acetylcholine receptor; Lymphocytes; Nicotinic c~-subunits; WF6 antibody; Immunostaining; In situ hybridisation; Neuroimmunology
Acetylcholine in the brain is believed to play a major role in cognitive funt'tions [12]. Postmortem studies revealed a decrease in the concentration of acetylcholine receptors in the brain of patients suffering from senile dementia of the Alzheimer type (SDAT) [6,18]. Adem et al. [1] reported that these changes in the brain are also reflected by peripheral blood cells. In lymphocytes from SDAT patients, the number of nicotinic binding sites was found to be lower than in blood cells of age-matched controis [1]. Human lymphocytes express receptors for several neurotransmitters including acetylcholine [2,9]. Binding studies with radioligands revealed predominantly muscarinic receptors [2,9], whereas data on the occurrence of nicotinic acetylcholine receptors are less conclusive. * Corresponding author. Tel.: +49 6131 177363; fax: +49 6131 176690. t This paper contains result.,~ of the M.D. thesis of M. Stolp.
0304-3940/96/$12.00
Most of the binding of nicotinic ligands was found to be non-specific and non-saturable [9] and the concentration of specific binding sites was very low. Immunodetection of acetylcholine receptors has so far not been performed in lymphocytes. Moreover, studies on distinct subunits of nicotinic acetylcholine receptors whose expression differs in the brain and in peripheral organs [4,7,12] are lacking. Using immunohistochemistry and in situ hybridisation, we demonstrated that ot-subunit genes of nicotinic acetylcholine receptors are expressed in human lymphocytes. The study included 10 healthy volunteers (5 female and 5 male, ages 23-40 years). They were all students or staff of the Department of Psychiatry, University of Mainz, Germany and participated in the trial on an informed consent basis. Blood (10 ml) was withdrawn from the antecubital vein in heparinised tubes. Peripheral blood lymphoid (PBL)
© 1996 Elsevier Science Ireland Ltd. All rights reserved
PII S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 12908-6
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c. Hiemke et al. / Neuroscience Letters 214 (1996) 171-174
cells were prepared by a standard Ficoll/Histopac procedure. The blood was mixed with 25 ml phosphate-buffered saline (PBS) and aliquoted into 50 ml tubes, underlaid with 10 ml Ficoll separating solution (Biochrom KG, Berlin, Germany) and centrifuged for 15 min at 900 g. Cells were washed three times to remove serum components and Ficoll solution. The final preparation contained 0.31.6 × 106 cells/ml. Using trypan blue exclusion staining revealed that at least 96% of the cells had remained viable. Twenty microliters of the cell suspension were applied on poly-L-lysine-coated slides and dried at room temperature (RT). The cells were fixed using acetone for 10 min at -20°C and subsequently washed twice at RT for 5 rain with PBS. To block endogenous peroxidases, the preparation was exposed to methanol containing 0.3% H202 for 20 min at RT. After incubation with 20% (w/v) swine serum albumin (Serva, Heidelberg, Germany) to reduce non-specific binding, the slides were incubated overnight at 4°C with the primary antibody WF6 (1:100) in a moist chamber. After washing in PBS (2 × 10 rain), the samples were incubated for 60 min with goat anti-mouse IgG conjugated to biotin (1:50; Amersham-Buchler, Braunschweig, Germany). After washing out unbound antibodies with PBS (2 x 10 min), a streptavidin-peroxidase complex (1:50; 60 min at RT; Amersham-Buchler) was applied. After another PBS wash (2 × 10 rain), the enzyme activity was visualised by reaction with diaminobenzidine (DAB; Sigma, Germany) (1 mg DAB and 0.1 mg H202/ml in Tris buffer 0.05 M; pH 7.4) in the dark at RT for 10 min. For final washing (2 × 5 min), Tris-HCl (0.05 M; pH 7.4) was used. The samples were dehydrated in a graded series of ethanols followed by xylene and mounted using commercial imbedding medium (Fluka, Neu-Ulm, Germany). Specificity controls were performed for each cell preparation by omitting primary and/or secondary antibodies and incubating the cells under otherwise unchanged conditions. For comparison of the binding of WF6 to blood cells, the WF6 antibody was replaced, under otherwise unchanged conditions, by an antibody to cluster differentiating type 4 antigen (CD4; diluted 1:100, Coulter, Krefeld, Germany), a glycoprotein expressed primarily on