A new inflammation marker of chronic obstructive pulmonary disease–adiponectin

Abstract

BACKGROUND:

This study was undertaken to measure the concentration of adiponectin (APN) in serum and induced sputum in patients with chronic obstructive pulmonary disease (COPD during acute exacerbation (AECOPD) and at stable stage and to determine the role of APN as a marker of inflammation in the pathogenesis of COPD.

METHODS:

All the patients in this prospective study were enrolled from October 2008 to October 2009, including 30 male AECOPD patients from the emergency department, 30 male stable COPD patients from the department of respiratory diseases, and 30 healthy non-smoking male controls from the department of medical examination. The serum and induced sputum were collected from each patient. All of the patients had normal weight (BMI range 18.5-24.9 kg/m²). Patients with severe bronchial asthma, bronchiectasis or autoimmune disease were excluded. Cell count and classification was performed for the induced sputum. The concentrations of APN, IL-8, IL-6 and TNF-α were measured by ELISA. Pulmonary function was tested among the three groups. Comparisons between the groups were conducted by Student’s t test, ANOVA analysis or nonparametric test. Correlation analysis was carried out by Pearson’s product-moment correlation coefficient test or Spearman’s rank-order correlation coefficient test.

RESULTS:

The concentrations of APN in the serum or induced sputum in AECOPD patients were significantly higher than those in stable COPD patients or healthy non-smoking controls (P<0.01). The concentration of APN in stable COPD patients was significantly higher than that in healthy non-smoking controls (P<0.01). For the AECOPD patients, APN was positively correlated with IL-8 and TNF-α in the serum and induced sputum (r=0.739, 0.734, 0.852, 0.857 respectively, P<0.05). For the stable COPD patients, APN was also positively correlated with IL-8 and TNF-α in the serum and induced sputum (r=0.751, 0.659, 0.707, 0.867 respectively, P<0.05). In addition, for the AECOPD patients, APN was positively correlated with the percentage of neutrophils in the induced sputum (r=0.439, P<0.05).

CONCLUSIONS:

APN is involved in the process of systematic and airway inflammation of COPD. This process is related to neutrophils in the airway, IL-8 and TNF-α. APN could be used as a new marker for inflammation of COPD.

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is considered to be as a chronic non-specific inflammation, which occurs in the airway, lung parenchyma and pulmonary vessels. This can cause the activation of inflammatory cells and the release of various inflammatory mediators, such as IL-8, IL-6, and TNF-α. They can destroy the lung structure and promote the inflammatory response of neutrophils.[1,2] Adiponectin (APN) is one of the cytokines mainly secreted by adipose tissue. [3-5] Miller et al[6] reported that the epithelial cells can also secrete APN, which played a potential role in regulating the inflammatory response by means of autocrine or paracrine.

At present, the mechanism of APN participation in the inflammation of COPD remains unknown. To find out its role in COPD inflammation, we measured the concentration of APN in the serum and induced sputum in patients, and explored its relationship with the percentage of neutrophils in the airway, IL-8, IL-6 and TNF-α.

METHODS

General information of patients

The patients were male, and they were admitted to Shanghai Fifth People’s Hospital between October 2008 and October 2009. Among them, 30 patients in the acute exacerbation stage of COPD aged on average (64.21 ± 2.84) years who had been admitted to the emergency department served as group A. Among them, 19 patients were smokers, with a mean smoking amount of 28 (10-40) packages/year, whereas the other 11 patients were non-smokers. They all suffered from aggravated cough, sputum, shortness of breath, wheezing or other acute exacerbation symptoms in a short term.

Another 30 patients, in the stage of COPD, aged on average (65.64 ± 2.55) years, were outpatients of the respiratory department and served as group B. Among them, 18 were smokers, and their mean smoking amount was 30 (10-40) packages/year whereas the other 12 patients were non-smokers. They were all relieved from cough, sputum, shortness of breath or other respiratory symptoms, and had been in stable status for at least 4 weeks.

All COPD patients were not given glucocorticoid or antibiotics in the latest one month, and met the diagnostic criteria for COPD.[7] Patients with severe bronchial asthma and bronchiectasis, serious heart, brain, kidney or vascular diseases, cancer, autoimmune diseases were excluded.

Thirty healthy non-smokers aged on average (65.60 ± 2.19) years who were from the department of medical examination served as a control group (group C). Since APN was proved to be affected by gender and weight, all the subjects in this study were male patients with normal weight (BMI range of 18.5 - 24.9 kg/m²). This study was approved by the Ethics Committee of Shanghai Fifth People’s Hospital Affiliated to Fudan University. All of the patients signed informed consent.

