Inhibition of granulocyte migration by tiotropium bromide

Abstract

Study objectives: Neutrophil influx into the airways is an important mechanism in the pathophysiology of the inflammatory process in the airways of patients with chronic obstructive pulmonary disease (COPD). Previously it was shown that anticholinergic drugs reduce the release of non-neuronal paracrine mediators, which modulate inflammation in the airways. On this basis, we investigated the ability of the long-acting anticholinergic tiotropium bromide to inhibit a) alveolar macrophage (AM)-mediated chemotaxis of neutrophils, and b) cellular release of reactive oxygen species (ROS).

Method: AM and neutrophils were collected from 71 COPD patients. Nanomolar concentrations of tiotropium bromide were tested in AM cultured up to 20 h with LPS (1 μg/ml). AM supernatant was tested for TNFα, IL8, IL6, LTB4, GM-CSF, MIPα/β and ROS. It was further used in a 96-well chemotaxis chamber to stimulate the migration of fluorescence labelled neutrophils. Control stimulants consisted of acetylcholine (ACh), carbachol, muscarine or oxotremorine and in part PMA (phorbol myristate acetate, 0.1 μg/ml). Potential contribution of M1-3-receptors was ascertained by a) analysis of mRNA transcription by RT-PCR, and b) co-incubation with selective M-receptor inhibitors.

Results: Supernatant from AM stimulated with LPS induced neutrophilic migration which could be reduced by tiotropium in a dose dependent manner: 22.1 ± 10.2 (3 nM), 26.5 ± 18,4 (30 nM), and 37.8 ± 24.0 (300 nM, p < 0.001 compared to non-LPS activated AM). Concomitantly TNFα release of stimulated AM dropped by 19.2 ± 7.2% of control (p = 0.001). Tiotropium bromide did not affect cellular IL8, IL6, LTB4, GM-CSF and MIPα/β release in this setting. Tiotropium (30 nM) reduced ROS release of LPS stimulated AM by 36.1 ± 15.2% (p = 0.002) and in carbachol stimulated AM by 46.2 ± 30.2 (p < 0.001). M3R gene expression dominated over M2R and M1R. Chemotaxis inhibitory effect of tiotropium bromide was mainly driven by M3R inhibition.

Conclusion: Our data confirm that inhibiting muscarinic cholinergic receptors with tiotropium bromide reduces TNFα mediated chemotactic properties and ROS release of human AM, and thus may contribute to lessen cellular inflammation.

Figure 1

Tiotropium inhibited chemotactic activity from LPS activated AM (test runs from left to right: n = 4, n = 9, n = 20 patients). Mean ± SD (standard deviation). * p < 0.001 compared with LPS activation without inhibition. Concentrations according to expected values in the airways after tiotropium inhalation http://www.rxlist.com/cgi/generic3/spiriva_cp.htm.

Figure 2

Cellular mRNA levels of muscarinic M1R (MRC1), M2R (MRC2), M3R (MRC3) subreceptors in alveolar macrophages from COPD patients (n = 11). Mean+SD.

Figure 3

Differences in muscarinic receptor (MRC1-3) expression profile in AM from n = 19 COPD patients depending on culture conditions. X-axis represents comparisons always after an incubation time of 20 h: carbachol vs. control, LPS vs. control, 20 h vs. 2 h, LPS+ACh vs. LPS. Mean ± SD. * p = < 0.001 (repeated measures One Way ANOVA).

Figure 4

Inhibition of AM induced chemotactic activity from tiotropium on neutrophils is predominantly driven by M3R-blockage (n = 10): Tiotropium 30 nM, 4-DAMP 100 nM (each: p < 0.01 vs. LPS stimulation alone), Ipratropium 30 nM, Gallamine 100 μM, Tubocurarine 100 μM, Telenzepine 10 nM. Doses were adapted from earlier studies and customized to our assay conditions [43,63,64].

Figure 5

Correlation of TNFα release of pre-cultured and LPS activated alveolar macrophages (AM) to the migration rate of neutrophils cultured with AM cell medium (Tiotropium 30 nM).

Figure 6

ROS (reactive oxygen species) release from LPS, PMA or carbachol activated alveolar macrophages (AM) from 16 patients. ROS production in unstimulated cells was defined as 100%, ROS in cell free medium was 0. LPS 1 μg/ml; Carb = carbachol in various concentrations; Dexam = Dexametasone 10^-7 M in LPS activated AM; PMA 0.1 μg/ml. Data expressed as mean ± SD. *p < 0.001 vs. LPS.

Figure 7

Inhibition of ROS release by tiotropium (3 × 10⁹ M) in LPS (1 μg/ml) or carbachol (10^-4 M) activated alveolar macrophages. *p < 0.001, both comparisons with LPS or carbachol alone. Mean ± SD.