the cell surface of T-helper cells [8]. For detection of c~4 and a3 mRNA, digoxigeninlabelled antisense and sense cRNA probes were prepared [17] from rat a4-1 and ot3-subunits, respectively. Rat and human ot3-subunits exhibit 93% amino acid sequence homology [4], and the ~4-1 variant 82% [7]. A previous investigation has shown that the rat cRNA probes are suitable for labelling nicotinic acetylcholine receptor subunit mRNAs in the human brain [17]. The protocol previously established for brain tissue [17] was also used for the detection of lymphocyte mRNAs, with a few minor modifications. Isolated mononuclear blood cell suspensions, containing 0.6-1.6 × 105 or 0.61.6 × 10 4 cells/ml were mounted on adhesive slides (Bio-Rad, Mtinchen, Germany) by cytocentrifugation
(Shandon-Elliot). The slides had been prewashed in absolute ethanol and dried before use. After 20 min drying at RT, the samples were stored frozen (-80°C) until assayed. Before hybridisation, the slides were brought to RT for 1.5 h, subsequently fixed in 4% (v/v) paraformaldehyde in PBS (5 min at RT) and washed again twice with 2 × SSC (0.3 M NaC1, 30 mM sodium citrate; pH 7.0) at RT, followed by incubation in triethanolamine, 0.1 M (pH 8.0) and acetic acid, 0.0026% (v/v) ( l x l 0 min) and two washes in 2x SSC (2 × 1 min at RT) and PBS (lxl min). Finally, the samples were incubated with 0.1 M glycine and 0.2 M Tris-HCl (pH 7.4) (30 min at RT) and then washed in 2 × SSC (2 x 5 min at RT). Prehybridisation and hybridisation were performed at 50°C with a content of 50% formamide as described in detail earlier [17]. Non-hybridised probes were removed by incubation at 52°C in 2 x SSC, containing 50% formamide. Detection of hybridised digoxigenin-labelled cRNA probes were achieved by incubation with an alkaline phosphatase, conjugated digoxigenin-antibody (Boehringer, Mannheim, Germany) and a colour substrate reaction using 5bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium chloride. Control in situ hybridisations were performed using labelled sense cRNA instead of antisense cRNA or hybridisation solution without probe. Isolated human lymphocytes showed good preservation after staining by routine histological methods and light microscopical inspection of the ceils (data not shown). The monoclonal antibody WF6, raised against the nicotinic acetylcholine receptor of Torpedo m a r m o r a f a [3], was bound specifically to the lymphocytes. Peroxidase staining was not detectable when primary and/or secondary antibody were omitted. Immunostaining was detected in the perinuclear/surface region of the lymphocytes (Fig. 1A-C) without allowing the exact localisation of the immunostaining on the subcellular level. When replacing the WF6 antibody by an antibody to CD4, the light microscopical results resembled closely that seen using WF6 (Fig. 1D). Using routine histological staining in addition to immunostaining, it seemed likely that the vast majority of lymphocytes bound WF6 (data not shown). Moreover, all volunteers studied for WF6 binding exhibited similar patterns of specific immunostaining. In situ hybridisation revealed that lymphocytes of all volunteers expressed at least one of the nAChR subunit genes tested (Fig. 2). Incubations with labelled sense cRNA (Fig. 2C,F) or under omission of the antisense cRNA yielded negative results. Using the ~4-1 probe, hybridisation was observed within 5 to 20 min after beginning of the enzyme reaction (Fig. 2A,B). Incubations with sense cRNA (Fig. 2C) or without cRNA probes were negative. Calculation of the number of or4 antisense-labelled cells indicated that about 80-90% of them expressed mRNA for this isoform. ~3-Subunit mRNA was detected in only eight of the 10 individuals (Fig. 2D,E). Moreover, the reaction times
C. Hiemke et al. / Neuroscience Letters 214 (1996) 171-174
qi*
173
¢b ~D~O
e
o D
cB~
~
....
o
q D
Fig. 1. Light microscopic demonstration of nicotinic acetylcholine receptor immunoreactivity on human lymphocytes using the monoclonal antibody WF6 in cells of a male (A,B) and female (C) subject. The typical cell surface protein on T helper-cells, CD4, was visualised using the WF6 immunostaining protocol and a CD4 antibody instead of WF6 (D). Magnification bar, (A) 50 #m; 5 /zm (B-D).
required for detection of phosphatase activity were 20-60 min which was three to five times longer than required for detection of ct4-subunits. Incubations with c~3 sense cRNA were negative (Fig. 2F).
