Dysregulated apoptosis and NFkappaB expression in COPD subjects

Abstract

Background: The abnormal regulation of neutrophil apoptosis may contribute to the ineffective resolution of inflammation in chronic lung diseases. Multiple signalling pathways are implicated in regulating granulocyte apoptosis, in particular, NFkappaB (nuclear factor-kappa B) signalling which delays constitutive neutrophil apoptosis. Although some studies have suggested a dysregulation in the apoptosis of airway cells in chronic obstructive pulmonary disease (COPD), no studies to date have directly investigated if NFkappaB is associated with apoptosis of airway neutrophils from COPD patients. The objectives of this study were to examine spontaneous neutrophil apoptosis in stable COPD subjects (n = 13), healthy smoking controls (n = 9) and non-smoking controls (n = 9) and to investigate whether the neutrophil apoptotic process in inflammatory conditions is associated with NFkappaB activation.

Methods: Analysis of apoptosis in induced sputum was carried out by 3 methods; light microscopy, Annexin V/Propidium iodide and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method. Activation of NFkappaB was assessed using a flow cytometric method and the phosphorylation state of IkappaBalpha was carried out using the Bio-Rad Bio-Plex phosphoprotein IkappaBalpha assay.

Results: Flow cytometric analysis showed a significant reduction in the percentage of sputum neutrophils undergoing spontaneous apoptosis in healthy smokers and subjects with COPD compared to non-smokers (p < 0.001). Similar findings were demonstrated using the Tunel assay and in the morphological identification of apoptotic neutrophils. A significant increase was observed in the expression of both the p50 (p = 0.006) and p65 (p = 0.006) subunits of NFkappaB in neutrophils from COPD subjects compared to non-smokers.

Conclusion: These results demonstrate that apoptosis is reduced in the sputum of COPD subjects and in healthy control smokers and may be regulated by an associated activation of NFkappaB.

Figure 1

(a) CRP levels in sputum from non-smokers, n = 7 (NS), healthy smokers, n = 9 (HS) and in COPD subjects, n = 12. (b) Log₁₀ IL-6 levels (pg/ml) in sputum from non-smokers, n = 5 (NS), healthy smokers, n = 5 (HS) and in COPD subjects, n = 12 (c) IL-8 levels (pg/ml) in sputum from non-smokers, n = 5 (NS), healthy smokers, n = 5 (HS) and in COPD subjects, n = 12 (d) Log₁₀ GM-CSF levels (pg/ml) in sputum from non-smokers, n = 5 (NS), healthy smokers, n = 5 (HS) and in COPD subjects, n = 12. Data are expressed as mean (SEM). p values shown is from Tukey's multiple comparison post-hoc analysis following one-way ANOVA.

Figure 2

Apoptotic sputum neutrophils identified using light microscopy. Typical apoptotic neutrophils displaying loss of chromatin filaments (heavy arrow) and shrinkage of the nucleus (thin arrow).

Figure 3

Annexin V/PI analysis in induced sputum. (a) Horizontal axis represents side scatter (SS) and vertical axis represents forward scatter (FS) (linear scale). Gated area, containing granulocytes, represented by area marked M (b) Horizontal axis represents intensity of staining for Av (Annexin V) (logarithmic scale) and vertical axis intensity of staining for PI (logarithmic scale). Typical dot plot representing populations of early apoptotic (AV⁺/PI^-), secondary necrotic (AV⁺/PI⁺), and necrotic granulocytes (AV⁺/PI^-) (c) Analysis of neutrophil apoptosis in sputum using the Annexin V/Propidium iodide assay. One way ANOVA showed a reduction in Av⁺/PI^- in healthy smokers (HS) and COPD subjects compared to non-smokers (NS), p < 0.001. % AV⁺/PI^-: early apoptosis, % AV^+/PI⁺: later stage apoptosis/secondary necrosis:% AV^-/PI⁺: necrosis.

Figure 4

Percent of apoptotic neutrophils in sputum by DNA strand breaks (Tunel) and morphological features of apoptosis. Data are displayed as median (interquartile range). Kruskal-Wallis test was used to determine any significant differences between the 3 groups, followed by post-hoc analysis (Dunn's Multiple Comparison test). p values shown are from post-hoc analysis.

Figure 5

sFas and sFasL levels in plasma from non-smokers (NS), healthy smokers (HS) and COPD subjects. Data are displayed as median (interquartile range).

Figure 6

NF[kappa]B analysis in induced sputum. (a) Identification of granulocyte nuclei (gated area shown) in sputum by comparing separated granulocyte nuclei from sputum sample (left) with whole sputum nuclei (right) (b) Sputum granulocyte nuclei: FL-3-peak versus FL-3 integral dot plot showing a singlet gate to exclude aggregates (c) Percentage of neutrophils expressing p50 and p65 (NFκB activation) in induced sputum in non-smokers (NS), healthy smokers (HS) and in COPD subjects. In COPD subjects closed symbols = current smokers and open symbols = ex-smokers. Mann-Whitney U test was used to determine any significant differences. COPD n = 12; HS n = 6; NS n = 8. Line represents median value.

Figure 7

Quantification of IκBα activation in induced sputum. Control non-smokers (NS, n = 5), healthy smokers (HS, n = 4) and in COPD subjects (n = 7). In COPD subjects closed symbols = current smokers and open symbols = ex-smokers. Line represents median value.