Background There is growing evidence that nervous and immune system communicate with each other through soluble mediators.1 Immune cells such as neutrophils express muscarinic acetylcholine receptors (mAChR), which are neuroimmune receptors and highly prevalent in the nervous system.2 Aberrant neutrophil functioning plays an important role in various autoimmune diseases. Dysregulation of neutrophil immune responses such as oxidative burst and migration is one of the key mechanisms leading to tissue damage in autoimmune diseases.3 However, the impact of mAChR activation on neutrophils remains contradictory.
Objectives We aimed to determine effects of muscarinic receptor activation on development and functions of neutrophils.
Methods Neutrophils were isolated from peripheral blood of healthy donors by dextran sedimentation. After one hour in the absence or presence of the natural ligand acetylcholine (Ach) (10nM-100μM) or the muscarinic agonist oxotremorine-m (oxo-m) (10nM-100μM), neutrophil respiratory burst was analyzed by dihydrorhodamine (DHR) flow cytometry assay and migration assessed by transwell assay in response to N-formylmethionyl-leucyl-phenylalanine (fMLP). Cells that migrated were quantified by flow cytometry. To analyze the effects mAChR activation on the development of neutrophils, HL-60 cells were incubated in the presence of DMSO (1%), oxo-m (100μM) or DMSO plus oxo-m. After 6 days, cells were harvested and expression of maturation markers (CD15, CD63 and CD16) as well as mAChR (M1-M5) were measured by flow cytometry.
Results We observed no effects of mAChR activation on the respiratory burst of neutrophils. However, both ACh and oxo-m inhibited neutrophil migration in a dose-dependent manner, but with peculiar differences. By increasing acetylcholine concentrations, we observed a reduction of neutrophil migration in a directly proportional manner. On the other hand, while the lowest dose (10nM) of oxo-m inhibited migration most effectively, the increase of oxo-m showed inversely proportional effects on neutrophil migration. Thus, we aimed to investigate, if the highest dose of oxo-m has a different effect on neutrophils ontogeny. In agreement with the results obtained with neutrophils, the incubation of HL-60 cells with the highest dose of oxo-m showed no effect on oxidative burst and migration and induced no changes in the expression of mAChRs (M1-M5), CD16 and CD63 in HL-60 cells. However, we observed that it resulted in significantly increased surface levels of the neutrophilic lineage marker CD15.
Conclusions Our data indicate a differential activation of mAChR affecting different steps of neutrophil ontogeny. Considering this finding, abnormalities in the activation of muscarinic receptors as have been observed in autoimmune diseases might contribute to neutrophil dysfunction and need further investigation.
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Milara et al. Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients. Respiratory Research (2016) 17:145.
Wright, H. L. et al. The multifactorial role of neutrophils in rheumatoid arthritis Nat Rev Rheumatol 10 593–601 (2014).
Disclosure of Interest None declared
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