Blockade of nicotinic and muscarinic receptors facilitates spontaneous migration of human peripheral granulocytes: Failure in cystic fibrosis Ignaz Wessler a, ⁎, Stefanie Neumann a , Michael Razen a , Fred Zepp b , Charles James Kirkpatrick a a Institute of Pathology, Johannes Gutenberg University Mainz, D-55101 Mainz, Germany b Children's Hospital, University Medical Center, Johannes Gutenberg University Mainz, D-55101 Mainz, Germany abstract article info Article history: Received 31 October 2011 Accepted 4 April 2012 Keywords: Non-neuronal acetylcholine Nicotinic and muscarinic receptors Peripheral granulocytes Resting state Control of migration Cystic fibrosis Aims: Circulating leucocytes express muscarinic (m) and nicotinic (n) receptors and synthesize acetylcholine (ACh) regulating various cell functions. Leucocytes from patients with cystic fibrosis contain less ACh; therefore it was tested whether the regulation of cellular functions like migration differed from healthy volunteers. Main methods: Peripheral blood (10–20 ml) was used, leucocytes were isolated by Ficoll® gradient and the commercial MIGRATEST® combined with flow cytometric analysis was applied (pore size 3 μm). Key findings: In the absence of test substances 4900 ± 1800 (n = 10) leucocytes migrated within a time period of 2 h. In the presence of tubocurarine (TC, 30 μM) the cell number increased to 7500±2700 [n=10] corresponding to an increase of 162±20% (mean of individual experiments; p b 0.02). Atropine (1 μM) was not effective (120±17%, n=7). Simultaneous application of atropine and TC produced a slightly higher effect than TC alone (185±23%; n=8); a 10-fold increase of TC and atropine resulted to a somewhat stronger effect (248±39%; n=8). When migration time was reduced to 30 min or the chemoattractant fMLP (0.05 μM) present neither atropine nor TC affected migration. Granulocytes isolated from patients with cystic fibrosis did not respond (2 h migration) to 30 μM TC (control: 5180±1400 cells [n=10]; TC: 5800±1400 [n= 10]). Also in the presence of atropine (1 μM) and TC (30 μM) a significant effect was not detected (5800 ± 1300 [n= 10]). Significance: Auto-paracrine acetylcholine limits the migration of unstimulated peripheral granulocytes. This effect is impaired in cystic fibrosis most likely because of a reduced endogenous cholinergic tone. © 2012 Elsevier Inc. All rights reserved. Introduction Synthesis of acetylcholine has been demonstrated in non-neuronal cells, for example epithelial, endothelial, mesothelial and immune cells as well as in smooth muscle fibres (Grando, 1997; Klapproth et al., 1997; Kawashima and Fujii, 2000, 2003; Wessler et al., 1998; Wessler and Kirkpatrick, 2007). In addition, n- and mAChRs are also expressed on these non-neuronal cells (for references see Wessler and Kirkpatrick, 2007). Migration of epithelial cells has been shown to be regulated by non-neuronal acetylcholine. For example the migration of bronchial epithelial cells in an ex vivo wound repair model is reduced by mecamylamine, a nAChRs antagonist (Tournier et al., 2006). In human skin cells random migration is also regulated by n- and mAChRs, the α7-subytpe appearing to reduce and the α3-subytpe to stimulate random migration (Grando et al., 2006). Likewise the migration of isolated peripheral human granulocytes could be modified by nAChRs antagonists. Tubocurarine and α-bungarotoxin stimulated the transwell migration of peripheral granulocytes, indicating an α7-subtype involved (Neumann et al., 2007). In the present experiments this finding was investigated in more detail, particularly with respect to the involvement of mAChRs and a possible difference in granulocytes isolated from patients with cystic fibrosis. Cystic fibrosis (CF) is the most frequent genetic disorder. The genetic basis of CF is well characterized, showing a mutation of the CF gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR; Riordan et al., 1989). CF is associated with enhanced infections and it has recently been reported that the content of ACh was significantly reduced in the airways and peripheral leucocytes of CF-patients (Wessler et al., 2007). Therefore, in the present study it was tested whether the cholinergic modulation of basal migration of granulocytes, primarily responsible for early unspecific defence, is impaired compared to healthy controls. TC was used as antagonist which does not discriminate between subtypes of nAChRs; the nAChRs involved had already been identified as α7-subtype (Neumann et al., 2007). Methods Blood donors The protocol for removing 10–20 ml peripheral blood (mixed with 1000 I.U. liquemin® for preventing coagulation) was approved by the Life Sciences 91 (2012) 1119–1121 ⁎ Corresponding author at: Institute of Pathology, Johannes-Gutenberg University Mainz, Langenbeckstr. 1, D-55101 Mainz, Germany. Tel.: + 49 6131 172824x7305; fax: +49 6131 176604. E-mail address: wessler@uni-mainz.de (I. Wessler). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2012.04.002 Contents lists available at SciVerse ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie