The aim of this study was to investigate whether changes in gene expressions for members of the Bcl-2 family of pro- and anti-apoptotic proteins were involved in the delayed neutrophil apoptosis seen in COPD. We found that, in peripheral blood neutrophils from subjects with stable mild to moderate COPD, mRNA expressions for two anti-apoptotic genes (Bcl-xl and Mcl-1) were upregulated and that for a pro-apoptotic gene (Bak) was downregulated (Figure ). Our findings showed that the abnormal expression of Bcl-2 family member genes was present in an early stage COPD and that this trend of abnormal gene expression was consistent with the delayed apoptosis seen in neutrophils from COPD patients (Figure ). We also found that these mRNA expression levels were significantly correlated with lung function parameters (Figure ).
In our study, after 36 h in unstimulated culture, the apoptosis rate of peripheral blood neutrophils of COPD patients was significantly delayed relative to neutrophils from healthy controls. For peripheral blood neutrophils, others have reported decreased apoptosis during COPD exacerbations, but not in stable COPD [8
]. Using cells from sputum samples, neutrophil apoptosis for COPD patients who were current smokers was reported to be only one-third of that seen in sputum samples from healthy non-smokers.12
This difference in results between neutrophils from peripheral blood and sputum is presumably due to the additional effect on sputum neutrophils of the chronic inflammation present in the lungs of COPD patients, particularly those who smoke.
In our study, the differences between COPD patients and controls in neutrophil mRNA levels of Bcl-2 family genes were greater than the differences seen in the percentages of apoptotic neutrophils. The mRNA levels for the two anti-apoptotic genes (Bcl-xl and Mcl-1) were twice as high as in controls, and the mRNA level of the pro-apoptotic gene (Bak) was 60% of that in controls’ neutrophils.
The Bcl family contains pro- and anti-apoptotic proteins that are found in the outer membranes of mitochondria. The balance between the pro- and anti-apoptotic members of this family is the central factor in the opening and closing of permeability transition (PT) pores [21
], which is a critical pathway for cytochrome c release from mitochondria [22
] and causes apoptosis by activating caspase-9. The increase in anti-apoptotic mRNA expression and decrease in pro-apoptotic mRNA expression seen in our study could tip the balance toward neutrophil survival and decrease the apoptosis rate.
However, it is the relative abundance of Bcl-2 family proteins, rather than their mRNA expression, that would ultimately regulate apoptosis, and protein levels do not always correspond to mRNA levels. This was reportedly the case for Bcl-xL. Moulding et al. [15
], who studied peripheral blood neutrophils from normal subjects, detected Bcl-xL mRNA in these cells, but could not detect the corresponding protein. They also found that blocking gene transcription with actinomycin decreased Mcl-1 mRNA and protein, but had virtually no effect on Bcl-xL mRNA levels. Therefore, Bcl-xL may not be involved in the control of apoptosis in human neutrophils.
Although gene transcription is important in determining mRNA levels, the mRNA half-life is also important. The half-life of the mRNA for Mcl-1, an anti-apoptotic protein, is relatively short and the half-life of the mRNA for Bak, a pro-apoptotic protein, is relatively long [15
]. Thus, Mcl-1, whose mRNA levels can change relatively rapidly, might be more important than Bak in the short-term control of apoptosis. A relationship between decreased Mcl-1 mRNA expression and an increase in neutrophil apoptosis has previously been reported for patients with sepsis [23
], but until now, research on COPD patients has been lacking.
We found only minimal differences in Bcl-2 family mRNA expression between healthy smokers and healthy non-smokers, and between smoking and non-smoking COPD patients. This suggests that the effects of smoking are not evident in neutrophils from peripheral blood as would be expected in cells from BALF, especially in patients with mild to moderate COPD like those included in the current study. An alternative explanation would be that Bcl-2 member expression is implicated in COPD pathogenesis, but not through a pathway that is affected by smoking. In addition, COPD is the result of the interactions between genes and the environment [24
]. Genes other than Bcl-2 family members could be affected by these factors.
More studies are needed to investigate the relationships between mRNA and protein levels, and to determine whether the results of Bcl family members in normal neutrophils apply to COPD patients. An in-depth study is also needed on the neutrophil apoptosis pathways that are influenced by cigarette smoking and the complex effects of nicotine on neutrophil apoptosis in vivo.