The impact of smoking on inflammatory biomarkers in patients with chronic obstructive pulmonary disease.

Yasser A. Korani ¹, Alaa T. Hassan ¹, Effat A. E. Tony ², Madleen Adel A. Abdou³

¹Departments of chest, ² Internal medicine and ³Clinical Pathology,

Faculty of Medicine, Assiut University, Assiut, Egypt.

ABSTRACT

Background: Chronic obstructive pulmonary disease (COPD) is a chronic progressive inflammatory disease characterized by limitations airflow that is not fully reversible (42). The pathophysiology of COPD is not completely understood. Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD). Elevated CRP has been increasingly used as a surrogate marker of systemic inflammation in diverse conditions. TNF-α, a powerful pro-inflammatory cytokine primarily produced by activated macrophages, is thought to a critical role in the pathogenesis of COPD [13,14].The aim of the work: to evaluate the impact of smoking on inflamatory biomarkers and relations between these biomarkers and the decline of lung function in COPD patients.Methods: This case –control observational prospective study was conducted on fifty-eight clinically stable COPD patients (26 non-smokers and 32 current smokers; at different stages ranged from mild to very severe), their mean age 53.1 ± 14.25 and 53.9 ± 5.95 years respectively), recruited from Chest Department, Assiut University Hospitals. All patients met the Global Initiative for Obstructive Lung Disease (GOLD) [4]. All participants were subjected to thorough history taking, full clinical examination, anthropometric measurements with spirometry and chest X-ray. Peripheral hemogram, liver function tests, kidney function tests, high sensitivity C-reactive protein (hs CRP) and serum level of TNF-α were measured for both patients and controls.Results: The concentrations of circulating hs-CRP and TNF-α, were highly significantly elevated in patients with COPD in comparison to the control group (3.74±0.2 vs. 1.30±0.14 for hs-CRP; 33.88 ±5.97 vs. 8.79 ±0. 57 for TNF-α with p < 0.0001 for each) and the levels of measured TNF-α were significantly increased with the increased degree and severity of COPD and increased severity of smoking status. Regarding the smoking status of COPD patients, there was a highly significantly difference for the measured TNF-α (53. 74±9.52 versus 12.73±1.20 with p < 0.0001) with no significant difference for the measured hs-CRP (3.87±0.29 versus 3.58±0.27 with p ˃ 0.05). Interestingly, there were significant negative correlations between the levels of TNF-α and hs- CRP, and FEV1 in stages II, III, IV of COPD.Conclusions: The circulating levels of the inflammatory markers hs- CRP and TNF-alpha are significantly elevated in patients with stable COPD and these biomarkers could be used as predictor factors for severity of inflammation in COPD patients.. Longitudinal studies evaluating the effects of smoking cessation on bronchial and systemic inflammation are needed to allow better understanding of these relationships and their consequences.

Keywords: COPD, FEV1, inflammatory markers; hs- CRP and TNF-alpha.

