Cholinergic mechanisms in an organic dust model simulating an acute exacerbation in patients with COPD

Figures & data

Figure 1 Study design.

Notes: (A) All participants visited the laboratory on three occasions: at baseline, for a methacholine challenge, and after 2 hours of exposure in a pig barn (in vivo exposure). The order of the two last visits was randomized. Outcome measures at each visit are given. At baseline and after exposure in the pig barn, blood was collected for ex vivo stimulations. (B) Ex vivo stimulations of isolated blood neutrophils were performed using dust collected in the pig barn, acetylcholine, and a combination of dust and acetylcholine. Stimulations were performed in the absence and presence of muscarinic receptor antagonists: an non-specific muscarinic receptor antagonist (tiotropium, three doses), a specific M₃-receptor antagonist (solifenacin), a specific M₁-antagonist (pirenzepine), and an acetylcholinesterase inhibitor (neostigmine).
Abbreviations: CAT, COPD Assessment Test; PEF, peak expiratory flow; SPT, skin-prick test.

Figure 2 FVC (A) and FEV₁ (B) at baseline and 7 hours after start of in vivo exposure in a pig barn in controls (healthy non-smokers) and smokers with COPD. Vertical lines indicate baseline difference between the groups and horizontal lines difference induced by exposure within each group. In vivo exposure induced decreases in FEV₁ and FVC greater in the COPD group than in the healthy non-smokers (P<0.0001). (C) Change in FEV₁ induced by in vivo exposure in a pig barn related to bronchial responsiveness to methacholine indicated by 20% decrease in FEV₁ (PD₂₀FEV₁) in 15 healthy non-smokers and 13 smokers (methacholine challenge was not performed in two COPD patients who experienced symptoms).

Table 1 Baseline characteristics of healthy non-smokers (controls) and smokers with COPD

	Controls, n=15	COPD, n=15	P-value
Female/male	9/6	11/4
Age (years), mean, range	60.5 (50–71)	63.3 (53–77)	0.22
Smoking (pack-years), mean, range		42.2 (22–95)
COPD Assessment Test (points), mean, range	3 (0–7)	18 (7–34)	<0.0001
FEV1 (L), mean (95% CI)	3.20 (2.63–3.78)	1.85 (1.67–2.03)	<0.0001
FEV1 (% predicted), mean (95% CI)	96 (87–104)	67 (61–74)	<0.0001
FVC (L), mean (95% CI)	3.89 (3.25–4.52)	2.84 (2.61–3.07)	0.002
FVC (% predicted), mean (95% CI)	88 (82–94)	80 (73–86)	0.05
FEV1/VC or FVC (%), mean (95% CI)	0.81 (0.77–0.85)	0.60 (0.56–0.64)	<0.0001
PD20FEV1 (mg), median (25th–75th percentile)	2.72 (0.59–11.1)	0.30 (0.06–0.66)	0.004

Notes: Lung function was measured 15 minutes after inhalation of 0.4 mg salbutamol. P-values refer to between-group comparisons and were calculated by mean of unpaired t-tests or Mann–Whitney U test (the cumulative dose causing a 20% decrease in FEV₁ [PD₂₀ FEV₁]).

Figure 3 Sputum analyses before and after in vivo exposure in COPD subjects and controls.

Notes: Horizontal lines indicate comparisons between pre- and postexposure values. Double arrows indicate comparisons between baseline (preexposure) values. Median values and 25th–75th percentiles indicated by bars and vertical lines.

Table 2 Expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT)

