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Immunohistochemical detection of nicotinic acetylcholine receptor subunits α9 and α10 in rat lung isografts and allografts
Life Sciences 80 (2007) 2286 – 2289 www.elsevier.com/locate/lifescie
Immunohistochemical detection of nicotinic acetylcholine receptor subunits α9 and α10 in rat lung isografts and allografts Simone Biallas a , Sigrid Wilker a , Katrin S. Lips b , Wolfgang Kummer b , Sergei A. Grando c , Winfried Padberg a , Veronika Grau a,⁎ a
b
Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, University of Giessen Lung Center, Justus-Liebig-University Giessen, Rudolf-Buchheim-Str. 7, D-35385 Giessen, Germany Institute for Anatomy and Cell Biology, University of Giessen Lung Center, Justus-Liebig-University Giessen, Aulweg 123, D-35385 Giessen, Germany c Department of Dermatology, University of California, Davis, 3301 C Street, Suite 1400, Sacramento, CA 95816, USA Received 23 October 2006; accepted 24 January 2007
Abstract The success of clinical lung transplantation is poor in comparison to other solid organ transplants and novel therapeutic approaches are badly needed. In the view of the recent discovery of anti-inflammatory pathways mediated via nicotinic acetylcholine receptors, we investigated changes in this system in pulmonary isografts and allografts by immunohistochemistry. Lung transplantation was performed in the isogeneic Lewis to Lewis rat strain combination. For allogeneic transplantation Dark Agouti rats were used as donors. Nicotinic α9 and α10 acetylcholine receptor subunits were detected on alveolar macrophages as well as in the lung parenchyma of native and transplanted lungs. The expression of both receptor subunits was up-regulated in the parenchyma of day 4 allografts. These allografts were characterized by accumulations of alveolar macrophages strongly expressing the α9 and the α10 receptor subunit. Therapeutic application of nicotinic agonists might down-modulate proinflammatory functions of alveolar macrophages and protect pulmonary transplants. © 2007 Elsevier Inc. All rights reserved. Keywords: Lung transplantation; Nicotinic acetylcholine receptor; Alveolar macrophage
Introduction Lung transplantation is the only curative therapy for an increasing population of patients suffering from end-stage pulmonary failure. In contrast to other organ transplants, the clinical outcome of lung allografts is poor. Up to 30% of the grafts are lost during the first months due to ischemia/ reperfusion damage, infections and acute rejection (Trulock et al., 2006; Wilkes et al., 2005). Later on, lung allografts are prone to chronic rejection which develops earlier and more frequently in lung transplants compared to other organ grafts (Trulock et al., 2006; Wilkes et al., 2005). New therapeutic strategies to prevent acute and chronic lung allograft rejection are required. The cholinergic system of the lung might represent a promising therapeutic target because acetylcholine regulates ⁎ Corresponding author. Tel.: +49 641 99 44791; fax: +49 641 99 44709. E-mail address: [email protected] (V. Grau). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.043
vital functions of the lung comprising bronchoconstriction, fluid and mucus secretion as well as ciliary beat (White, 1994). Furthermore, acetylcholine regulates cells of the immune system: On the one hand, T lymphocytes and mast cells are activated via muscarinic receptors (Kawashima and Fujii, 2003), on the other hand, stimulation of nicotinic receptors results in a silencing of pro-inflammatory macrophages (Pavlov et al., 2003). Alveolar macrophages play a pivotal role in pulmonary allograft rejection (Wilkes et al., 1998; Sekine et al., 1997). The expression of acetylcholine receptors, the molecular targets of cholinergic therapeutics, has not been investigated after lung transplantation. Nicotinic acetylcholine receptor (nAChR) subunits α9 and α10 are expressed in the nervous system and by numerous nonneuronal cells including cells of the immune system (Lips et al., 2002; Peng et al., 2004). The α9 nAChR subunit forms homopentamers or heteropentamers with α10 subunits which function as ligand gated Ca++ channels. Here we investigate the expression of the nAChR subunits α9 and α10 in an
S. Biallas et al. / Life Sciences 80 (2007) 2286–2289
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Fig. 1. Immunohistochemical detection of the nicotinic acetylcholine receptor (nAChR) subunits α9 and α10 on paraffin sections of the parenchyma of left transplanted lungs and right native lungs of pulmonary graft recipient rats. Isogeneic (iso) LEW to LEW and allogeneic (allo) DA to LEW transplantations were performed and the recipients were sacrificed on day 2 (d2) or day 4 (d4) posttransplantation. A: The nAChR subunits α9 and α10 are stained in brown using a peroxidase-coupled detection system, and the sections are mildly counterstained with hemalum. The arrows point to immunopositive alveolar macrophages. B: The specificity of the antibody-binding was tested by adding excess amounts of the peptides used to raise the antisera. The peptides strongly decreased the staining intensity of both anti-α9 and anti-α10 antisera. C: Double-staining experiments using the monoclonal antibody ED1 binding to a CD68-like antigen expressed by monocyte/ macrophages indicate that alveolar macrophages express both α9 and α10 nAChR subunits. ED1 is stained in red, bound antibodies to the nAChR subunits are stained in blue using an alkaline phosphatase-coupled detection system.