To our knowledge, this is the first report demonstrating that human lymphocytes express c~-subunit genes of nicotinic acetylcholine receptors. Immunostaining was performed with WF6, a monoclo-
Fig. 2. Expression of a4- and u3-subunits mRNA of nicotinic acetylcholine receptors in human lymphocytes. Transcripts were visualised using digoxigenin-labelled cRNA antisense probes for ~4 (A,B) and a3-subunits (D,E). Control samples using sense RNA probes for a4- (C) or a3-subunits were negative and visualised by phase contrast microscopy (F). Magnification bar, (A,D) 100/zm; (B,C,E,F) 10 #m.
174
C. Hiemke et al. / Neuroscience Letters 214 (1996) 171-174
nal antibody raised in mice against the purified nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [3]. This antibody recognises the acetylcholine binding domain of some nicotinic acetylcholine receptor isoforms [3,16] in tissues from various species including man [10,14]. The staining observed here indicates that a protein with the antigenic properties of nicotinic acetylcholine receptor ot-subunits is synthesised by human lymphocytes. In regard to the subcellular localisation of the WF6 binding site, light microscopic inspection does not suffice to differentiate between the cell surface and the cytoplasm. Since CD4 staining yielded results similar to those obtained with WF6 it was concluded that WF6 immunoreactivity was localised preferentially on the surface of the lymphocytes. Further evidence for the synthesis of nicotinic acetylcholine receptors by human lymphocytes is provided by the in situ hybridisation study, mRNAs of two ot-subunit isoforms were identified using cRNA antisense probes for o~4- and ct3-isoforms. It seemed likely that a4-subunits were more abundant than ot3-subunits, since staining was more pronounced. Since the c~4-subunit is the predominant subunit in the human brain [12,13], it may be concluded that lymphocytes express a composition of o~-isoforms similar to the brain. This suggestion, however, needs further clarification. Moreover, it has to be shown if/3subunit genes are expressed because coexpression of c¢ and /3-subunits is necessary to encode for a functional acetylcholine receptor [12]. A functional role of these receptors is supported by pharmacological studies demonstrating that acetylcholine receptor agonists affect immune functions of lymphocytes [5,11,15]. Our morphological findings that used two different approaches, i.e. immunohistochemistry and in situ hybridisation, and functional studies suggest that nicotinic acetylcholine receptors are present on human lymphocytes. It therefore seems promising to look for changes of nicotinic acetylcholine receptor properties on lymphocytes in connection to demential diseases, since changes in the expression of nicotinic acetylcholine receptors on lymphocytes might reflect alterations of cholinergic neurotransmission in the brain as suggested for SDAT and other diseases accompanied by cognitive dysfunctions [1]. This work was supported by the Deutsche Forschungsgemeinschaft (grants Schr 283•8-2 and Ma 599/18-1). We thank Marita W6rsdfrfer for helpful technical advise, Gabriele Stroba for skilful technical assistance, Ursula Disque-Kaiser for photographic work and Michaela Jahnke for typing the manuscript.
