Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression

Zhang Jisong, He Jian, Xia Jingwen, Chen Zhen, Chen, Chen Xiaodong

Description

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

Sujets

• Apoptosis
• Bcl-2
• Chronic obstructive pulmonary disease
• Spirometry
• Neutrophil granulocyte

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 22
• Langue: English

Extrait

Zhang et al. Diagnostic Pathology 2012, 7:65
http://www.diagnosticpathology.org/content/7/1/65
RESEARCH Open Access
Delayed apoptosis by neutrophils from COPD
patients is associated with altered bak, bcl-xl,
and mcl-1 mRNA expression
2† 1† 1 1 1*Jisong Zhang , Jian He , Jingwen Xia , Zhen Chen and Xiaodong Chen
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
Keywords: Apoptosis, Bcl-2, Chronic obstructive pulmonary disease, Lung function, Neutrophils
Introduction [2]. An inflammatory pulmonary infiltrate comprised
priChronic obstructive pulmonary disease (COPD) is charac- marily of neutrophils is important in COPD pathogenesis.
terized by irreversible airflow limitation [1]. COPD is also Delayed cellular apoptosis can prolong the life of
neutrocharacterized by an abnormal inflammatory response to phils and can lead to their accumulation, resulting in a
pernoxious gases, especially cigarette smoke, and these abnor- sistent inflammation in the lungs and airway, which is
mal inflammatory responses exacerbate airflow obstruction considered to be a critical step in COPD pathogenesis [3].
Aggregation of neutrophils in the lungs and airway is
another characteristic of COPD [4-6].
* Correspondence: [e-mail]
† Although a number of studies have confirmed the
Equal contributors
1 delayed apoptosis of neutrophils in the peripheral circu-Department of Pulmonology, Huashan Hospital, Fudan University, Shanghai
200040, China lation, and in induced sputum and bronchoalveolar
Full list of author information is available at the end of the article
© 2012 Zhang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.Zhang et al. Diagnostic Pathology 2012, 7:65 Page 2 of 8
http://www.diagnosticpathology.org/content/7/1/65
lavage fluid (BALF) samples during the development diseases. No subjects had had an infection of the
respiraof COPD [7-10], the mechanisms underlying this tory tract for at least 3 months, and none were currently
phenomenon are not clear. One report found no rela- being treated with antibiotics or steroids. The study was
tionship between serum or sputum cytokines and the approved by the ethics review committee of Huashan
rate of neutrophil apoptosis during COPD exacerbations Hospital, Fudan University and was conducted in
[11]. However, Brown et al. found that neutrophil apop- accordance with the Declaration of Helsinki and Good
tosis was reduced in both stable COPD subjects and in Clinical Practice guidelines. All patients gave written
healthy controls who were smokers, which might have informed consent.
been attributed to the activation of NFκB [12].
Some genes may increase the risk of COPD develop- Lung function determinations
ment in certain populations. Ning et al. found 6 genes Lung function was determined in the Lung Function
whose expressions were different in mild COPD patients Laboratory according to standard protocols using a
who were smokers and in smokers without lung injury pneumotachograph (Jaeger MS Diffusion, Würzburg,
[13]. Thus, we hypothesized that delayed neutrophil Germany). A subject breathed smoothly several times,
apoptosis was caused by differences in the expressions of and then inspired forcibly until total lung capacity was
genes involved in the apoptotic regulatory pathway. achieved. Subsequently, the subject expired forcibly until
Members of the Bcl-2 protein family are part of the reaching his/her residual capacity. Then, the subject
mitochondrial intrinsic apoptotic pathway, and it has been inspired until total lung capacity was achieved followed
suggested that regulationofneutrophil apoptosis primarily by expiration. Results for the % predicted forced
expirainvolves members of this family [14]. Human neutrophils tory volume in one second (FEV1) and the forced vital
express many members of the Bcl-2 family, including the capacity (FVC) were generated automatically.
pro-apoptotic proteins Bax, Bid, Bak, and Bad, and the COPD patients were asked to stop taking long-acting
anti-apoptotic proteins Mcl-1, A1, and,Bcl-xl[15,16]. aminophylline 48 h or oral aminophylline 12 h before the
We hypothesized that a reason for the delayed neutro- examination and stopped using any inhaled β−agonist 6 h
phil apoptosis observed in COPD is the abnormal before the examination. If the FEV1/FVC was<70%,
expression of Bcl-2 family members. For this study, we patients were recommended to inhale salbutamol
(Ventoselected genes for the pro-apoptotic protein Bak and the lin) (200 μg×2 puffs), and spirometry was repeated 15 min
anti-apoptotic proteins Bcl-xl and Mcl-1, as they have later. An increase in FEV1/FVC of ≥12% and an absolute
been considered to be critical genes in regulating neu- increase of FEV1≥200 ml were regarded as positive for
trophil apoptotic pathways [14,15]. We examined the bronchodilation.
mRNA expressions of these genes in unstimulated
peripheral blood neutrophils from patients with mild to Isolation of peripheral blood neutrophils
moderate COPD and healthy volunteers to investigate Neutrophils were isolated from peripheral blood
samthe relationship between Bcl-2 family gene regulation, ples collected in sterile heparinised tubes by a two-step
delayed neutrophil apoptosis, and COPD pathogenesis. procedure. First, erythrocytes were removed by
sedimentation using dextran (T500, Pharmacia). Next,
Methods granulocytes were isolated using discontinuous Percoll
Subjects gradient centrifugation [17]. We used 0.9% saline to
All COPD patients in this study were in a stable stage prepare 60% and 70% (by volume) Percoll solutions
and were recruited from the out-patient clinic of our (densities of 1.079 g/ml and 1.091 g/ml, respectively)
hospital. These patients had mild-to-moderate COPD from a stock solution of 1.130 g/ml (100% fine grade
(total: n=47; smokers: n=24). The categorization of Percoll, Pharmacia). After centrifugation, the density of
mild-to-moderate COPD was based on the Global Initia- peripheral blood granulocytes, primarily neutrophils, in
tive on Chronic Obstructive Lung Disease (GOLD) the Percoll solution was between 1.080 to 1.085 g/ml.
criteria (http://www.goldcopd.org). Patients who were The isolated cells were determined to be>98%
neutropossibly asthmatic were excluded based on the criteria phils by Giemsa staining.
of the Global Initiative on Asthma (GINA; http://www.
ginaasthma.org). Neutrophil apoptosis assay
We also recruited age-matched healthy controls (total:ils were suspended to a concentration of
5
n=47; smokers: n=24). For both COPD patients and 5×10 cells/ml in RPMI 1640 medium supplemented
healthy controls, smoking history was recorded in pack- with 10% FCS and 100 U/ml of penicillin-streptomycin,
years. Exclusion criteria for all subjects were history of and 200 μl of this suspension was pipetted into each well
allergy, tuberculosis, neoplasm, asthma, thoracic or of a 36 well flat-bottom microtest plate. Unstimulated
abdominal surgery, or other serious concomitant cells were cultured at 37°C in a 5% CO atm for 4, 24, or2Zhang et al. Diagnostic Pathology 2012, 7:65 Page 3 of 8
http://www.diagnosticpathology.org/con