Introduction

Chronic obstructive pulmonary disease (COPD) is a syndrome characterized and defined by a single physiological parameter: limitation of expiratory air-flow which, most often, is slowly progressive over the years According to the widely accepted definition from Global Initiative on Obstructive Lung Disease (GOLD) COPD is "a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases" [Agarwal R et al 2013]. COPD as a condition characterized by an abnormal inflammatory response beyond the lungs with evidence of low-grade systemic inflammation which causes systemic manifestations such as weight loss, skeletal muscle dysfunction, an increased risk of cardiovascular disease, osteoporosis and depression, among others [Garrod R etal 2007 and Samy N etal 2010]. Several studies have shown systemic inflammation in COPD patients with increased neutrophil, macrophage, and T- lymphocyte numbers and high concentrations of inflammatory mediators in peripheral blood (C-reactive protein (CRP) and TNF-α) [Pinto- Plata VM et al 2006 and Quint JK and Wedzicha JA 2007 ]. Airway inflammatory markers are higher in more severe disease and increase during COPD exacerbations. However, no exhaled biomarker has been widely used in clinical trials in COPD (Smith et al, 2005). Kostikas et al., 2008 stated that systemic inflammation is present in stable COPD and the intensity of the inflammatory process relates to the severity of the underlying disease. In the interest of improving the diagnosis of COPD, several types of biomarker have been measured that are related to disease pathophysiology and the inflammatory and destructive process in the lung. However, there is little information about biomarkers reproducibility and the relationship to disease development, severity, or progression (Franciosi et al., 2006). Several inflammatory markers such as C-reactive protein (CRP), fibrinogen and IL-6, are increased in patients with COPD in both stable disease and exacerbations, with CRP being the most studied biomarker (Dahl et al., 2007). One of the inflammatory markers which is increasingly evaluated in COPD patients is CRP. CRP is an acute phase protein synthesized predominantly by the hepatocytes in response to tissue damage or inflammation. It has been accepted that levels of CRP relate to the presence of airflow obstruction [Agarwal R et al 2013]. Elevated CRP has been known to be used as a surrogate marker of systemic inflammation in diverse conditions. TNF-α (tumor necrosis factor- α) is a glycoprotein, a strong inflammatory cytokine which has a key role in inflammation primarily produced by activated macrophages. TNF-α is thought to play a critical role in the pathogenesis of COPD by promoting and maintaining the expression and release of various proinflammatory mediators which lead to tissue damage and remodelling. [Marevic S. et al 2008]. Very little is known about the mechanism of increased TNF-α concentration in the plasma of COPD patients and its relationship with disease severity and active smoking has not been established [Franciosi LG et al 2006 and Mukhopadhyay S et al 2006]. Tobacco smoking is the main risk factor of chronic obstructive pulmonary disease (COPD) but not all smokers develop the disease. An

abnormal pulmonary and systemic inflammatory response to smoking is thought to play a major pathogenic role in COPD, but this has never been tested directly. Yet, only a proportion of smokers, so called ‘‘susceptible smokers’’, develop the disease [Vestbo J, et al. 2013 and Rosa Faner et al 2014]. The genetic and epigenetic background of each smoker is likely to regulate the type and intensity of his/her inflammatory response to smoking [DeMeo DL et al 2005]. In ‘‘susceptible smokers’’, this response is thought to be ‘‘enhanced’’, both in the lungs and in the systemic circulation, and is believed to drive disease progression [Agusti A, et al 2012]. However, despite the wide acceptance of this notion , no previous study has actually studied the ‘‘response’’ to smoking (i.e., the specific inflammatory changes that occur before and after smoking) in susceptible smokers (i.e., patients with COPD) and resistant smokers (i.e., smokers with normal spirometry) [Rosa Faner et al 2014]. Long-term smoking causes airway inflammation characterized by neutrophil, macrophage, and activated T lymphocyte infiltration and by increased cytokine concentrations such as tumour necrosis factor-alpha (TNF-α), interleukins (IL)-6 and IL-8 [Battaglia S et al 2007 and Gamble E etal 2007]. Although nearly all smokers show some evidence of lung and systemic cellular and/or humoral inflammation, only a few will suffer an amplified response and develop COPD. The aim of this study was to identify whether the inflammatory process in stable COPD patients is powerful enough to produce significant changes in the selected inflammatory markers (TNF- α and hs CRP). The role and levels of TNF- α and hs CRP in the peripheral circulation of COPD patients and the relationship between these biomarkers with prognostic factors in COPD will be investigated and as we hypothesized that smoking exposure will induce a different inflammatory signature, the impact of smoking on inflammatory biomarkers in patients with chronic obstructive pulmonary disease will be evaluated. With the development of many new drugs that target inflammation in COPD, there is a pressing need to identify reliable biomarkers that may indicate whether an anti inflammatory therapy is likely to have clinical benefit. A major problem is the lack of any gold standard anti inflammatory therapy that is effective in COPD, as a yardstick to compare potential therapies.