Sample preparation

The induced sputum[8] was collected from the subjects at 8 AM after the lung function test. All of the subjects must be confirmed that they rinsed their mouths with clear water and blew their noses before the collection. Ultrasonic aerosol was inhaled with 3% hypertonic saline for 15 minutes. Then the sputum was forcefully coughed to the petri dish. The sputum was mixed up with 0.1% dithiothreitol (Shanghai Huamei Biotechnology Company) by the volume ratio of 1:4. After the 37 °C water bath for 10 minutes and centrifugation, the sputum supernatant was placed in a -70 °C refrigerator, preserved for measurement of cytokines. For the precipitation layer, the total number of cells was counted. Those with more than 25 leucocytes and less than 10 squamous epithelial cells per low-power field were considered as qualified specimens. Smears were produced for cell count and classification, and the percentage of neutrophils was calculated. In addition, we took 5ml fasting venous blood per subject and separated the serum.

Cytokines tests

The kit used in this study was produced by R & D Company, USA. Double antibody sandwich ABC-ELISA was performed to detect the concentrations of APN, IL-8, IL-6 and TNF-α in the serum and induced sputum.

Pulmonary function test

We measured FEV₁ % and FEV₁/FVC% among all of the three groups.

Statistical analysis

For the data with normal distribution, the result was described as mean ±SD. Compared of the groups, one way ANOVA was performed with the homogeneity of variance; while a non-parametric test (Kruskal-Wallis H) was performed with heterogeneity of variance, comparison was made between the two groups. The data with non-normal distribution were described as the median (interquartile range). A non-parametric test (Kruskal-Wallis H) and comparison between the groups were performed. Pearson’s product-moment correlation coefficient test or Spearman’s rank-order correlation coefficient test was used for correlation analysis. Data analysis was carried out with SPSS 15.0. The difference was considered to be statistically significant when P<0.05.

RESULTS

Comparison of general data

There were no significant differences in age and BMI between the three groups (P>0.05). The disease duration was also not significantly different between groups A and B (P>0.05); however, FEV₁ % and FEV₁/ FVC% between the three groups were significantly different (P<0.01). The differences of WBC count and the percentage of neutrophils were also significant among the three groups, and they were higher in group A than in group B or group C, and higher in group B than in group C (P<0.01)(Table 1).

Table 1.

Baseline and clinical characteristics of subjects in the three groups (mean ±SD)

Levels of APN, IL-8, IL-6 and TNF-α in the serum and induced sputum among the three groups

In serum, APN, IL-8, IL-6 and TNF-α among different groups were statistically significant (χ² values were 54.83, 56.34, 57.43, and 69.08, respectively, P<0.01 for all); comparison between two groups showed that their concentrations were significantly higher in group A than in group B or in group C (P<0.01 for all), and also significantly higher in group B than in group C (P<0.01 for all). Meanwhile, in the induced sputum, the differences of APN, IL-8, IL-6 and TNF-α among different groups were also statistically significant (χ² values were 57.38, 65.82, 42.43, and 61.94, respectively, P<0.01 for all); comparison between two groups also indicated that their concentrations were significantly higher in group A than in group B or in group C (P<0.01 for all), and significantly higher in group B than in group C as well (P<0.01 for all) (Table 2).

Table 2.

The concentrations of APN, IL-8, IL-6 and TNF-α in the serum and induced sputum among all subjects

Correlation analysis

In the serum, APN in group A was positively correlated with IL-8 and TNF-α (r values were 0.739, and 0.734, P<0.05); APN in group B was also positively correlated with IL-8, TNF-α (r values were 0.751, and 0.659, P<0.01); while in the induced sputum, APN in group A was positively correlated with IL-8, TNF-α and the percentage of neutrophils (r values were 0.852, 0.857, and 0.439, P<0.05); APN in group B was also positively correlated with IL-8, TNF-α (r values were 0.707, and 0.867, P<0.01). There were no significant correlations between APN and FEV₁ %, FEV₁/FVC% or IL-6.

