Blockade of nicotinic and muscarinic receptors facilitates spontaneous migration of human peripheral granulocytes: failure in cystic fibrosis

Abstract

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<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 (2h 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.