[1] Adem, A., Nordberg, A., Bucht, G. and Winblad, A., Extraneural cholinergic markers in Alzheimer's and Parkinson's disease, Prog. Neuro-Psychophannacol. Biol. Psychiatry, 10 (1986) 247-257. [2] Atweh, S.F., Grayhack, M.S. and Richman, D.P., A cholinergic receptor site on murine lymphocytes with novel binding characteristics, Life Sci., 35 (1984) 2459-2469. [3] Fels, G., Pliirner-Wilk, R., Schreiber, M. and Maelicke, A., A monoclonal antibody interfering with binding and response of the acetylcholine receptor, J. Biol. Chem., 33 (1986) 15746-15754. [4] Fomasari, D., Chini, B., Tarroni, P. and Clementi, F., Molecular cloning of human neuronal nicotinic receptor ~3 subunit, Neurosci. Lett., 111 (1990) 351-356. [5] Geng, Y.M., Savage, S.M., Johnson, L.J., Seagrave, J. and Sopori, M.L., Effects of nicotine on the immune response. 1. Chronic exposure to nicotine impairs antigen receptor-mediated signal transduction in lymphocytes, Toxicol. Appl. Pharmacol., 135 (1995) 268-278. [6] Giacobini, E., Cholinergic receptors in human brain: effects of aging and Alzheimer's disease, J. Neurosci. Res., 27 (1991) 548-560. [7] Goldman, D., Deneris, E., Luyten, W., Kochhar, A., Patrick, J. and Heineman, S., Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system, Cell, 48 (1987) 965-977. [8] Janeway, C.A. Jr., Carding, S., Jones, B., Murray, J., Portoles, P., Rasmussen, R., Rojo, J., Saizawa, K., West, J. and Bottomly, K., CD4 ÷ T cells: specificity and function, Immunol. Rev., 101 (1988) 39-80. [9] Maslinski, W., Cholinergic receptors of lymphocytes, Brain Behav. Immunol., 3 (1989) 1-14. [10] Reuss, S., Schr0der, B., SchrOder, H.J. and Maelicke, A., Nicotinic cholinoceptors in the rat pineal gland as analyzed by Western blot, light- and electron microscopy, Brain Res., 573 (1992) 114-118. [11] Rinner, I., Felsner, P., Falus, A., Skreiner, E., Kukulansky, T., Globerson, A., Hirokawa, K. and Schauenstein, K., Cholinergic signals to and from the immune system, Immunol. Lett., 44 (1995) 217-220. [12] Sargent, P.B., The diversity of neuronal nicotinic acetyicholine receptors, Annu. Rev. Neurosci., 16 (1993) 403-443. [13] Schr6der, H.J., Immunohistochemistry of cholinergic receptors, Anat. Embryol., 186 (1992) 407-429. [14] Schrtider, H.J., Zilles, K., Maelicke, A. and Hajos, F., Immunohisto- and cytochemical localization of cortical nicotinic cholinoceptors in rat and man, Brain Res., 502 (1989) 287-295. [15] Strom, T.B., Sytkowski, A.J., Carpenter, C.B. and Merrill, J.P., Cholinergic augmentation of lymphocyte-mediated cytotoxicity. A study of the cholinergic receptor of cytotoxic T lymphocytes, Proc. Natl. Acad. Sci. USA, 4 (1974) 1330-1333. [16] Watters, D. and Maelicke, A., Organization of ligand binding sites at the acetylcholine receptor: a study with monoclonal antibodies, Biochemistry, 22 (1983) 1811-1819. [17] Wevers, A., Jeske, A., Lobron, C., Birtsch, C., Heinemann, S., Maelicke, A., Schr0der, R. and SchrOder, H., Cellular distribution of nicotinic acetylcholine receptor subunit mRNAs in the human cerebral cortex as revealed by non-isotopic in situ-hybridisation, Mol. Brain Res., 24 (1994) 122-128. [18] Whitehouse, P.J., Martino, A.M., Wagster, M.V., Price, D.L., Mayeux, R., Atack, J.R. and Kellar, K.J., Reductions in [3H]nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study, Neurology, 38 (1988) 720-723.
Neuroscience Letters 214 (1996) 171-174
NIUIIOSHHg LETTERS
Expression of alpha subunit genes of nicotinic acetylcholine receptors in human lymphocytes 1 C. Hiernke a'*, M. Stolp a, S. Reuss b, A. Wevers d, S. Reinhardt c, A. Maelicke c, S.