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experimental model of acute lung allograft rejection on days 2 and 4 after transplantation. Day 2 was chosen because the histopathological changes were about the same in isografts and allografts and probably due to ischemia/reperfusion injury (Schmidt et al., 2007). In contrast, day 4 allografts are heavily infiltrated due to acute rejection whereas isografts are almost unimpaired (Schmidt et al., 2007). Apart from the infiltrate the allograft lung parenchyma is still intact on day 4 but irreversibly destroyed on days 5 and 6. Materials and methods Young adult male LEW(RT1ℓ) and DA(RT1av1) rats were purchased from Harlan Winkelmann (Borchen, Germany) and kept under conventional conditions. Animal care and animal experiments were performed following the current version of the German Law on the Protection of Animals as well as the NIH “Principles of laboratory animal care”. Lung transplantation and fixation was performed as described previously (Schmidt et al., 2007). Briefly, the left lung was transplanted orthotopically using a cuff technique for the vascular anastomosis and sutures for the bronchial anastomosis. No immunosuppression was applied. The graft and the right native lungs of allograft and isograft recipients (n = 4 each) were fixed on day 2 and day 4 posttransplantation by instillation of 4% buffered paraformaldehyde and embedded in paraffin for histological investigations. Sections were dewaxed, rehydrated and pretreated with 0.5 mg/ml Protease Type XIV (Sigma-Aldrich, Taufkirchen, Germany) in 50 mM Tris-HCl buffer pH 7.6, 0.9% NaCl for 15 min at room temperature followed by 1% H2O2 in PBS for 30 min. After washing in PBS pH 7.2, the sections were incubated for 30 min with PBS, pH 7.2, 1% BSA (Serva, Heidelberg, Germany), 0.1% NaN3 (p.a. Merck, Darmstadt, Germany). Polyclonal antisera raised in rabbits against peptides specific for α9 and α10 nAChR subunits have been characterized before (Lips et al., 2002; Nguyen et al., 2000). Incubation with these antisera diluted in PBS/BSA/NaN3 was performed over night at 4 °C. Bound primary antibodies were detected using the anti-rabbit EnVision peroxidase system (DAKO, Hamburg, Germany) and 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) followed by a slight counterstaining with hemalum. To confirm the specificity of the staining, excess amounts of the peptides (100 μg/ml for α9 and 200 μg/ ml for α10) used to produce the antibodies were added to the primary antibody dilution. Specificity of secondary antibodies was further tested by omission of primary antibodies on control sections. In double-staining experiments, the nAChR subunits were labeled in blue using the anti-rabbit/anti-mouse EnVision alkaline phosphatase system (DAKO) containing 5% normal rat serum and the chromogen Fast Blue. To prepare the sections for the second staining step, antigen retrieval in 0.01 M sodium citrate buffer pH 6.0 was performed for 15 min at 120 °C and 1.1 bar. By doing so, bound antibodies and enzymes from the first staining are completely removed. Macrophages were detected in red with monoclonal antibody ED1 binding to a
