Dysregulated apoptosis and NFkappaB expression in COPD subjects.
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.


CONCLUSIONS
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.
Brown
Vanessa
V
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
Elborn
J Stuart
JS
Bradley
Judy
J
Ennis
Madeleine
M
D000368:Aged; D017209:Apoptosis; D002097:C-Reactive Protein / Q000032:analysis; D016022:Case-Control Studies; D016207:Cytokines / Q000032:analysis; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D008875:Middle Aged; D000072000:NF-KappaB Inhibitor alpha; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D009504:Neutrophils / Q000276:immunology* / Q000473:pathology; D010766:Phosphorylation; D029424:Pulmonary Disease, Chronic Obstructive / Q000276:immunology* / Q000473:pathology; D012907:Smoking / Q000276:immunology / Q000473:pathology; D013183:Sputum / Q000166:cytology / Q000276:immunology; D051996:Transcription Factor RelA / Q000378:metabolism*; D015854:Up-Regulation
D016207:Cytokines; D020672:I-kappa B Proteins; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; C000606204:NFKBIA protein, human; C498143:RELA protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D002097:C-Reactive Protein