Effect of Nicotinic Acetylcholine Receptor Ligands on Adhesive Properties of Murine Bone Marrow Granulocytes During Inflammation

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Abstract

The first stage of mature neutrophil granulocytes leaving the bone marrow into the blood and migration to inflammatory center is attachment to vascular endothelium. 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 study 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 using 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 was negligible, possibly due to low expression of α3 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.

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About the authors

E. A. Jirova

Institute of Cell Biophysics of the Russian Academy of Sciences

Email: safronova@icb.psn.ru
Russian Federation, Pushchino, Moscow oblast, 142290

D. A. Serov

Institute of Cell Biophysics of the Russian Academy of Sciences; Prokhorov General Physics Institute of the Russian Academy of Sciences

Email: safronova@icb.psn.ru
Russian Federation, Pushchino, Moscow oblast, 142290; Moscow, 119991

E. V. Fedorova

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences

Email: safronova@icb.psn.ru
Russian Federation, Pushchino, Moscow oblast, 142290

V. G. Safronova

Institute of Cell Biophysics of the Russian Academy of Sciences

Author for correspondence.
Email: safronova@icb.psn.ru
Russian Federation, Pushchino, Moscow oblast, 142290

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2. Fig. 1. Effect of nAChR ligands on CM-granulocyte adhesion of control animals and animals with inflammation. The effects of 100 μM nicotine (a) or one of the antagonists and antagonist together with nicotine on the adhesion of intact cells are shown: 10 nM α-CTX (b), 10 nM GIC (c), 5 nM MII (d), 200 nM MII (e), 10 nM RgIA (f) or 50 nM Vc1.1 (g). The single effect of toxins on CM-granulocyte adhesion of control mice and animals with inflammation is represented by the two columns on the left, respectively; the effect of nicotine (100 μM) in the presence of one of the antagonists is represented by the two columns on the right in each group of diagrams (b-j); the effect of nicotine on intact cells is shown for comparison (a). The presence or absence of a substance is indicated by "+" or "-", respectively. * - Significant difference from the parameter taken as 100%; # - difference between the effect of the agent in the control and "inflammatory" groups; & - difference between the effect of antagonist and jointly antagonist + nicotine on intact cells, p < 0.05.

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3. Fig. 2. Visualization of the α7 nAChR subunit on BM granulocytes. Microphotographs of mouse bone marrow granulocytes: a, b - fluorescent signals of NucRed and AF488-α-CTX, respectively; c - visible light image; d - overlay of images. Examples of granulocytes are shown with arrows. Scale bar: 10 μm.

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4. Fig. 3. Visualization of α10 nAChR subunit on CM-granulocytes. Microphotographs of cells after treatment with primary anti-α10 nAChR and secondary AF488-anti-IgG antibodies: a, b - NucRed and AF488-α-CTX fluorescent signals, respectively; c - visible light image; d - overlay of images. Examples of granulocytes are shown with arrows. Scale bar: 10 μm.

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5. Fig. 4. Effect of nicotine and pertussis toxin (PTX) on CM-granulocyte adhesion. Cells were incubated with nicotine, PTX or both substances for 2 h. Shown are the effects of: 10 and 100 μM nicotine (a); 300 ng/mL PTX (b); together 10 μM nicotine and PTX (c). Denotations: "+" and "- " - presence or absence of substance, respectively; * - difference from the parameter of intact cells; # - difference between the parameter of cells of control animals and animals with inflammation; n = 4, p < 0.05.

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6. Fig. 5. Action of nicotine on adhesion in the presence of inhibitors of intracellular signaling components. a - Comparison of the effect of inhibitors on adhesion of CM-granulocytes of control group mice (black bars) and mice with inflammation (gray bars). The effect of each inhibitor in the groups was calculated by the ratio of the ΔOD value from cells treated with one of the inhibitors to the ΔOD value from intact cells taken as 100%. b - Shown are the effects of: 100 μM nicotine on intact cells (1) and 100 μM nicotine together with 10 nM staurosporine (2); 50 nM tyrphostin 51 (3), 10 nM wortmannin (4) or 140 nM Y27632 (5). The effect of nicotine in each group was calculated: a - as the ratio of the ΔOD value from nicotine-treated cells to the ΔOD value from intact cells, taken as 100%; b - as the ratio of the ΔOD value from cells treated with nicotine + one of the inhibitors to the ΔOD value from cells treated with the same inhibitor, taken as 100%. 5-16 independent measurements were performed. * - Significant difference from the parameter taken as 100%; # - difference between the parameter of cells from control animals and animals with inflammation; & - difference from the effect of nicotine on intact cells, p < 0.05.

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7. Fig. 6. Example of the result of PCR products electrophoresis in agarose gel. The synthesized amplicons of nAChR subunits α3 (Chrna3), α7 (Chrna7), α9 (Chrna9), α10 (Chrn10), β2 (Chrnb2) and β-actin (Actb) in BALB/c mouse bone marrow granulocyte samples (Gr), mouse brain sample (Br) and mouse thymus sample (Th) are shown. Mouse thymus and mouse brain were taken as positive controls. M - DNA length marker (bp).

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