CD68-like lysosomal protein typical for monocytes/macrophages (Damoiseaux et al., 1994) and EnVision alkaline phosphatase containing 5% normal rat serum and Fast Red. Controls omitting each of the primary antibodies separately as well as both primary antibodies were included as controls. Results Pulmonary allografts transplanted in the Dark Agouti (DA) to Lewis (LEW) rat strain combination are vigorously rejected within 5 to 6 days after transplantation (Schmidt et al., 2007). As described previously, the allografts were heavily infiltrated by macrophages and T lymphocytes at day 4 (Schmidt et al., 2007). In contrast, in LEW to LEW isografts only marginal histopathological changes were obvious. We investigated nAChR subunits α9 and α10 in left lung isografts (iso) and allografts (allo) on day 2 and day 4 after transplantation. As an internal control, the right native lungs of graft recipients were always included (Fig. 1A). The α9 and α10 nAChR subunits were detected by immunohistochemistry on alveolar macrophages as well as in the parenchyma of native lungs. Isogeneic and allogeneic transplantation led to a slight global increase in the α9 subunit expression on days 2 and 4 after surgery. The overall expression of α10 nAChR subunits appeared to be identical in the graft and in the native lung of isograft recipients. However, in the parenchyma of day 4 allografts, labelling for the α10 receptor subunit was more intense than in the right native lung. Day 4 allografts were characterized by accumulations of alveolar macrophages strongly expressing the α9 and the α10 receptor subunit (Fig. 1A). Additionally, perivascular and peribronchiolar mononuclear infiltrates expressed both nAChR subunits. Antibody binding was specific, because the staining intensity for both receptor subtypes was strongly decreased when the specific peptide which was used to produce the antisera was added to the primary antibody solution (Fig. 1B). Double-staining experiments on sections of day 4 lung allografts with the monoclonal antibody ED1 confirmed the expression of both receptor subunits by alveolar macrophages (Fig. 1C). Discussion Our data indicate that the nAChR subunits α9 and α10 are expressed by the lung parenchyma and by alveolar macrophages of isogeneic lung transplants. Their expression increases in pulmonary allografts during rejection. The mechanisms involved in the up-regulation of these receptors in lung transplants are unknown. Since both hypoxia and mechanical injury can influence the expression of nAChR subunits (Verbois et al., 2002; Heeschen et al. 2002; Del Signore et al., 2004), the changes observed in isografts and allografts might be due to ischemia/reperfusion or mechanical damage that unavoidably occur in the process of lung transplantation. Additionally, the denervation of the lung might play a role, similar to the known changes in α-subunit composition of skeletal muscle nAChR induced by denervation
S. Biallas et al. / Life Sciences 80 (2007) 2286–2289
(Romano et al., 1997; Fischer et al., 1999). However, as the strongest increase in the receptor expression was seen in day 4 allografts, the up-regulation is probably triggered by inflammatory mediators (cf. Poea-Guyon et al., 2005). In conclusion, α9 and α10 nAChR subunits are up-regulated by experimental allogeneic lung transplantation. As alveolar macrophages express both receptor subunits, therapeutic application of nicotinic agonists might dampen lung allograft rejection by down-modulation of their pro-inflammatory functions. Acknowledgements The authors are grateful to Renate Plaβ for excellent technical support and to Sandra Iffländer for caring for the experimental animals. This study was supported by the DFG (FE 287/6–1). References Damoiseaux, J.G.M.C., Döpp, E.A., Calame, W., Chao, D., MacPherson, G.G., Dijkstra, C.D., 1994. Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83, 140–147. Del Signore, A., Gotti, C., Rizzo, A., Moretti, M., Paggi, P., 2004. Nicotinic acetylcholine receptor subtypes in the rat sympathetic ganglion: pharmacological characterization, subcellular distribution and effect of pre- and postganglionic nerve crush. Journal of Neuropathology and Experimental Neurology 63 (2), 138–150. Fischer, U., Reinhardt, S., Albuquerque, E.X., Maelicke, A., 1999. Expression of functional α7 nicotinic acetylcholine receptor during mammalian muscle development and denervation. European Journal of Neuroscience 11, 2856–2864. Heeschen, C., Weis, M., Aicher, A., Dimmler, S., Cooke, J.P., 2002. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. Journal of Clinical Investigation 110 (4), 527–536. Kawashima, K., Fujii, T., 2003. The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sciences 74, 675–696. Lips, K.S., Pfeil, U., Kummer, W., 2002. Coexpression of α9 and α10 nictotinic acetylcholine receptors in rat dorsal root ganglion neurons. Neuroscience 115 (1), 1–5. Nguyen, V.T., Ndoye, A., Grando, S.A., 2000. Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by
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Pemphigus vulgaris autoimmunity. American Journal of Pathology 157 (4), 1377–1391. Pavlov, V.A., Wang, H., Czura, C.J., Friedman, S.G., Tracey, K.J., 2003. The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Molecular Medicine 9 (5–8), 125–134. Peng, H., Ferris, R.L., Matthews, T., Hiel, H., Lopez-Albaitero, A., Lustig, L.R., 2004. Characterization of the human nicotinic acetylcholine receptor subunit alpha (α) 9 (CHRNA9) and alpha (α) 10 (CHRNA10) in lymphocytes. Life Sciences 76 (3), 263–280. Poea-Guyon, S., Christadoss, P., Le Panse, R., Guyon, T., De Baets, M., Wakkach, A., Bidault, J., Tzartos, S., Berrih-Aknin, S., 2005. Effects of cytokines on acetylcholine receptor expression: implications for myasthenia gravis. Journal of Immunology 174 (10), 5941–5949. Schmidt, A., Sucke, J., Fuchs-Moll, G., Freitag, P., Hirschburger, M., Kaufmann, A., Padberg, W., Grau, V., 2007. Macrophages in experimental lung isografts and allografts: infiltration and proliferation in situ. Journal of Leukocyte Biology 81, 186–194. Romano, S.J., Pugh, P.C., McIntosh, J.M., Berg, D.K., 1997. Neuronal-type acetylcholine receptors and regulation of α7 gene expression in vertebrate skeletal muscle. Journal of Neurobiology 32, 69–80. Sekine, Y., Bowen, L.K., Heidler, K.M., Van Rooijen, N., Cummings, O.W., Wilkes, D.S., 1997. Role of passenger leukocytes in allograft rejection: effect of depletion of donor alveolar macrophages on the local production of TNF-alpha, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation. Journal of Immunology 159, 4084–4093. Trulock, E.P., Edwards, L.B., Taylor, D.O., Boucek, M.M., Keck, B.M., Hertz, M.I., 2006. Registry of the International Society for Heart and Lung Transplantation: twenty-third official adult lung and heart–lung transplant report—2006. Journal of Heart and Lung Transplantation 25 (8), 880–892. White, S.R., 1994. Functional autonomic innervation of the airways: the cholinergic and adrenergic systems. In: Kaliner, M.A., Barnes, P.J., Kunkel, G.H.H., Baraniuk, J.N. (Eds.), Neuropeptides in respiratory medicine. Marcel Dekker Inc., New York, pp. 21–55. Wilkes, D.S., Thompson, L.K., Cummings, O.W., Bragg, S., Heidler, K.M., 1998. Instillation of allogeneic lung macrophages and dendritic cells cause differential effects on local IFN-gamma production, lymphocytic bronchitis, and vasculitis in recipient murine lungs. Journal of Leukocyte Biology 64, 578–586. Wilkes, D.S., Egan, T.M., Reynolds, H.Y., 2005. Lung transplantation — opportunities for research and clinical advancement. American Journal of Respiratory and Critical Care Medicine 172, 944–955. Verbois, S.L., Scheff, S.W., Pauly, J.R., 2002. Time-dependent changes in rat brain cholinergic receptor expression after experimental brain injury. Journal of Neurotrauma 19 (12), 1569–1585.
