The Effect of Nicotine Acetylcholine Receptor Ligands on the Adhesive Properties of Murine Bone Marrow Granulocytes in Inflammation

Attachment to the vascular endothelium is the first step of the release of mature neutrophil granulocytes from the bone marrow into the blood and subsequent migration to the inflammatory center. Disturbance of neutrophil adhesiveness is critical for many diseases with inflammatory components. Endo- and exogenous factors modify the cell ability to adhere via different receptors, including nicotinic acetylcholine receptors (nAChRs). However, the involvement of nAChRs in the regulation of bone marrow (BM) granulocyte adhesion and the role of signaling components in the action of nicotine are poorly understood. In this work the role of different types of nAChRs in the regulation of murine BM granulocyte adhesion during acute inflammation was studied. The work was performed on BM granulocytes of the BALB/c mouse strain using static adhesion assay, confocal microscopy, inhibitor assay, and reverse transcription PCR (RT-PCR). The role of nAChR types was assessed applying selective antagonists: 10 nM α-CTX (α7), 10 nM GIC and 5 nM MII (α3β2), 200 nM MII (α3β2 and α7), RgIA and Vc1.1 (α9α10). The number of attached BM granulocytes did not differ between animals with and without inflammation. Nicotine (0.01–100 µM, 30 min) significantly increased cell adhesion in both groups. Toxins (α-CTX, RgIA, Vc1.1) enhanced cell adhesion in both groups, as 200 nM MII did in controls. Fluorescence labelling assays showed expression of α7 and α10 nAChR subunits on cytoplasmic membrane of native BM granulocytes. Using inhibitors, we showed that the effect of nicotine on BM granulocyte adhesion was mediated by heterotrimeric G-proteins, PKC, PI3K, and ROCK both normally and in the presence of inflammation. α7 and α9α10 nAChRs were predominantly involved in regulation of BM granulocyte adhesion, and participation of α3β2/α3α6*β2 was negligible, possibly due to low expression of α3/α6* subunits. In the regulation of cell adhesion by nicotine, the development of inflammation in the body enhanced the role of α7 nAChRs, which are conventionally expressed on the membrane of BM granulocytes.