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Increased leptin/leptin receptor pathway affects systemic and airway inflammation in COPD former smokers

Background Leptin, a hormone produced mainly by adipose tissue, regulates food intake and energy expenditure. It is involved in inflammatory diseases such as chronic obstructive pulmonary disease (COPD) and its deficiency is associated with increased susceptibility to the infection. The leptin receptor is expressed in the lung and in the neutrophils. Methods We measured the levels of leptin, tumor necrosis factor alpha (TNF-α) and soluble form of intercellular adhesion molecule-1 (sICAM-1) in sputum and plasma from 27 smoker and former smoker patients with stable COPD using ELISA methods. Further we analyzed leptin and its receptor expression in sputum cells from 16 COPD patients using immunocytochemistry. Results In plasma of COPD patients, leptin was inversely correlated with TNF-α and positively correlated with the patient weight, whereas the levels of sICAM-1 were positively correlated with TNF-α. In sputum of COPD patients leptin levels were correlated with forced expiratory volume in 1 second/forced vitality capacity. Additionally, increased levels of sputum leptin and TNF-α were observed in COPD former smokers rather than smokers. Further the expression of leptin receptor in sputum neutrophils was significantly higher in COPD former smokers than in smokers, and the expression of leptin and its receptor was positively correlated in neutrophils of COPD former smokers. Conclusion Our findings suggest a role of leptin in the local and systemic inflammation of COPD and, taking into account the involvement of neutrophils in this inflammatory disease, describe a novel aspect of the leptin/leptin receptor pathway in the regulation of host defense after smoking cessation.


Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and is associated with an abnormal inflammatory response to inhaled noxious particles, including cigarette smoke. Current or previous exposure to cigarette smoke is usually reported by COPD patients, and smoking cessation is the single most effective intervention to reduce the risk of developing COPD and stopping its progression. 1 Clinical and functional heterogeneity is a hallmark of COPD, and the forced expiratory volume in 1 second (FEV 1 ) value and the FEV 1 /forced vitality capacity (FVC) ratio are considered the most important altered parameters of lung function in COPD patients. 2 In addition, patients with COPD exhibit a chronic inflammatory response of the airways, with a persistent inflammation within the proximal airways mainly characterized by an endoluminal influx of neutrophils and overproduction of mucus and proinflammatory cytokines. 3 The airways of these patients are often colonized by mucoid bacteria attached to epithelium by a biofilm and purulent sputum is strongly associated with bacterial growth in COPD exacerbations. 4 Leptin, initially discovered as a regulator of food intake and energy expenditure, is emerging as a pleiotropic cytokine involved in the recruitment, activation and survival of inflammatory cells. 5 In particular, via short and long isoforms of its receptor, it is able to regulate a variety of cell types including neutrophils, eosinophils, T-lymphocytes, and monocytes, [6][7][8] and to activate neutrophils by the release of reactive oxygen species (ROS). 9,10 Studies performed in age-and gendermatched patients with stable COPD have demonstrated that plasma soluble form of intercellular adhesion molecule-1 (sICAM-1) can be considered a marker of inflammation 11 and that its levels are positively correlated with body mass index (BMI) and tumor necrosis factor alpha(TNF-α). 12 Moreover, temporary disturbances in the energy balance are present during an acute exacerbation of COPD and are related to increased leptin concentrations and to the systemic inflammatory response. 13 Accordingly, leptin and TNF-α serum levels are significantly higher in the patients experiencing exacerbation than in stable COPD patients and controls. 14 However, a number of studies have addressed the presence of leptin and its receptor in the lung. 15,16 Leptin deficiency might be associated with increased susceptibility to infections 17,18 and an assessed role of leptin in bacterial pneumonia in mice 19 suggests a protective effect of this adipokine against infections. Leptin is detectable in induced sputum of COPD patients, and it is positively correlated with the inflammatory markers C-reactive protein and TNF-α in sputum, indicating that leptin is involved in the local inflammatory responses in COPD. 20 Furthermore, increased levels of leptin expression were observed in the submucosa of bronchial biopsies from COPD and it is inversely correlated with the apoptosis of inflammatory cells, 15 suggesting that leptin might regulate the inflammatory cell infiltration of the submucosa in COPD. Moreover, the effect of smoking cessation in the leptin/leptin receptor pathway is largely unexplored. Based on this, the aim of the present work is to explore whether plasma and induced sputum concentrations of leptin are related to inflammatory markers, such as TNF-α and sICAM-1 and neutrophilic airway inflammation in stable COPD patients current and former smokers.

