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Dysregulated apoptosis and NFkappaB expression in COPD subjects.

Brown V, Elborn JS, Bradley J, Ennis M - Respir. Res. (2009)

Bottom Line: 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.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Respiratory Research Group, Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, UK. v.brown@qub.ac.uk

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.

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Related in: MedlinePlus

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

Mentions: The results from the three different methods used to assess apoptosis are summarised in Table 3. Using one-way ANOVA test, the percentage of apoptotic cells were significantly different between all 3 assays (p < 0.0001) with post-hoc test showing a higher percentage of Tunel positive cells, Av+/PI+ and Av-/PI+ cells compared to the percentage of neutrophils showing morphological features of apoptosis (Figure 2) (p < 0.001, p < 0.001 and p < 0.05 respectively). Also there was a higher percentage of apoptotic neutrophils detected by the Tunel assay compared with those cells that were Av+/PI- (p < 0.0.5). There was a significant correlation between Av+/PI- neutrophils (early apoptotic neutrophils) and the percentage of neutrophils displaying morphological features of apoptosis (r = 0.67, p < 0.001, Pearson correlation).


Dysregulated apoptosis and NFkappaB expression in COPD subjects.

Brown V, Elborn JS, Bradley J, Ennis M - Respir. Res. (2009)

Apoptotic sputum neutrophils identified using light microscopy. Typical apoptotic neutrophils displaying loss of chromatin filaments (heavy arrow) and shrinkage of the nucleus (thin arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667166&req=5

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).
Mentions: The results from the three different methods used to assess apoptosis are summarised in Table 3. Using one-way ANOVA test, the percentage of apoptotic cells were significantly different between all 3 assays (p < 0.0001) with post-hoc test showing a higher percentage of Tunel positive cells, Av+/PI+ and Av-/PI+ cells compared to the percentage of neutrophils showing morphological features of apoptosis (Figure 2) (p < 0.001, p < 0.001 and p < 0.05 respectively). Also there was a higher percentage of apoptotic neutrophils detected by the Tunel assay compared with those cells that were Av+/PI- (p < 0.0.5). There was a significant correlation between Av+/PI- neutrophils (early apoptotic neutrophils) and the percentage of neutrophils displaying morphological features of apoptosis (r = 0.67, p < 0.001, Pearson correlation).

Bottom Line: 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.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Respiratory Research Group, Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, UK. v.brown@qub.ac.uk

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.

Show MeSH
Related in: MedlinePlus