Methods: This case –control observational prospective study was conducted on fifty-eight clinically stable COPD patients (26 non-smokers and 32 current smokers; at different stages ranged from mild to very severe one), their ages mean age 53.1 ± 14.25 and 53.9 ±5.95 years respectively), recruited from Chest Department, Assiut University Hospitals. All patients met the Global Initiative for Obstructive Lung Disease (GOLD) [4] recommended spirometric criteria for COPD (ratio of post-bronchodilator forced expiratory volume in one second [FEV1] to forced vital capacity [FVC] less than 70%) [17]. The COPD patients were subdivided according to their treatment into: thirteen patients use only short acting bronchodilator as needed, the remaining forty five patients were using long-term bronchodilators from those 45 patients, twenty seven (13 COPD current smokers and 14 non-smokers) patients were regularly using inhaled corticosteroid (800 mcg of budesonide) , sixteen patients of them had been on stable oxygen flow

therapy for the last six months. No patients were medicated with theophylline or leukotriene modifiers.

Patients on oral steroid use or exacerbations in the last three months before enrolment in the study, or diagnosed with other respiratory disease like asthma, bronchiectasis, bronchiolitis or tuberculosis, or presence of other chronic diseases like diabetes, renal failure, cancer were excluded. In addition, age, sex and BMI matched apparently healthy subjects with no evidence of COPD or using regular medications for other diseases from medical stuff and families who underwent health examination at Assuit university hospitals were enrolled in the study as control group. .the controls were subdivided into fifteen current smoker controls (at least 10 pack-years) and fifteen never-smoker controls. All participants were subjected to thorough history taking, full clinical examination, anthropometric measurements with chest X-ray and spirometry. Forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC) were obtained from the flow- volume curve using a spirometer [Conventional spirometry was a 10-Lclosed circuit Spirographs (Zan 300, Sensor Medics MGA USB, Germany)] to study patients and control groups. Static lung volumes were measured by closed-circuit helium dilution method. The reference values used were those of the American Thoracic Society standards) before and 20 minutes after β-agonist (fenoterol 400 mcg) inhalation. The highest value of at least three measurements was selected and expressed as a percentage of reference values [18]. Venous blood samples were collected from participants under standardized conditions. samples were centrifuged (3,000 g for 10 min.) and serum samples were divided and stored in aliquots at – 20 ºC until analysis. Peripheral hemogram, liver function tests, kidney function tests, High sensitivity C-reactive protein (hs-CRP) measurement was done by Accu-Bind ELISA Microwells (Monobind Inc., USA code 3125-300A lot EIA- 31KIC2). Assay of serum levels of TNF-α was performed using AviBion Human TNF-α ELISA Kit (Orgenium, Finland, Rev 02.10). Smoking history was calculated in pack-years as the product of tobacco use (i current smoker n years) and the average number of cigarettes smoked per day/20 (years x cig. per day/20). The study was approved by the Institutional Ethics Committee of Faculty of Medicine, Assiut University and written informed consent was obtained from each participant.

Statistical analysis: Data were analyzed with Statistical package for social sciences (SPSS) version 17 Chicago.USA. Data were expressed as means ±SD or means ±SE and frequencies according if they are quantitative or qualitative respectively. Non- parametric tests were used in the current study.

1. Mann-Whitney Test (equivalent to independent student t test in parametric tests) was used for comparison of quantitative variables among two independent groups

2. Wilcoxon Signed Ranks Test (equivalent to paired t test in parametric tests) was used for comparison of quantitative variables among two dependent groups.

3. Chi-Square Test was used for comparison of distribution of qualitative variables among different groups.

4. Comparisons between groups were achieved by Kruskal-Wallis Test (equivalent to one way ANOVA test).

5. Correlation between continuous variables was performed using Spearman's correlation coefficient.

P value <0.05 is considered statistically significant. Multiple regression analysis was used to dete-rmine independent factors affecting dependent variables.

Results: Demographics, baseline lung function, and body mass index of COPD and controls according to their smoking status smokers were presented in Table 1 where it showed COPD patients tended to have a nearly same age to controls regardless their smoking status with similar smoking history (pack-years) in current smoker COPD patients and controls. As expected, COPD patients had significant severe airflow limitation and decreased SpO2 with highly significant decline in FEV1 compared to controls.

Table (1): Demographic data, Pulmonary functions and blood gases among COPDpatients compared to controls according to their smoking status.

Variables

Control

Non-smoker

(N=15)

Control - smoker

(N=15)

COPD

Non-smoker

(N=26)

COPD

Smoker

(N=32)

P-value Significa

nce

FVC% pred 94.6 ± 8.47 91.8 ± 7.5 73.2 ±3.6 71.5 ± 6.3 0.009*

RV% pred 92.3 ± 4.2 88.3 ± 4.2 129.33 ± 8.4 133.5 ± 12.4 0.004*

TLC% pred 91.7 ± 6.8 85.2 ± 3.4 127.8 ± 5.1 131.1 ± 10.6 0.003*

FEV1% pred 83.2 ± 3.1 81.5 ± 2.0 54.23 ± 23.4 51.6 ± 22.2 0.007*

FEV/FVC % 79.56 ± 9.1 76.4 ± 6.42 63.9 ± 5.46 59.2 ± 10.7 0.011*

Pa O2 mmHg 88.3 ± 6.3 82.6 ± 5.4 67.3 ± 4.9 61.5 ± 8.7 0.041*

Pa CO2 mmHg 39.1 ± 3.6 40.5 ± 3.8 58.5 ± 8.4 61.3 ± 11.9 0.037*

Age (years) 49.37±14.37 52.56±8 53.1±14.25 53.9±5.95 0.746

BMI(kg/m2 24.1±5.0 24.2±4.51 22.8±4.9 21.4±5.7 0.149

Kruskal-Wallis Test * Statistical significant difference

PatientsControl

53.74

12.7310.7

6.88

70

60

50

40

30

20

10

0Smoker Non-smoker

Tables 2 (a , b) , Tables 3 (a, b )and Figures (1 and 2) : showed highly significant elevated levels of circulating TNF-α and hs-CRP in COPD patients in comparison to the control group regardless their smoking status with significant highest levels in current smokers patients and controls .

Table (2a): TNF-alpha (pg/ml)

Parameters Patients(n= 58) Controls(n=30)P-value

Mean ± SE 35.88 ± 5.97 8.79 ± 0.570.000*

Range 3.7 – 265.5 3.7 – 14

Mann-Whitney Test * Statistical significant difference (P < 0.05)

Table (2b): TNF-alpha (pg/ml)

Parameters Smoker Non-smokerP-value

Mean ± SE Mean ± SE

Patients 53.74 ± 9.52 12.73 ± 1.20 0.000*

Controls 10.70 ± 0.60 6.88 ± 0.69 0.001*

Mann-Whitney Test * Statistical significant difference (P < 0.05)

Figure (1) levels of TNF-alpha in COPD patients and controls (pg/ml) according to their smoking status.

Mea

n ±

PatientsControl

3.87

3.58

1.96

0.64

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

Smoker Non-smoker

Table (3a): hs-CRP (ug/ml)

Parameters Patients

(n= 58)

Controls

(n= 30)P-value

Mean ± SE 3.74 ± 0.20 1.30 ± 0.140.000*

Range 1.2 – 8.0 0.2 – 2.5

Mann-Whitney Test * Statistical significant difference (P < 0.05)

Table (3b): hs-CRP (ug/ml)

Parameters Smokers Non-smokersP-value

Mean ± SE Mean ± SE

Patients 3.87 ± 0.29 3.58 ± 0.27 0.764

Controls 1.96 ± 0.14 0.64 ± 0.06 0.000*

Mann-Whitney Test * Statistical significant difference (P < 0.05)

Figure (2): Levels of hs-CRP in COPD patients and controls (ug/ml) according to their smoking status.

Mea

n ±

Table (4): The mean levels of circulating TNF- alpha according to increased the degree of COPD, the severity of dyspnea and the steroid inhalation therapy in COPD patients.

Parameters Mean ± SE P-value

Degree of COPD:

0.000*

Mild 10.16 ± 0.92

Moderate 21.86 ± 0.92

Severe 46.63 ± 4.39

Very severe 149.90 ± 33.04

Dyspnea:

0.000*Grade II 17.06 ± 1.49

Grade III 38.76 ± 5.09

Grade IV 137.26 ± 30.66

Steroid inhalation therapy:

0.077Yes 29.61 ± 12.33

No 46.16 ± 9.29

Kruskal-Wallis Test * Statistical significant difference (P < 0.05)

Table 4: showed the significant increased levels of the TNF-α with the increased severity of COPD and increased grade of dyspnea. Moreover, non significant differences for the measured TNF-α were found between the patients according to the use of the inhaled corticosteroids therapy.

Table (5): Regression models with robust standard errors for TNF-α and CRP in COPD Patients.

ParametersTNF-alpha(pg/ml) Hs CRP(ug/ml)

r-value P-value r-value P-value

Smoking index 0.850 0.000* 0.614 0.000*

Degree of COPD 0.936 0.000* 0.198 0.122

Grades of dyspnea 0.708 0.000* 0.179 0.163

Spearman correlation * Statistical significant difference (P < 0.05)

The regression results showed a high significant positive association among the measured TNF-α with smoking index, increased degree of dyspnea and degree of COPD, while a non significant positive association among the measured hs-CRP with increased degree of dyspnea and degree of COPD with a high significant positive association among the measured hs-CRP with smoking index were found in Table 5

Table (6) : The correlation between the measured TNF-alpha and hs-CRP versus smoking index in COPD patients and control groups.

VariablesSmoking Index

r-value P-value

Patient smokersTNF-alpha 0.788 0.000*

Hs CRP 0.614 0.000*

Control smokersTNF-alpha 0.889 0.000*

Hs CRP 0.858 0.000*

Spearman correlation * Statistical significant difference (P < 0.05)

Table 6 showed high significant positive associations among the measured TNF-α and hs-CRP with smoking index in COPD patients and current smoker controls.

Table (7): The correlation between the measured TNF-alpha and hs-CRP versus FEV1 in COPD patients and controls regarding to their smoking status.

VariablesFEV1

r-value P-value

COPD smokersTNF-alpha -0.843 0.000*

hsCRP -0.501 0.002*

COPD non-smokersTNF-alpha -0.464 0.015*

hsCRP -0.067 0.741

Control smokersTNF-alpha -0.881 0.000*

hsCRP -0.848 0.000*

Control non-smokersTNF-alpha -0.879 0.000*

hsCRP -0.857 0.000*

Spearman correlation * Statistical significant difference (P < 0.05)

Table 7 showed high significant positive associations among the measured TNF-α levels with FEV1 in COPD patients and controls regardless to their smoking status. However, Regarding to smoking status in COPD patients, there was a high significant positive association among current smokers with no significant difference among the non smokers between FEV1with the measured hs-CRP. In controls regardless to their smoking status high significant positive associations among the measured hs-CRP with FEV1were detected.

Discussion:

Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease induced primarily by smoking tobacco. Other aetiological factors of COPD, like air pollution, poor nutrition, professional risk at work and genetic factors may be probably participated. The pathophysiology of COPD is not completely understood. (Samy N etal, 2010). According to Global Initiative on Obstructive Lung Disease (GOLD) definition , The airflow limitation in COPD is not fully reversible and is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases" (Agarwal R et al 2013). In the interest of improving the diagnosis of COPD, several types of biomarker have been measured that are related to disease pathophysiology and the inflammatory and destructive process in the lung. The aim of this study was to to identify whether the inflammatory process in stable COPD patients is powerful enough to produce significant changes in the selected inflammatory markers (TNF- α and hs CRP) and to evaluate the relationship between systemic inflammation and smoking status in COPD patients.

CRP is an acute phase protein synthesized predominantly by the hepatocytes in response to tissue damage or inflammation. It has been accepted that levels of CRP relate to the presence of airflow obstruction .Elevated CRP has been known to be used as a surrogate marker of systemic inflammation in diverse conditions (Agarwal R et al 2013). Interestingly , Our study in fact , showed that the serum level of hs CRP was highly significantly elevated in COPD patients compared with controls regardless to their smoking status .This result confirms that the higher levels of circulating hs CRP may be thus regarded as a valid biomarker of low- grade systemic inflammation in the COPD. Our results were in agreement with Nillawar AN et al 2013 who reported that COPD patients had higher levels of hs-CRP due to the fact of the earlier response of hs-CRP, as it is secreted as acute phase reactants by the liver in response to IL-6. Moreover, Gan WQ et al 2004, Pinto- Plata VM et al 2006 and Quint JK and Wedzicha JA 2007 stated that systemic inflammation in COPD patients is associated with increased neutrophil , macrophage, and T-lymphocyte numbers and high concentrations of inflammatory mediators in peripheral blood (C-reactive protein (CRP) and TNF-α) . Their study showed a significantly higher levels of circulating hs CRP in COPD patients than in smoking and non smoking controls. However, the level of hs CRP in the smoking COPD population remained non significantly higher than in the non- smoking patients .We suggest that , although cigarette smoking has a role in promoting inflammatory process in COPD patients , it is not the leading cause of increased inflammatory markers. it should be noticed that only some cases develop inflammatory reaction following cigarette smoking, and this can be due to genetic differences. In contrast to the above findings, Agarwal R et al 2013 , showed the level of hs CRP in the smoking COPD population remained significantly higher than in the non- smoking patients.

TNF-α, a powerful pro-inflammatory cytokine primarily produced by activated macrophages, is thought to a critical role in the pathogenesis of COPD by promoting and maintaining the expression and release of various proinflammatory mediators which lead to tissue damage and remodelling (Higashimoto Y et al 2008 ). Very little is known about the mechanism of increased TNF-α concentration in the plasma of COPD patients and its relationship with disease severity and active smoking has not been established (Franciosi LG et al 2006 and Mukhopadhyay S et al 2006 ). However, Marevic S. et al 2008 reported that ttobacco smoking induces the release of TNF-α and has an important role in the pathophysiology of COPD. TNF-α influences the gene expression of interleukin-8 and endothelin-1 (ET-1) in human endothelial cells, and participates in bronchoconstriction through secondary release of ET-1.

Our study showed that the level of serum inflammatory marker ; TNF-α was highly significantly elevated in COPD patients regardless to their smoking status indicating a persistent systemic inflammation in subjects with COPD with significant highest levels of TNF-α in current COPD smokers .These results suggest that smoking may be associated with higher TNF-α mediated systemic inflammation in COPD patients. These findings were in agreement with Higashimoto Yet al.2008 who reported the increased levels of serum TNF-α, IL-6 and tissue inhibitors of metalloproteinase-1 in asthma and COPD patients with highest levels in current smoker COPD patients and controls. Moreover, our results were in consistent with Pinto- Plata VM et al 2006 , Quint JK and Wedzicha JA 2007, Marevic S. et al 2008 and Samy N et al 2010 and who reported significant increase in serum inflammatory markers TNF-α, hs CRP and interlukin-6 in different stages of COPD patients and this increment was significantly higher than those of the controls. We may thus speculate the association of smoking with higher systemic TNF-α levels observed in this study may partially explain some of benefits associated with smoking cessation. In contrast to our findings, Vernooy JH et al2002 reported increase in the concentration of soluble TNF-α receptors , while the concentration of TNF-α was within the reference range for healthy individuals . possible explanation for unchanged concentration of TNF-α is its local and short term effect, its degradation and its half life of approximately 6-7 minutes, as well as its binding to receptors and renal clearance.

FEV1 is easily measured and is a spirometric predictor of mortality in patients with COPD. Factors that affect the decline of FEV1 are , therefore , of prognostic importance in COPD. (Samy N etal , 2010) . Our study revealed the increased TNF-α levels in COPD patients with increased airway obstruction severity , increased and degree of dyspnoea with a significant negative correlation between TNF- α with FEV1 .These results clearly confirm the fact of that the intensity of the inflammatory process in COPD could be related to the severity of the underlying disease. Our findings were in agreement with Wagner PD , 2008 who stated the higher serum TNF-α levels in COPD patients was associated with FEV1 < 30% in comparison with

mild-moderate COPD patients. Furthermore , Kostikas et al., 2008 and Samy N et al , 2010 who reported TNF-α levels increased according to the stage of the COPD. Interestingly , non significant higher hs-CRP concentrations have been associated with increased severity of airway obstruction and increased degree of dyspnoea were observed in our study. These findings were in agreement with Godoy I et al 2003 and Mukhopadhyay S et al 2006 who reported that higher hs-CRP concentration has been associated with airway obstruction severity, increased resting energy expenditure, and impaired exercise capacity and quality of life in COPD patients Agarwal R et al 2013 concluded that the circulating levels of the inflammatory marker hs-CRP are significantly elevated in patients with COPD, supporting the view that COPD is in part an inflammatory disorder. Hs-CRP levels in stable COPD patients are best correlated with FEV1 and 6-minute walk distance (6MWD) . Clearly, in the our study hs CRP levels were inversely correlated with FEV1 in stable COPD patients. The strongest negative association between FEV1 and CRP and higher CRP levels over time were associated with a faster FEV1 decline. these results were in consistent with Man SF et al 2006 and Agarwal R et al 2013 who stated CRP levels associated with accelerated decline in FEV1and mortality in patients with mild to moderate COPD, indicating that CRP measurements might enable identification of patients at high risk of disease progression and mortality .