Materials and methods Patients
Twenty-seven stable COPD patients, matched for age and BMI as a marker of nutritional status, were enrolled. BMI was expressed as kg/m 2 . To eliminate the effects of gender differences, all patients were male. Diagnosis of COPD was based on the combination of clinical history and functional data. 1 All patients were in a stable condition, as defined by the absence, for at least 4 weeks, of clinical signs or symptoms of acute exacerbation. All subjects were under treatment with long-acting beta-adrenergic agonist (salmeterol 50 µg twice daily).
Exclusion criteria were history of COPD with severe comorbidities (tumors, end-stage New York Heart Association III/IV heart failure classes, severe renal failure, liver diseases, dementia). All patients had a history of cigarette smoking, 20 having quit smoking for at least 2 years and 7 still smoking. Informed consent was obtained from the patients before enrolment into the study.

Blood samples
Blood was collected in an EDTA tubes vacutainer (Becton-Dickinson) in fasting patients in the morning before sputum induction. Blood samples were centrifuged at 1000 g for 15 minutes and plasma was stored at −70°C until analysis.

cytokines assays
Leptin, sICAM-1, and TNF-α were measured in plasma and induced sputum supernatant by commercially available specific enzyme immunoassay kits ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer instructions. Lower detection limits were 7.8 pg/mL, 0.35 ng/mL, and 0.12 pg/mL respectively. Plasma leptin and sICAM-1 are expressed as ng/mL and TNF-α as pg/mL. In induced sputum, leptin and TNF-α are expressed as pg/g of sputum and sICAM-1 as ng/g of sputum.
sputum induction and processing Sputum was induced by inhalation of 3% sterile hypertonic saline by a De Vilbiss Ultraneb 99 ultrasonic nebulizer (Healthcare Inc, Somerset, PA) through a mouthpiece without using valves or nose clips, as previously described. 21,22 Sputum was processed according to the methods of the plugs. 23 Briefly, the selected plugs were diluted with 4 volumes of phosphate-buffered saline (PBS 1X; Gibco). The resulting suspension was vortexed for 30 seconds and then centrifuged at 1000 g for 20 minutes. The supernatant was collected and stored at -70°C until analysis. The pellet was resuspended in 4 volumes of fresh 0.1% dithiothreitol (DTT) (Sigma-Aldrich, St Louis, MO) in PBS and processed as previously described. 24 Cytospins were prepared on aptex (3-aminopropiltryetoxisilane) -coated slides by adding 100 µL of cell suspension (about 5 × 10 5 cells/mL) into a Shandon II cytocentrifuge at 180 g for 5 minutes. Differential cell counts were performed by May-Gruenwald-Giemsa staining. In all cases 400 nonsquamous cells were counted by 2 blind observers and results were expressed as percentage of total nonsquamous cells. Air-dried slides for immunocytochemistry were fixed in periodate-lysine-paraformal for 30 minutes and in 15% sucrose in Dulbecco's phosphate-buffered saline for 30 minutes 25,26 and stored at −70°C until immunocytochemical staining.

Immunocytochemical staining
Slides were incubated with a rabbit polyclonal antibody Ob anti-leptin (A-20, 1:20 dilution in antibody diluent, 1 hour at room temperature), and with a goat-polyclonal antibody Ob-R antileptin receptor against the common part of the short and long isoforms (M-18, 1:15 dilution in antibody diluent, overnight, 4°C). 27 Both antibodies were from Santa Cruz Biotechnology, CA. The reaction was revealed by LSAB KIT phosphatase method according to the manufacturer's instructions. Both antibody diluent reagent and LSAB KIT were from DAKO Glostrup Denmark. Control slides for leptin were prepared as described: the immunocomplex was obtained by immunoprecipitation (A/G plus-agarose; Santa Cruz Biotechnology), and by incubation with the rabbit-polyclonal antibody Ob (2 µg/mL) and human recombinant leptin (20 µg/mL) (Sigma-Aldrich) overnight at 4°C. Control slides for leptin receptor were prepared by using an irrelevant mouse antibody of the same isotype and at the same concentration of the specific primary mAb (Dako). The cell nuclei were stained for 1 minute with hematoxylin (Dako). Slides were evaluated using a Leica (Wetzlar, Germany) microscope at 400 × magnification. Cell identification was based on cell morphology under light microscopy (400 × final magnification), carefully referring to the cell type distribution in corresponding Diff-Quik-stained slides; red staining identified positive cells. Two independent observers counted a minimum of 600 cells, and the mean value of the 2 observations was used (r = 0.91). The results were expressed as positively staining cells as a percentage of the total cell number.

statistical analysis
Medians and 25% to 75% percentiles of measured parameters were calculated to perform descriptive analysis of population. A nonparametric Mann-Whitney test was applied to test the differences between the two groups of subjects. Correlations were determined using a Spearman rank correlation. Values of P , 0.05 were considered statistically significant.

