Delayed apoptosis by neutrophils from COPD patients is associated with altered Bak, Bcl-xl, and Mcl-1 mRNA expression

Abstract

Background: Delayed neutrophil apoptosis may be an important factor in the persistent inflammation associated with chronic obstructive pulmonary disease (COPD). Bcl-2 family proteins are important regulators of neutrophil apoptosis. We determined the mRNA levels of pro-apoptotic Bak and anti-aptototic Bcl-xl and Mcl-1 members of the Bcl-2 family in unstimulated peripheral blood neutrophils from patients with mild to moderate COPD and compared these to neutrophils from healthy controls.

Methods: Neutrophils were isolated from peripheral blood samples of 47 COPD patients (smokers: N = 24) and 47 healthy controls (smokers: N = 24). Percentages of apoptotic cells were determined at 4, 24, and 36 h for unstimulated neutrophils cultured in vitro. Neutrophil mRNA expression of Bak, Bcl-xl, and Mcl-1 was determined by real-time polymerase chain reaction (PCR). FEV1 (% predicted) and FVC were determined by spirometry and correlations between mRNA levels and lung function parameters were determined.

Results: The percentages of apoptotic cells among unstimulated neutrophils from COPD patients were significantly lower compared to cells from controls after 4, 24, and 36 h in culture; smoking history had only a minimal effect on these differences. Unstimulated neutrophils from COPD patients had significantly lower Bak mRNA expression and higher expressions of Bcl-xl and Mcl-1 mRNA than cells from healthy controls. Again, smoking history had only a minimal effect on these trends. Bak mRNA expression was significantly positively correlated with both % predicted FEV1 and the FEV1/FVC ratio, while Bcl-xl and Mcl-1 mRNA expressions were significantly negatively correlated with %predicted FEV1 and the FEV1/FVC ratio.

Conclusions: The genes for pro-apoptotic Bak, and anti-apoptotic Bcl-xl and Mcl-1 may be important in regulating the delayed neutrophil apoptosis observed in COPD, which may contribute to COPD pathogenesis.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1605269445677066.

Figure 1

Comparisons of percentages of apoptotic neutrophils from healthy controls and COPD patients. Peripheral blood neutrophils were isolated from healthy controls and COPD patients; these groups were sub-divided into those with and without smoking histories. As described in Methods, unstimulated peripheral blood neutrophils were cultured in vitro for 4, 24, or 36 h, after which the percent of apoptotic cells was determined. Results are means ± SEMs for: COPD/smokers (n = 24); COPD/non-smokers (n = 23); Healthy/smokers (n = 24); and Healthy/non-smokers (n = 23). Group comparisons were by Mann–Whitney U test. * significant difference as compared with Healthy/non-smokers (p < 0.05); † significant difference as compared with Healthy/smokers (p < 0.05); ‡ significant difference as compared with COPD/non-smokers (p < 0.05).

Figure 2

Expression of Bak, Bcl-xl, and Mcl-1 mRNA’s in peripheral blood neutrophils. Real-time PCR was used to determine the expression of (A) Bak, (B) Bcl-xl, and (C) Mcl-1 mRNAs in peripheral blood neutrophils. The ΔΔCt value for each mRNA was normalized to the value of the GADPH housekeeping gene mRNA. Results are means ± SEMs for: COPD/smokers (n = 24); COPD/non-smokers (n = 23); Healthy/smokers (n = 24); and Healthy/non-smokers (n = 23). Groups were compared using two-sample t-test for Bak and Mcl-1 mRNA’s, and a Mann–Whitney U test for Bcl-x1 mRNA due to its non-normal distribution. * significant difference as compared with Healthy/non-smokers (p < 0.05); † significant difference as compared with Healthy/smokers (p < 0.05).

Figure 3

Correlations between mRNA levels of Bak, Bcl-xl, and Mcl-1 and lung function parameters. COPD patients are represented by solid circles and healthy controls by open circles (N = 47 for each group) Spearman correlation analysis was used to assess associations between variables. (A) Bak mRNA level was positively correlated with lung function. FEV1/FVC%: r = 0.72; P < 0.001; FEV1% reference: r = 0.70; P < 0.001. (B) Bcl-xl mRNA level was negatively correlated with lung function. FEV1/FVC%: r = -0.82; P < 0.001; FEV1% reference: r = -0.86; P < 0.001. (C) Mcl-1 mRNA level was negatively correlated with lung function. FEV1/FVC%: r = -0.79; P < 0.001; FEV1% reference: r = -0.79; P < 0.001.