Immunohistochemical detection of nicotinic acetylcholine receptor subunits α9 and α10 in rat lung isografts and allografts Simone Biallas a , Sigrid Wilker a , Katrin S. Lips b , Wolfgang Kummer b , Sergei A. Grando c , Winfried Padberg a , Veronika Grau a,⁎ a
b
Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, University of Giessen Lung Center, Justus-Liebig-University Giessen, Rudolf-Buchheim-Str. 7, D-35385 Giessen, Germany Institute for Anatomy and Cell Biology, University of Giessen Lung Center, Justus-Liebig-University Giessen, Aulweg 123, D-35385 Giessen, Germany c Department of Dermatology, University of California, Davis, 3301 C Street, Suite 1400, Sacramento, CA 95816, USA Received 23 October 2006; accepted 24 January 2007
Abstract The success of clinical lung transplantation is poor in comparison to other solid organ transplants and novel therapeutic approaches are badly needed. In the view of the recent discovery of anti-inflammatory pathways mediated via nicotinic acetylcholine receptors, we investigated changes in this system in pulmonary isografts and allografts by immunohistochemistry. Lung transplantation was performed in the isogeneic Lewis to Lewis rat strain combination. For allogeneic transplantation Dark Agouti rats were used as donors. Nicotinic α9 and α10 acetylcholine receptor subunits were detected on alveolar macrophages as well as in the lung parenchyma of native and transplanted lungs. The expression of both receptor subunits was up-regulated in the parenchyma of day 4 allografts. These allografts were characterized by accumulations of alveolar macrophages strongly expressing the α9 and the α10 receptor subunit. Therapeutic application of nicotinic agonists might down-modulate proinflammatory functions of alveolar macrophages and protect pulmonary transplants. © 2007 Elsevier Inc. All rights reserved. Keywords: Lung transplantation; Nicotinic acetylcholine receptor; Alveolar macrophage
Introduction Lung transplantation is the only curative therapy for an increasing population of patients suffering from end-stage pulmonary failure. In contrast to other organ transplants, the clinical outcome of lung allografts is poor. Up to 30% of the grafts are lost during the first months due to ischemia/ reperfusion damage, infections and acute rejection (Trulock et al., 2006; Wilkes et al., 2005). Later on, lung allografts are prone to chronic rejection which develops earlier and more frequently in lung transplants compared to other organ grafts (Trulock et al., 2006; Wilkes et al., 2005). New therapeutic strategies to prevent acute and chronic lung allograft rejection are required. The cholinergic system of the lung might represent a promising therapeutic target because acetylcholine regulates ⁎ Corresponding author. Tel.: +49 641 99 44791; fax: +49 641 99 44709. E-mail address: [email protected] (V. Grau). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.043
vital functions of the lung comprising bronchoconstriction, fluid and mucus secretion as well as ciliary beat (White, 1994). Furthermore, acetylcholine regulates cells of the immune system: On the one hand, T lymphocytes and mast cells are activated via muscarinic receptors (Kawashima and Fujii, 2003), on the other hand, stimulation of nicotinic receptors results in a silencing of pro-inflammatory macrophages (Pavlov et al., 2003). Alveolar macrophages play a pivotal role in pulmonary allograft rejection (Wilkes et al., 1998; Sekine et al., 1997). The expression of acetylcholine receptors, the molecular targets of cholinergic therapeutics, has not been investigated after lung transplantation. Nicotinic acetylcholine receptor (nAChR) subunits α9 and α10 are expressed in the nervous system and by numerous nonneuronal cells including cells of the immune system (Lips et al., 2002; Peng et al., 2004). The α9 nAChR subunit forms homopentamers or heteropentamers with α10 subunits which function as ligand gated Ca++ channels. Here we investigate the expression of the nAChR subunits α9 and α10 in an
S. Biallas et al. / Life Sciences 80 (2007) 2286–2289
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Fig. 1. Immunohistochemical detection of the nicotinic acetylcholine receptor (nAChR) subunits α9 and α10 on paraffin sections of the parenchyma of left transplanted lungs and right native lungs of pulmonary graft recipient rats. Isogeneic (iso) LEW to LEW and allogeneic (allo) DA to LEW transplantations were performed and the recipients were sacrificed on day 2 (d2) or day 4 (d4) posttransplantation. A: The nAChR subunits α9 and α10 are stained in brown using a peroxidase-coupled detection system, and the sections are mildly counterstained with hemalum. The arrows point to immunopositive alveolar macrophages. B: The specificity of the antibody-binding was tested by adding excess amounts of the peptides used to raise the antisera. The peptides strongly decreased the staining intensity of both anti-α9 and anti-α10 antisera. C: Double-staining experiments using the monoclonal antibody ED1 binding to a CD68-like antigen expressed by monocyte/ macrophages indicate that alveolar macrophages express both α9 and α10 nAChR subunits. ED1 is stained in red, bound antibodies to the nAChR subunits are stained in blue using an alkaline phosphatase-coupled detection system.