S c h l e g e l a,
H. S c h r 6 d e r d aDepartment of Psychiatry, University of Mainz, Untere Zahlbacher Strausse 8, D-55101 Mainz, Germany bDepartment of Anatomy, University of Mainz, Mainz, Germany CDepartmentof Physiological Chemistry, University of Mainz, Mainz, Germany dDepartment of Anatomy, University of K6ln, Ki~ln, Germany Received 13 May 1996; revised version received 17 July 1996; accepted 17 July 1996
Abstract Using immunohistochemistry and in situ hybridisation, we have studied whether c~-subunits of nicotinic acetylcholine receptors (nAChRs) are expressed in human lymphocytes. Cells were isolated by differential low speed gradient centrifugation from heparinised venous blood of 10 healthy volunteers. Receptor sites were visualised using the monoclonal antibody WF6 which specifically recognises c~-isoforms from several species including man. For visualisation of transcripts, digoxigenin-labeUed cRNA probes for a4- and t~3subunits were used. Immunostaining revealed specific binding of WF6 to isolated human lymphoid cells. The antibody was bound to most cells and concentraled preferentially in the perinuclear/surface region. The immunoreactivity resembled that observed after application of an antibody recognising CD4 surface proteins which was conducted for comparison. In situ-hybridisation revealed that the t~4-subunit genes of nAChRs was expressed in lymphocytes of all probands. The a3-subunit was found~ with lower intensity than t~4-transcripts, in eight of the 10 individuals. Control incubations with corresponding sense cRNAs were negative. It is concluded that human lymphocytes are able to express c~-subunit genes of nAChRs.
Keywords: Acetylcholine receptor; Lymphocytes; Nicotinic c~-subunits; WF6 antibody; Immunostaining; In situ hybridisation; Neuroimmunology
Acetylcholine in the brain is believed to play a major role in cognitive funt'tions [12]. Postmortem studies revealed a decrease in the concentration of acetylcholine receptors in the brain of patients suffering from senile dementia of the Alzheimer type (SDAT) [6,18]. Adem et al. [1] reported that these changes in the brain are also reflected by peripheral blood cells. In lymphocytes from SDAT patients, the number of nicotinic binding sites was found to be lower than in blood cells of age-matched controis [1]. Human lymphocytes express receptors for several neurotransmitters including acetylcholine [2,9]. Binding studies with radioligands revealed predominantly muscarinic receptors [2,9], whereas data on the occurrence of nicotinic acetylcholine receptors are less conclusive. * Corresponding author. Tel.: +49 6131 177363; fax: +49 6131 176690. t This paper contains result.,~ of the M.D. thesis of M. Stolp.
0304-3940/96/$12.00
Most of the binding of nicotinic ligands was found to be non-specific and non-saturable [9] and the concentration of specific binding sites was very low. Immunodetection of acetylcholine receptors has so far not been performed in lymphocytes. Moreover, studies on distinct subunits of nicotinic acetylcholine receptors whose expression differs in the brain and in peripheral organs [4,7,12] are lacking. Using immunohistochemistry and in situ hybridisation, we demonstrated that ot-subunit genes of nicotinic acetylcholine receptors are expressed in human lymphocytes. The study included 10 healthy volunteers (5 female and 5 male, ages 23-40 years). They were all students or staff of the Department of Psychiatry, University of Mainz, Germany and participated in the trial on an informed consent basis. Blood (10 ml) was withdrawn from the antecubital vein in heparinised tubes. Peripheral blood lymphoid (PBL)
© 1996 Elsevier Science Ireland Ltd. All rights reserved
PII S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 12908-6
172
c. Hiemke et al. / Neuroscience Letters 214 (1996) 171-174
cells were prepared by a standard Ficoll/Histopac procedure. The blood was mixed with 25 ml phosphate-buffered saline (PBS) and aliquoted into 50 ml tubes, underlaid with 10 ml Ficoll separating solution (Biochrom KG, Berlin, Germany) and centrifuged for 15 min at 900 g. Cells were washed three times to remove serum components and Ficoll solution. The final preparation contained 0.31.6 × 106 cells/ml. Using trypan blue exclusion staining revealed that at least 96% of the cells had remained viable. Twenty microliters of the cell suspension were applied on poly-L-lysine-coated slides and dried at room temperature (RT). The cells were fixed using acetone for 10 min at -20°C and subsequently washed twice at RT for 5 rain with PBS. To block endogenous peroxidases, the preparation was exposed to methanol containing 0.3% H202 for 20 min at RT. After incubation with 20% (w/v) swine serum albumin (Serva, Heidelberg, Germany) to reduce non-specific binding, the slides were incubated overnight at 4°C with the primary antibody WF6 (1:100) in a moist chamber. After washing in PBS (2 × 10 rain), the samples were incubated for 60 min with