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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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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.
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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.

Mentions: Localisation of single granulocyte nuclei allowed for the quantification of NFκB activation in sputum from COPD subjects, healthy smoking controls and non-smoking controls (Figure 6a and 6b). p50 and p65 subunit expression in sputum neutrophil nuclei showed a significant increase in the expression of both NFκB subunits in COPD subjects (p50, p = 0.006 and p65: p = 0.006) compared to non-smokers (Figure 6c). Although there was a higher expression of p50 and p65-positive neutrophil nuclei in healthy smokers compared to non-smokers, this did not reach significance. An association was observed between sFasL in plasma from non-smokers and activation of p65 in sputum (r = -0.90, p = 0.002, Spearman's correlation).


Dysregulated apoptosis and NFkappaB expression in COPD subjects.

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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.
Mentions: Localisation of single granulocyte nuclei allowed for the quantification of NFκB activation in sputum from COPD subjects, healthy smoking controls and non-smoking controls (Figure 6a and 6b). p50 and p65 subunit expression in sputum neutrophil nuclei showed a significant increase in the expression of both NFκB subunits in COPD subjects (p50, p = 0.006 and p65: p = 0.006) compared to non-smokers (Figure 6c). Although there was a higher expression of p50 and p65-positive neutrophil nuclei in healthy smokers compared to non-smokers, this did not reach significance. An association was observed between sFasL in plasma from non-smokers and activation of p65 in sputum (r = -0.90, p = 0.002, Spearman's 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