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experimental model of acute lung allograft rejection on days 2 and 4 after transplantation. Day 2 was chosen because the histopathological changes were about the same in isografts and allografts and probably due to ischemia/reperfusion injury (Schmidt et al., 2007). In contrast, day 4 allografts are heavily infiltrated due to acute rejection whereas isografts are almost unimpaired (Schmidt et al., 2007). Apart from the infiltrate the allograft lung parenchyma is still intact on day 4 but irreversibly destroyed on days 5 and 6. Materials and methods Young adult male LEW(RT1ℓ) and DA(RT1av1) rats were purchased from Harlan Winkelmann (Borchen, Germany) and kept under conventional conditions. Animal care and animal experiments were performed following the current version of the German Law on the Protection of Animals as well as the NIH “Principles of laboratory animal care”. Lung transplantation and fixation was performed as described previously (Schmidt et al., 2007). Briefly, the left lung was transplanted orthotopically using a cuff technique for the vascular anastomosis and sutures for the bronchial anastomosis. No immunosuppression was applied. The graft and the right native lungs of allograft and isograft recipients (n = 4 each) were fixed on day 2 and day 4 posttransplantation by instillation of 4% buffered paraformaldehyde and embedded in paraffin for histological investigations. Sections were dewaxed, rehydrated and pretreated with 0.5 mg/ml Protease Type XIV (Sigma-Aldrich, Taufkirchen, Germany) in 50 mM Tris-HCl buffer pH 7.6, 0.9% NaCl for 15 min at room temperature followed by 1% H2O2 in PBS for 30 min. After washing in PBS pH 7.2, the sections were incubated for 30 min with PBS, pH 7.2, 1% BSA (Serva, Heidelberg, Germany), 0.1% NaN3 (p.a. Merck, Darmstadt, Germany). Polyclonal antisera raised in rabbits against peptides specific for α9 and α10 nAChR subunits have been characterized before (Lips et al., 2002; Nguyen et al., 2000). Incubation with these antisera diluted in PBS/BSA/NaN3 was performed over night at 4 °C. Bound primary antibodies were detected using the anti-rabbit EnVision peroxidase system (DAKO, Hamburg, Germany) and 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) followed by a slight counterstaining with hemalum. To confirm the specificity of the staining, excess amounts of the peptides (100 μg/ml for α9 and 200 μg/ ml for α10) used to produce the antibodies were added to the primary antibody dilution. Specificity of secondary antibodies was further tested by omission of primary antibodies on control sections. In double-staining experiments, the nAChR subunits were labeled in blue using the anti-rabbit/anti-mouse EnVision alkaline phosphatase system (DAKO) containing 5% normal rat serum and the chromogen Fast Blue. To prepare the sections for the second staining step, antigen retrieval in 0.01 M sodium citrate buffer pH 6.0 was performed for 15 min at 120 °C and 1.1 bar. By doing so, bound antibodies and enzymes from the first staining are completely removed. Macrophages were detected in red with monoclonal antibody ED1 binding to a