Tobacco smoking is the main risk factor of chronic obstructive pulmonary disease (COPD) but not all smokers develop the disease. An abnormal pulmonary and systemic inflammatory response to smoking is thought to play a major pathogenic role in COPD, but this has never been tested directly. Long-term smoking causes airway inflammation characterized by neutrophil, macrophage, and activated T lymphocyte infiltration and by increased cytokine concentrations such as tumour necrosis factor- alpha (TNF-α), interleukins (IL)-6 and IL-8 (Battaglia S etal 2007 and Gamble E et al 2007). Although nearly all smokers show some evidence of lung and systemic cellular and/or humeral inflammation, only a few will suffer an amplified response and develop COPD .

Interestingly , highly significant higher concentrations of TNF-α and hs-CRP in COPD patients have been associated with increased severity of smoking status were observed in our study. The correlations of TNF-α and hs CRP concentration with smoking habit (pack/years) supports the assumption that tobacco smoke has an influence on systemic inflammation. Hs-CRP and TNF-α indicate that COPD is a systemic inflammatory disease, and they could serve as predictors for exacerbations. However, it is unclear whether established inflammation in smokers returns to normal after smoking cessation . Smoking cessation is the only management measure associated with reduced FEV1decline in COPD patients; however ,to date , no reduction in airway inflammation has been demonstrated when ex-smokers are compared to current smokers by Gamble E et al 2007 who did not find any differences in cell counts or inflammatory markers (CD8+, CD4+, CD 68+, or TNF-α)

in bronchial biopsies of COPD current smokers and COPD ex-smokers. Nevertheless, Barnes PJ et al 2006 reported that neutrophil and lymphocyte numbers, and IL-8 concentration in the sputum of COPD patients were increased one year after smoking cessation.

Unfortunately , in spite of our COPD patients were classified according to GOLD classes and use of LTOT or inhaled corticosteroids , we could not revealed the influence of inhaled corticosteroids on systemic inflammation in COPD patients owing to smaller sized sample used , poor power to detect potential differences and longer time observation was needed to confirm these results .

Conclusions:

The circulating levels of the inflammatory markers hs- CRP and TNF-alpha are significantly elevated in patients with COPD, supporting the view that COPD is in part an inflammatory disorder. These circulating biomarkers in stable COPD patients are best correlated with FEV1 suggesting that these circulating biomarkers are good candidates as predictors for rapid decline of FEV1 in COPD, although a larger size study with longer term observation is needed to confirm these findings. It is likely that in the future biomarkers will become increasingly required to aid in determining those patients who will benefit from a given drug therapy to improve risk and/or cost benefit, especially since it is becoming more widely considered in COPD. Smoking is associated with higher levels of TNF-alpha and hs- CRP mediated systemic inflammation in patients with or without COPD, therefore active smoking maybe associated with even higher degree of the systemic inflammation. Longitudinal studies evaluating the effects of smoking cessation on bronchial and systemic inflammation are needed to allow better understanding of these relationships and their consequences.

Abbreviations:

BMI: Body Mass Index; COPD: Chronic Obstructive Pulmonary Disease; Hs-CRP: Highly sensitive C - reactive protein; FEV1: Forced Expiratory Volume in one second; FEV1/FVC: Forced Expiratory Volume in one second/Forced Vital Capacity; BMI: Body Mass Index; FVC: Forced Vital Capacity; GOLD: Global Initiative for Chronic Obstructive Lung Disease; IL: Interleukin; SpO2: Peripheral Oxygen Saturation; TNF-α: Tumor Necrosis Factor alpha.

Competing interests

The authors declare that they have no competing interests.

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