goat anti-mouse IgG conjugated to biotin (1:50; Amersham-Buchler, Braunschweig, Germany). After washing out unbound antibodies with PBS (2 x 10 min), a streptavidin-peroxidase complex (1:50; 60 min at RT; Amersham-Buchler) was applied. After another PBS wash (2 × 10 rain), the enzyme activity was visualised by reaction with diaminobenzidine (DAB; Sigma, Germany) (1 mg DAB and 0.1 mg H202/ml in Tris buffer 0.05 M; pH 7.4) in the dark at RT for 10 min. For final washing (2 × 5 min), Tris-HCl (0.05 M; pH 7.4) was used. The samples were dehydrated in a graded series of ethanols followed by xylene and mounted using commercial imbedding medium (Fluka, Neu-Ulm, Germany). Specificity controls were performed for each cell preparation by omitting primary and/or secondary antibodies and incubating the cells under otherwise unchanged conditions. For comparison of the binding of WF6 to blood cells, the WF6 antibody was replaced, under otherwise unchanged conditions, by an antibody to cluster differentiating type 4 antigen (CD4; diluted 1:100, Coulter, Krefeld, Germany), a glycoprotein expressed primarily on the cell surface of T-helper cells [8]. For detection of c~4 and a3 mRNA, digoxigeninlabelled antisense and sense cRNA probes were prepared [17] from rat a4-1 and ot3-subunits, respectively. Rat and human ot3-subunits exhibit 93% amino acid sequence homology [4], and the ~4-1 variant 82% [7]. A previous investigation has shown that the rat cRNA probes are suitable for labelling nicotinic acetylcholine receptor subunit mRNAs in the human brain [17]. The protocol previously established for brain tissue [17] was also used for the detection of lymphocyte mRNAs, with a few minor modifications. Isolated mononuclear blood cell suspensions, containing 0.6-1.6 × 105 or 0.61.6 × 10 4 cells/ml were mounted on adhesive slides (Bio-Rad, Mtinchen, Germany) by cytocentrifugation
(Shandon-Elliot). The slides had been prewashed in absolute ethanol and dried before use. After 20 min drying at RT, the samples were stored frozen (-80°C) until assayed. Before hybridisation, the slides were brought to RT for 1.5 h, subsequently fixed in 4% (v/v) paraformaldehyde in PBS (5 min at RT) and washed again twice with 2 × SSC (0.3 M NaC1, 30 mM sodium citrate; pH 7.0) at RT, followed by incubation in triethanolamine, 0.1 M (pH 8.0) and acetic acid, 0.0026% (v/v) ( l x l 0 min) and two washes in 2x SSC (2 × 1 min at RT) and PBS (lxl min). Finally, the samples were incubated with 0.1 M glycine and 0.2 M Tris-HCl (pH 7.4) (30 min at RT) and then washed in 2 × SSC (2 x 5 min at RT). Prehybridisation and hybridisation were performed at 50°C with a content of 50% formamide as described in detail earlier [17]. Non-hybridised probes were removed by incubation at 52°C in 2 x SSC, containing 50% formamide. Detection of hybridised digoxigenin-labelled cRNA probes were achieved by incubation with an alkaline phosphatase, conjugated digoxigenin-antibody (Boehringer, Mannheim, Germany) and a colour substrate reaction using 5bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium chloride. Control in situ hybridisations were performed using labelled sense cRNA instead of antisense cRNA or hybridisation solution without probe. Isolated human lymphocytes showed good preservation after staining by routine histological methods and light microscopical inspection of the ceils (data not shown). The monoclonal antibody WF6, raised against the nicotinic acetylcholine receptor of Torpedo m a r m o r a f a [3], was bound specifically to the lymphocytes. Peroxidase staining was not detectable when primary and/or secondary antibody were omitted. Immunostaining was detected in the perinuclear/surface region of the lymphocytes (Fig. 1A-C) without allowing the exact localisation of the immunostaining on the subcellular level. When replacing the WF6 antibody by an antibody to CD4, the light microscopical results resembled closely that seen using WF6 (Fig. 1D). Using routine histological staining in addition to immunostaining, it seemed likely that the vast majority of lymphocytes bound WF6 (data not shown). Moreover, all volunteers studied for WF6 binding exhibited similar patterns of specific immunostaining. In situ hybridisation revealed that lymphocytes of all volunteers expressed at least one of the nAChR subunit genes tested (Fig. 2). Incubations with labelled sense cRNA (Fig. 2C,F) or under omission of the antisense cRNA yielded negative results. Using the ~4-1 probe, hybridisation was observed within 5 to 20 min after beginning of the enzyme reaction (Fig. 2A,B). Incubations with sense cRNA (Fig. 2C) or without cRNA probes were negative. Calculation of the number of or4 antisense-labelled cells indicated that about 80-90% of them expressed mRNA for this isoform. ~3-Subunit mRNA was detected in only eight of the 10 individuals (Fig. 2D,E). Moreover, the reaction times
C. Hiemke et al. / Neuroscience Letters 214 (1996) 171-174
qi*
173
¢b ~D~O
e
o D
cB~
~
....