Demographic characteristics of the patients
Demographic characteristics of the patients are reported in Table 1. Smoker and former smoker COPD patients were classified as stable GOLD II. No statistical differences were detected between smokers and former smokers for pulmonary functional parameters and BMI and body weight.

cytokines concentrations and correlations in plasma and sputum
A positive correlation was found between plasma leptin levels and BMI (Rho = 0.63; P = 0.001) ( Figure 1A) and between plasma leptin levels and patient weight (Rho = 0.69; P = 0.0006) ( Figure 1B), whereas plasma leptin levels were inversely correlated with plasma TNF-α levels (Rho = −0.44, P = 0.02) ( Figure 1C). Plasma TNF-α levels were positively correlated with plasma sICAM-1 levels (Rho = 0.47, P = 0.02) ( Figure 1D). No correlation was found between plasma leptin and sICAM-1 levels (data not shown). A significant positive correlation was present between sputum leptin levels and FEV 1 /FVC (Rho = 0.39, P = 0.04) (Figure 2A) in all COPD patients. Significantly increased concentrations of leptin were present in sputum of former smokers compared with current smokers (Table 2). Additionally, we observed a nonsignificant trend toward increased concentrations of TNF-α in sputum of former smokers in comparison with current smokers (Table 2). Furthermore, no statistically significant differences were found between the two groups of COPD patients for leptin, TNF-α, and sICAM-1 in plasma (Table 2).
Total and differential cell counts in sputum Total and differential cell counts of induced sputum cells were performed in all COPD patients, both smokers and former smokers. No statistical significant differences were found between smokers and former smokers in either of these cell counts (Table 3).

Leptin and leptin receptor expression by induced sputum cells
The expression of leptin and its receptor was evaluated in sputum cells from both smoker and former smoker COPD patients. The expression of leptin receptor was significantly increased in neutrophils of former smokers compared with current smokers (Figure 2A; Figure 3), but no difference was observed for leptin. No differences were detected for both leptin and leptin receptor expression between the two COPD groups in macrophages, lymphocytes, and eosinophils (Table 3). Additionally in former smokers, leptin and leptin receptor were positively correlated (Rho = 0.74, P = 0.02) (Figure 4).

Discussion
COPD is a pulmonary disease recognized to have important systemic and airway inflammatory effects. 28 The present study underlines that leptin is involved in local and systemic inflammation of smoker and former smoker COPD patients, matched for age, gender, pulmonary function, and BMI and body weight. Additionally, taking into account the crucial role of neutrophils in this inflammatory disease, the study suggests that the leptin/leptin receptor pathway may contribute to increase the host defense by neutrophils in COPD patients after smoking cessation, driving the neutrophil function. Therefore, because of this study's small sample size we recommend an explorative study. Leptin is proinflammatory adipokine involved in different inflammatory diseases such as rheumatoid arthritis. 29 It is produced mainly by fat tissue and is always correlated with BMI and TNF-α and is also higher in obese individuals. Accordingly, this study and previous studies show that stable COPD patients have a positive correlation between plasma lep-tin levels and patient BMI 30,31 as well as with plasma sICAM-1 and plasma TNF-α. 12 In addition, we show that plasma leptin levels are positively correlated with body weight, but inversely correlated with plasma TNF-α levels. We did not identify a positive correlation between plasma leptin and TNF-α levels, or between leptin and sICAM-1 levels in stable COPD, in accord with previous studies showing that serum leptin and serum TNF-α levels are significantly higher in patients experiencing exacerbation than in stable patients and controls. 20   Additionally, a previous study showed that leptin levels are lower and TNF-α levels are higher in stable patients than in patients experiencing exacerbation, 14 strongly supporting the negative correlation between plasma leptin and TNF-α levels identified in our patients. Furthermore, sICAM-1 and leptin concentrations are significantly higher in patients with higher levels of BMI 31-33 than in COPD patients enrolled in our study. Clinical and functional FEV 1 /FVC ratio is considered the most important altered parameter of lung function in COPD patients. A previous study has already shown that respiratory pathogens isolated from the sputum are associ-ated with severe airflow obstruction (FEV 1 /FVC , 60%). 34 We demonstrate here that sputum leptin levels were positively correlated with the FEV 1 /FVC values in COPD patients studied, hence it is possible to consider a possible relationship between leptin and the resolution of airway infection in COPD patients. However, since we selected patients in a stable condition, we observed only little correlation. COPD patients exhibit a chronic inflammatory response of the airways with a persistent inflammation characterized by an increased influx of neutrophils at the site of inflammation. 3 The role of neutrophils in COPD is very important because they play a pivotal role in the defense against infections and are critically involved in the innate defense mechanisms. 34 The infections are critical events that complicate COPD and lead to the progressive decrease of lung function. 35 Also, smoking attenuates the oxidative burst of inflammatory cells and 3 weeks of abstinence normalizes the oxidative burst. 36 These results suggest that smoking cessation improves the function and activity of inflammatory cells. We have demonstrated that sputum leptin and TNF-α levels are higher in sputum of former smoker than in current smoker COPD patients. We found these differences only in the airway leptin system of stable COPD patients due to a direct action of the cigarette smoke in the airways. To our knowledge, only one study has assessed the relationship between leptin and other cytokines in sputum of COPD patients, suggesting a specific role of this adipokine in the local inflammatory response in COPD 20

Smokers (n = 6)
Ex-smokers (n = 10) but not clarifying the specific role of cigarette smoke in these mechanisms. Our results strengthen the hypothesis that quitting smoking might increase the leptin action in host defense mechanism from respiratory pathogens mediated by sputum neutrophils. Indeed, we show here for the first time the expression of leptin together with its receptor in sputum neutrophils in COPD patients and that the expression of leptin and its receptor is higher in former smoker than in current smoker COPD subjects and that these parameters are positively correlated only in former smokers. In addition the increased levels of leptin in the induced sputum from former smokers together with higher levels of TNF-α might be associated with the capacity of TNF-α to increase the levels of leptin expression 37,38 or to the increased appetite after smoking cessation. 39 Neutrophils express the short form of leptin receptors 7 and despite lacking the STAT3 docking site, the short leptin receptor isoform is still able to bind and activate Jak2, which subsequently activates the MAPK pathway in leptin-stimulated neutrophils. 40 The MAPK pathway is important for cytoskeletal processes, such as those involved in the transfer of CD11b from cytoplasmic granules to the plasma membrane, and for the generation of reactive oxygen species. 41,42 Leptin is able to stimulate neutrophils by increasing CD11b expression indirectly via monocyte-derived TNF-α 9 and since a leptin-induced ICAM-1 expression was observed in eosinophils 43 one of the mechanisms by which leptin affects neutrophil activity might be associated with the induction of the expression of ICAM on neutrophils of COPD patients. Additionally, it has been reported that reactive oxygen production is the consequence of a direct stimulation of neutrophils by leptin 10 and that exogenous leptin administration in vivo or in vitro could induce the phagocytic activity in leptin-deficient purified mouse neutrophils. 17 Together, this evidence suggests that leptin and its receptor are strongly involved in the activation of neutrophils, as supported by our results showing a positive correlation between leptin and its receptor in sputum neutrophils of former smokers. Indeed our patients, well matched for BMI and body weight, did not show differences in neutrophils in sputum cells from COPD and COPD former smokers, in contrast with previous results 44 obtained in a study population of COPD not matched for nutritional status. TNF-α is a proinflammatory cytokine with pleiotropic effects produced in the lung mainly by activated macrophages in response to inflammatory stimuli such as a chemoattractant for inflammatory cells. 45,46 TNF-α levels are higher in patients experiencing COPD exacerbation 14 and TNF-α receptors are significantly elevated in sputum from ex-smoker compared with current smoker COPD patients. 47 We did not identify significant differences between TNF-α levels in sputum and plasma from COPD former smokers and smokers, although some of our patients reached higher levels of TNF-α due to the fact that our patients were in a stable condition.
Although our study has some limitations because of the small number of patients, it is the first study to analyze the expression of the leptin/leptin receptor pathway in sputum cells from COPD patients in relation to the smoking habit of the patients. However our results, in accord with those of a previous study, 20,48 underline the fact that the levels of leptin together with TNF-α, in the inflammatory context of COPD, are more involved in the local inflammatory response of the lung than in the systemic circulation. Finally, our findings underline the correlation between leptin sputum levels and FEV 1 /FVC ratio in COPD patients together with the increased leptin levels in patients who have quit smoking, supporting the concept of a protective role of leptin in COPD with a deleterious effect of cigarette smoke. Our observation is further supported by the fact that the expression of leptin is correlated positively with the expression of its receptor in sputum neutrophils from former smoker COPD patients, leading us to suppose that cigarette smoke might affect the leptin/leptin receptor-mediated neutrophil activation. All these data could explain a long-term follow-up improvement of the pulmonary function in COPD patients after smoking cessation mediated by leptin neutrophils activation.
Taken together these findings strongly suggest that leptin plays a role in the activation of neutrophils in the airways of COPD patients and that leptin is involved in the protection of the airways of these patients. This effect is likely exerted via the role played by neutrophils in innate immune mechanisms against infections and is counteracted by the persistence of cigarette smoke exposure. To better identify the role of leptin in sputum neutrophils from COPD former smokers further studies are needed to analyze COPD patients before and after smoking cessation.