	Healthy non-smokers (controls)			Smokers with COPD			Baseline, COPD vs controls	Change, COPD vs controls
	Before in vivo exposure (baseline)	After in vivo exposure	P-value	Before in vivo exposure (baseline)	After in vivo exposure	P-value	P-value	P-value
Sputum
Neutrophils
AChE	2.04 (1.70–2.66)	1.69 (1.29–1.91)	0.60	2.29 (1.79–2.84)	2.58 (2.42–3.05)	1.00	0.59	0.073
ChAT	1.80 (1.58–2.95)	1.97 (1.56–2.40)	1.00	2.36 (1.69–2.75)	2.27 (2.01–2.76)	1.00	0.53	0.72
Monocytes
AChE	2.09 (1.45–3.08)	1.91 (1.33–2.61)	0.58	2.25 (2.05–2.62)	2.65 (2.29–2.93)	0.10	0.68	0.051
ChAT	2.53 (1.51–3.45)	1.63 (1.18–1.95)	0.096	1.84 (1.69–2.43)	1.64 (1.37–3.05)	1.00	0.33	0.28
Lymphocytes
AChE	2.24 (1.40–2.68)	1.41 (1.15–1.60)	0.27	2.38 (1.99–3.78)	2.47 (2.06–3.08)	0.77	0.36	0.57
ChAT	2.12 (1.71–2.77)	1.46 (1.11–1.81)	0.58	1.81 (1.54–3.17)	1.47 (1.40–1.93)	0.27	0.81	0.72
Blood
Neutrophils
AChE	2.27 (1.94–3.07)	1.91 (1.54–2.44)	0.30	2.12 (1.75–2.50)	1.86 (1.73–2.58)	1.00	0.51	0.48
ChAT	2.17 (1.78–3.24)	2.18 (1.87–2.54)	1.00	2.17 (1.70–2.55)	2.18 (1.58–2.57)	0.30	0.66	0.97
Monocytes
AChE	2.93 (2.63–3.40)	2.85 (2.50–3.75)	0.58	3.47 (2.93–4.20)	2.94 (2.44–3.71)	0.18	0.08	0.23
ChAT	1.71 (1.53–2.42)	1.92 (1.83–2.36)	0.30	2.08 (1.60–2.39)	1.96 (1.50–2.30)	0.79	0.62	0.18
Lymphocytes
AChE	3.00 (2.50–3.13)	1.85 (1.46–2.95)	0.04	2.83 (2.44–3.38)	2.67 (2.00–3.37)	0.79	0.98	0.32
ChAT	1.65 (1.44–1.80)	1.25 (1.08–1.54)	0.02	1.74 (1.38–2.00)	1.67 (1.35–2.10)	0.79	0.76	0.12

Notes: Expression on neutrophils, monocytes, and lymphocytes in sputum and blood before and after in vivo exposure (2 hours of exposure in a pig barn) in controls (healthy non-smokers) and smokers with COPD. Enzyme expression assessed by flow cytometry and presented as relative median fluorescence intensity. Values expressed as mean (95% CI).

Figure 4 Blood analyses before and after in vivo exposure in a pig barn in COPD subjects and controls.

Notes: Horizontal lines indicate comparisons between pre- and post-exposure values. Double arrows indicate comparisons between baseline (pre-exposure) values. Horizontal brackets indicate comparisons between exposure-induced changes between the groups. Median values and 25th–75th percentiles indicated by bars and vertical lines.

Figure 5 CXCR1, CXCR2 and ChAT expression.

Notes: Isolated blood neutrophils stimulated ex vivo with organic dust and the effect of tiotropium (Tio) in healthy controls and patients with COPD. Neutrophils were collected before and 7 hours after in vivo exposure in a pig barn. As the results were not significantly influenced by in vivo exposure in either group, only data before in vivo exposure are included in the figure. Data presented as relative median fluorescence intensity (rMFI) and mean (SEM). (A) CXCR1 expression on neutrophils was decreased by dust in controls (P<0.0003), but not in the COPD group (P=0.015). (B) Dust increased the expression of CXCR2 in both groups (P≤0.002). Tio did not significantly influence CXCR1 or CXCR2 expression before (A, B) or after in vivo exposure (data not shown) in either group. (C) Dust induced increased expression of choline acetyltransferase (ChAT) on neutrophils in controls (P<0.0001) and COPD (P=0.0003) before in vivo exposure. Similar findings were obtained after in vivo exposure (data not shown). There was no difference between the groups, nor did Tio alter the outcome.

Figure 6 (A) Before in vivo exposure, the release of TNFα from isolated blood neutrophils increased following ex vivo stimulation with dust in controls (P=0.0002) and COPD (P=0.0014). A similar increase was observed in controls (P=0.0069) and COPD (P=0.0014) after in vivo exposure (data not shown). There was no difference between the groups (P≥0.20). Dust-induced TNFα release was not significantly altered by tiotropium (Tio). (B) Dust by itself did not influence LTB₄ release in either group before (and after, data not shown) in vivo exposure. (C, D) Acetylcholine (Ach; 100 µM) induced a significant decrease in LTB₄ release from neutrophils before in vivo exposure (C) in controls (P=0.0017) and after in vivo exposure (D) in the COPD group (P=0.0002). Tio did not significantly alter the response induced by Ach. (E) Before in vivo exposure, dust increased LTB₄ release in the presence of pirenzepine in controls (P=0.0036) and COPD (P=0.0092). Similar findings were observed after in vivo exposure (P≤0.0022; data not shown). The combination of dust and neostigmine induced increased LTB₄ release in controls (P=0.0008) and COPD (P=0.0003) before in vivo exposure. Similar findings were observed after in vivo exposure (P≤0.0021; data not shown). Data presented as mean (SEM).