o
q D
Fig. 1. Light microscopic demonstration of nicotinic acetylcholine receptor immunoreactivity on human lymphocytes using the monoclonal antibody WF6 in cells of a male (A,B) and female (C) subject. The typical cell surface protein on T helper-cells, CD4, was visualised using the WF6 immunostaining protocol and a CD4 antibody instead of WF6 (D). Magnification bar, (A) 50 #m; 5 /zm (B-D).
required for detection of phosphatase activity were 20-60 min which was three to five times longer than required for detection of ct4-subunits. Incubations with c~3 sense cRNA were negative (Fig. 2F).
To our knowledge, this is the first report demonstrating that human lymphocytes express c~-subunit genes of nicotinic acetylcholine receptors. Immunostaining was performed with WF6, a monoclo-
Fig. 2. Expression of a4- and u3-subunits mRNA of nicotinic acetylcholine receptors in human lymphocytes. Transcripts were visualised using digoxigenin-labelled cRNA antisense probes for ~4 (A,B) and a3-subunits (D,E). Control samples using sense RNA probes for a4- (C) or a3-subunits were negative and visualised by phase contrast microscopy (F). Magnification bar, (A,D) 100/zm; (B,C,E,F) 10 #m.
174
C. Hiemke et al. / Neuroscience Letters 214 (1996) 171-174
nal antibody raised in mice against the purified nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [3]. This antibody recognises the acetylcholine binding domain of some nicotinic acetylcholine receptor isoforms [3,16] in tissues from various species including man [10,14]. The staining observed here indicates that a protein with the antigenic properties of nicotinic acetylcholine receptor ot-subunits is synthesised by human lymphocytes. In regard to the subcellular localisation of the WF6 binding site, light microscopic inspection does not suffice to differentiate between the cell surface and the cytoplasm. Since CD4 staining yielded results similar to those obtained with WF6 it was concluded that WF6 immunoreactivity was localised preferentially on the surface of the lymphocytes. Further evidence for the synthesis of nicotinic acetylcholine receptors by human lymphocytes is provided by the in situ hybridisation study, mRNAs of two ot-subunit isoforms were identified using cRNA antisense probes for o~4- and ct3-isoforms. It seemed likely that a4-subunits were more abundant than ot3-subunits, since staining was more pronounced. Since the c~4-subunit is the predominant subunit in the human brain [12,13], it may be concluded that lymphocytes express a composition of o~-isoforms similar to the brain. This suggestion, however, needs further clarification. Moreover, it has to be shown if/3subunit genes are expressed because coexpression of c¢ and /3-subunits is necessary to encode for a functional acetylcholine receptor [12]. A functional role of these receptors is supported by pharmacological studies demonstrating that acetylcholine receptor agonists affect immune functions of lymphocytes [5,11,15]. Our morphological findings that used two different approaches, i.e. immunohistochemistry and in situ hybridisation, and functional studies suggest that nicotinic acetylcholine receptors are present on human lymphocytes. It therefore seems promising to look for changes of nicotinic acetylcholine receptor properties on lymphocytes in connection to demential diseases, since changes in the expression of nicotinic acetylcholine receptors on lymphocytes might reflect alterations of cholinergic neurotransmission in the brain as suggested for SDAT and other diseases accompanied by cognitive dysfunctions [1]. This work was supported by the Deutsche Forschungsgemeinschaft (grants Schr 283•8-2 and Ma 599/18-1). We thank Marita W6rsdfrfer for helpful technical advise, Gabriele Stroba for skilful technical assistance, Ursula Disque-Kaiser for photographic work and Michaela Jahnke for typing the manuscript.
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