CD68-like lysosomal protein typical for monocytes/macrophages (Damoiseaux et al., 1994) and EnVision alkaline phosphatase containing 5% normal rat serum and Fast Red. Controls omitting each of the primary antibodies separately as well as both primary antibodies were included as controls. Results Pulmonary allografts transplanted in the Dark Agouti (DA) to Lewis (LEW) rat strain combination are vigorously rejected within 5 to 6 days after transplantation (Schmidt et al., 2007). As described previously, the allografts were heavily infiltrated by macrophages and T lymphocytes at day 4 (Schmidt et al., 2007). In contrast, in LEW to LEW isografts only marginal histopathological changes were obvious. We investigated nAChR subunits α9 and α10 in left lung isografts (iso) and allografts (allo) on day 2 and day 4 after transplantation. As an internal control, the right native lungs of graft recipients were always included (Fig. 1A). The α9 and α10 nAChR subunits were detected by immunohistochemistry on alveolar macrophages as well as in the parenchyma of native lungs. Isogeneic and allogeneic transplantation led to a slight global increase in the α9 subunit expression on days 2 and 4 after surgery. The overall expression of α10 nAChR subunits appeared to be identical in the graft and in the native lung of isograft recipients. However, in the parenchyma of day 4 allografts, labelling for the α10 receptor subunit was more intense than in the right native lung. Day 4 allografts were characterized by accumulations of alveolar macrophages strongly expressing the α9 and the α10 receptor subunit (Fig. 1A). Additionally, perivascular and peribronchiolar mononuclear infiltrates expressed both nAChR subunits. Antibody binding was specific, because the staining intensity for both receptor subtypes was strongly decreased when the specific peptide which was used to produce the antisera was added to the primary antibody solution (Fig. 1B). Double-staining experiments on sections of day 4 lung allografts with the monoclonal antibody ED1 confirmed the expression of both receptor subunits by alveolar macrophages (Fig. 1C). Discussion Our data indicate that the nAChR subunits α9 and α10 are expressed by the lung parenchyma and by alveolar macrophages of isogeneic lung transplants. Their expression increases in pulmonary allografts during rejection. The mechanisms involved in the up-regulation of these receptors in lung transplants are unknown. Since both hypoxia and mechanical injury can influence the expression of nAChR subunits (Verbois et al., 2002; Heeschen et al. 2002; Del Signore et al., 2004), the changes observed in isografts and allografts might be due to ischemia/reperfusion or mechanical damage that unavoidably occur in the process of lung transplantation. Additionally, the denervation of the lung might play a role, similar to the known changes in α-subunit composition of skeletal muscle nAChR induced by denervation
S. Biallas et al. / Life Sciences 80 (2007) 2286–2289
(Romano et al., 1997; Fischer et al., 1999). However, as the strongest increase in the receptor expression was seen in day 4 allografts, the up-regulation is probably triggered by inflammatory mediators (cf. Poea-Guyon et al., 2005). In conclusion, α9 and α10 nAChR subunits are up-regulated by experimental allogeneic lung transplantation. As alveolar macrophages express both receptor subunits, therapeutic application of nicotinic agonists might dampen lung allograft rejection by down-modulation of their pro-inflammatory functions. Acknowledgements The authors are grateful to Renate Plaβ for excellent technical support and to Sandra Iffländer for caring for the experimental animals. This study was supported by the DFG (FE 287/6–1). References Damoiseaux, J.G.M.C., Döpp, E.A., Calame, W., Chao, D., MacPherson, G.G., Dijkstra, C.D., 1994. Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83, 140–147. Del Signore, A., Gotti, C., Rizzo, A., Moretti, M., Paggi, P., 2004. Nicotinic acetylcholine receptor subtypes in the rat sympathetic ganglion: pharmacological characterization, subcellular distribution and effect of pre- and postganglionic nerve crush. Journal of Neuropathology and Experimental Neurology 63 (2), 138–150. Fischer, U., Reinhardt, S., Albuquerque, E.X., Maelicke, A., 1999. Expression of functional α7 nicotinic acetylcholine receptor during mammalian muscle development and denervation. European Journal of Neuroscience 11, 2856–2864. Heeschen, C., Weis, M., Aicher, A., Dimmler, S., Cooke, J.P., 2002. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. Journal of Clinical Investigation 110 (4), 527–536. Kawashima, K., Fujii, T., 2003. The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sciences 74, 675–696. Lips, K.S., Pfeil, U., Kummer, W., 2002. Coexpression of α9 and α10 nictotinic acetylcholine receptors in rat dorsal root ganglion neurons. Neuroscience 115 (1), 1–5. Nguyen, V.T., Ndoye, A., Grando, S.A., 2000. Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by
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Pemphigus vulgaris autoimmunity. American Journal of Pathology 157 (4), 1377–1391. Pavlov, V.A., Wang, H., Czura, C.J., Friedman, S.G., Tracey, K.J., 2003. The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Molecular Medicine 9 (5–8), 125–134. Peng, H., Ferris, R.L., Matthews, T., Hiel, H., Lopez-Albaitero, A., Lustig, L.R., 2004. Characterization of the human nicotinic acetylcholine receptor subunit alpha (α) 9 (CHRNA9) and alpha (α) 10 (CHRNA10) in lymphocytes. Life Sciences 76 (3), 263–280. Poea-Guyon, S., Christadoss, P., Le Panse, R., Guyon, T., De Baets, M., Wakkach, A., Bidault, J., Tzartos, S., Berrih-Aknin, S., 2005. Effects of cytokines on acetylcholine receptor expression: implications for myasthenia gravis. Journal of Immunology 174 (10), 5941–5949. Schmidt, A., Sucke, J., Fuchs-Moll, G., Freitag, P., Hirschburger, M., Kaufmann, A., Padberg, W., Grau, V., 2007. Macrophages in experimental lung isografts and allografts: infiltration and proliferation in situ. Journal of Leukocyte Biology 81, 186–194. Romano, S.J., Pugh, P.C., McIntosh, J.M., Berg, D.K., 1997. Neuronal-type acetylcholine receptors and regulation of α7 gene expression in vertebrate skeletal muscle. Journal of Neurobiology 32, 69–80. Sekine, Y., Bowen, L.K., Heidler, K.M., Van Rooijen, N., Cummings, O.W., Wilkes, D.S., 1997. Role of passenger leukocytes in allograft rejection: effect of depletion of donor alveolar macrophages on the local production of TNF-alpha, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation. Journal of Immunology 159, 4084–4093. Trulock, E.P., Edwards, L.B., Taylor, D.O., Boucek, M.M., Keck, B.M., Hertz, M.I., 2006. Registry of the International Society for Heart and Lung Transplantation: twenty-third official adult lung and heart–lung transplant report—2006. Journal of Heart and Lung Transplantation 25 (8), 880–892. White, S.R., 1994. Functional autonomic innervation of the airways: the cholinergic and adrenergic systems. In: Kaliner, M.A., Barnes, P.J., Kunkel, G.H.H., Baraniuk, J.N. (Eds.), Neuropeptides in respiratory medicine. Marcel Dekker Inc., New York, pp. 21–55. Wilkes, D.S., Thompson, L.K., Cummings, O.W., Bragg, S., Heidler, K.M., 1998. Instillation of allogeneic lung macrophages and dendritic cells cause differential effects on local IFN-gamma production, lymphocytic bronchitis, and vasculitis in recipient murine lungs. Journal of Leukocyte Biology 64, 578–586. Wilkes, D.S., Egan, T.M., Reynolds, H.Y., 2005. Lung transplantation — opportunities for research and clinical advancement. American Journal of Respiratory and Critical Care Medicine 172, 944–955. Verbois, S.L., Scheff, S.W., Pauly, J.R., 2002. Time-dependent changes in rat brain cholinergic receptor expression after experimental brain injury. Journal of Neurotrauma 19 (12), 1569–1585.