DISCUSSION

The inflammation in the airway leads to the main pathological process of COPD. In this process, a large number of inflammatory cells accumulate in the airway, including neutrophils and macrophages. They release various inflammatory mediators, causing pulmonary damage.[9-12] COPD is also a kind of systemic inflammatory disease,[13,14] and is characterized with the abnormal activation of inflammatory cells and the abnormal increase of circulating cytokines, including CRP, IL-8, TNF-α, IL-6 and leptin. APN is a newly discovered cytokine,[15,16] and its concentration is elevated in some immune and inflammatory diseases such as diabetes and rheumatoid diseases. IL-8, TNF-α and APN were highly expressed in the bronchoalveolar lavage fluid (BALF) of COPD patients, among whom the level of APN was the highest. APN in the BALF of COPD patients was 3.45 times that in the healthy control subjects. Because of the unacceptance of fiber optic bronchoscopy in getting BALF, induced sputum was collected instead in this study. Another study reported that COPD patients had increased APN level in plasma, and the level was related to elevated TNF-α.[17,18] Our study not only explored the relationship between APN and TNF-α, but also analyzed the correlation of APN and IL-8 in serum, the changes of APN in induced sputum and its relationship with IL-8, TNF-α and IL-6.

Neutrophils are the main inflammatory cells in the airway of COPD patients, especially in AECOPD patients. By the action of various inflammatory factors, neutrophils rapidly move to the airway through the transepithelial transport, releasing inflammatory mediators when they are activated, and leading to repeated injury and repair of the airway, finally causing airway obstruction.[19,20] IL-8 is a potent neutrophil chemokine and activator, which can induce the migration of neutrophils to the airway and promote neutrophils’ degranulation.[21,22] TNF-α is able to increase the expression of C3bi receptor / adhesion glycoprotein on the surface of neutrophils, so that the adhesion between endothelium and neutrophils can be strengthened, and then neutrophils migrate to the airway under function of the chemokines.[23] Our study revealed that for the COPD patients, APN, IL-8, TNF-α and neutrophils were all increased in the induced sputum. Besides, APN was positively correlated with IL-8 TNF-α, and the percentage of neutrophils in the AECOPD patients. It suggests that APN may be able to regulate the function of neutrophils through IL-8 and TNF-α, and indirectly involved in airway inflammation of COPD.

The main cytokines involved in the inflammation of COPD are IL-8, TNF-α and IL-6. Among them, IL-8 is the most important one, which can activate neutrophils and cause degranulation. [24,25] Various inflammatory mediators are released in the degranulation, promoting inflammatory reaction. Epithelial cells in the airway will stimulate the expression of IL-8 under APN,[26,27] suggesting a potential pro-inflammatory role of APN. [28] TNF-α is an important inflammatory mediator produced by several kinds of cells, playing a role in activating the neutrophils and stimulating the release of IL-8.[29,30] Its over-expression can lead to the chronicity of inflammatory response and the aggravation of lung injury. APN is able to inhibit macrophages producing TNF-α and reduce its synthesis and biological activity, suggesting that APN has some anti-inflammatory effects. [31] IL-6 is an important inducible factor of CRP and other protein produced in the acute phase of inflammation, and involved in the systemic inflammation of COPD. APN is found to be able to inhibit the synthesis of IL-6 and plays an anti-inflammatory role. However, our study found that there was no significant correlation between APN and IL-6, which was consistent with the results of Tomooda. Thus their relationship needs to be further studied. In this study, the elevated concentrations of APN, IL-8, TNF-α and IL-6 in the serum and induced sputum of the COPD patients and the positive correlation between APN, IL-8, and TNF-α both indicated that APN and the inflammatory mediators were inter-regulated, and that APN could play both pro-inflammatory and anti-inflammatory roles, thus limiting the inflammatory response within a certain range. It suggests that APN could be a new marker of COPD inflammation.

In the pathogenesis of COPD, IL-8, TNF-α and IL-6 are mainly secreted in the airway by the inflammatory cells.[32] However, APN is mainly secreted by fat cells and released into blood, and its secretion by airway epithelial cells plays a less important role.[2] The results of this study showed that the ratios of APN level in serum to induced sputum were 2.85 in AECOPD patients and 3.03 in stable COPD patients, while for IL-8, TNF-α and IL-6, the ratios were all less than 1, which indicated that the concentration of APN in blood could better reflect systemic inflammation of COPD, while the concentrations of IL-8, TNF-α and IL-6 in induced sputum could better reflect airway inflammation.

APN is a new marker of inflammation among COPD patients, with elevated level both in serum and induced sputum. The ratio of APN in serum to induced sputum is greater than 1. It is also positively correlated with the percentage of neutrophils in the airway, IL-8, and TNF-α. Limited by sample size, this study includes only the COPD patients with normal weight. In order to test the consistence of the results among the patients with low or high weight, further study needs to be conducted.