Levels of IL-32 in Serum, Induced Sputum Supernatant, and Bronchial Lavage Fluid of Patients with Chronic Obstructive Pulmonary Disease.

Interleukin-32 (IL-32) is a newly described cytokine which is expected to have an important role in autoimmune disorders. It was shown that chronic obstructive pulmonary disease (COPD) has a component of autoimmunity, though the role of IL-32 in its pathogenesis is not known. The aim of this study was to estimate IL-32 concentrations in serum, induced sputum (IS) supernatant and bronchoalveolar lavage (BAL) fluid from patients with COPD, and to compare asthma patients with and healthy subjects. Outpatients with COPD (63.7 ± 8.4 years, n = 51), asthma (58.3 ± 12.4 years, n = 31), and healthy subjects (59.8 ± 8.2 years, n = 9) were studied. The levels of IL-32 in serum, BAL fluid, and IS supernatant samples were analyzed by ELISA. Concentrations of IL-32 were higher in all the studied materials from patients with COPD (BAL 22.46 ± 2.48 pg/ml, IS 19.66 ± 1.69 pg/ml, serum 26.77 ± 2.56 pg/ml) in comparison with patients with asthma (BAL 6.25 ± 1.08 pg/ml, IS 5.82 ± 1.15 pg/ml, serum 6.09 ± 1.16 pg/ml, p < 0.05 respectively) as well as healthy subjects (BAL 4.21 ± 1.13 pg/ml, IS 3.59 ± 0.66 pg/ml, serum 4.63 ± 1.03 pg/ml, p < 0.05 respectively). Moreover, the level of IL-32 was higher in COPD smokers than in COPD ex-smokers in investigated respiratory tissue compartments and serum, and correlated with smoking history. Increased level of IL-32 in serum, IS supernatant, and BAL fluid from patients with COPD in comparison with asthma patients and healthy subjects suggest that IL-32 may play an important role in the pathogenesis of COPD, which depends on the smoking history.

The prognostic influence of tumor infiltrating Foxp3(+)CD4(+), CD4(+) and CD8(+) T cells in resected non-small cell lung cancer.

BACKGROUND: Different subsets of tumor infiltrating T lymphocytes are believed to play essential role in the immune response to cancer cells. The data of these cells in NSCLC are relatively rare and controversial therefore we aimed to evaluate the infiltration patterns of Foxp3 + CD4+, CD4+ and CD8+ T cells in NSCLC and to analyze their relations to survival. METHODS: Lung tissue specimens from 80 newly diagnosed and untreated patients who underwent surgery for NSCLC (stages I-III), and 16 control group subjects, who underwent surgery due to recurrent spontaneous pneumothorax, were analyzed. Foxp3 + CD4+, CD4+ and CD8+ T cells in tumor stroma and islets were evaluated immunohistochemically. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 20.0. RESULTS: Tumor infiltrating CD4+, CD8+ T cells were associated with neither overall survival nor disease-free survival. The presence of high tumor stroma infiltrating Foxp3 + CD4+ T cells was independently associated with improved NSCLC patients overall survival (P < 0.05). CONCLUSIONS: Our study demonstrated that tumor infiltrating Foxp3 + CD4+ T cells are associated with improved NSCLC patients' survival. In addition our findings highlight a tendency of high CD4+/CD8+ and CD8+/Foxp3 + CD4+ T cells ratio in prolonged NSCLC patients' survival.

p-STAT6, PU.1, and NF-κB are involved in allergen-induced late-phase airway inflammation in asthma patients.

BACKGROUND: Previous in vitro and animal studies demonstrated that transcription factors p-STAT6 and PU.1 are required to induce interleukin (IL)-9 secretion by T helper (Th) 9 cells. It is believed that n factor-kappaB (NF-κB) plays a role in eosinophil survival. The importance of these transcription factors in the pathogenesis of allergic asthma (AA) in humans is poorly understood. We evaluated p-STAT6 and PU.1 expression in peripheral blood Th9 cells and NF-κB expression in eosinophils during late-phase airway inflammation in AA patients. METHODS: Nineteen adults with AA and 14 adult healthy individuals (HI) were examined. Peripheral blood collected 24 h before (baseline) and 24 h after bronchial allergen challenge. CD4(+) cells and eosinophils were isolated by high-density gradient centrifugation and magnetic separation. The percentage of Th9 cells and apoptotic eosinophils was estimated by flow cytometry. p-STAT6 and PU.1 expression was expressed as mean fluorescence intensity (MFI) in Th9 cells. NF-κB levels were expressed as MFI in peripheral blood eosinophils. Serum IL-9 and IL-5 levels were determined by enzyme-linked immunosorbent assay. RESULTS: At baseline, MFI of p-STAT6 and PU.1 in peripheral blood Th9 cells and MFI of NF-κB in eosinophils and, serum IL-5 and IL-9 levels were greater in AA patients (P < 0.05). Decreased eosinophil apoptosis was seen in the AA group compared with HI (P < 0.05). MFI of p-STAT6, PU.1, and NF-κB and serum levels of IL-5 and IL-9 were increased in the AA group 24 h after challenge compared with baseline (P < 0.05). In the AA group, a correlation between serum IL-9 and Th9 cells (r = 0.7, P = 0.001) and MFI of PU.1 (r = 0.6, P = 0.01) 24 h after bronchial allergen challenge was observed. A correlation between Th9 cells and MFI of p-STAT6 (r = 0.45, P = 0.03) as well as MFI of PU.1 (r = 0.5, P = 0.02) 24 h after challenge was only observed in AA patients. A correlation between the MFI of NF-κB and eosinophil apoptosis was observed in AA patients 24 h before (r = - .46, P = 0.02) and after (r = -0.5, P = 0.02) challenge. DISCUSSIONS: p-STAT6 and PU.1 may be associated with Th9 cells and IL-9 production, whereas NF-κB and IL-5 may be associated with reduced eosinophil apoptosis in allergen-induced late-phase airway inflammation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02214303.

Functional activity of peripheral blood eosinophils in allergen-induced late-phase airway inflammation in asthma patients.

OBJECTIVE: We aimed to investigate peripheral blood eosinophil chemotaxis, generation of spontaneous reactive oxygen species (ROS), and apoptosis in patients with allergic asthma after bronchial allergen challenge. MATERIAL AND METHODS: A total of 18 patients with allergic asthma (AA), 14 with allergic rhinitis (AR), and 10 healthy subjects (HS) underwent bronchial challenge with a specific allergen extract. Eosinophils from peripheral blood were isolated 24 h before as well as 7 and 24 h after bronchial allergen challenge. Chemotaxis, spontaneous ROS production in eosinophils, and apoptosis were analyzed by flow cytometry. Serum and induced sputum IL-5 levels were measured by ELISA; the cell count in sputum was analyzed by the May-Grünwald-Giemsa method. RESULTS: Before bronchial allergen challenge, peripheral blood eosinophil chemotaxis, spontaneous ROS production was enhanced and eosinophil apoptosis was reduced in the patients with AA as compared with AR patients and HS (P < 0.05). Meanwhile, eosinophil chemotaxis and ROS generation markedly increased in the patients with AA 7 h and 24 h after challenge compared with other groups and baseline values (P < 0.05). The percentage of apoptotic eosinophils in the patients with AA decreased at 7 h as well as 24 h after challenge when compared with other groups and the baseline values (P < 0.05). There was a significant correlation between the migrated peripheral blood eosinophil count and the sputum eosinophil count (Rs = 0.89, P < 0.0001) and the sputum IL-5 level (Rs = 0.68, P = 0.002) at 24 h after bronchial challenge only in the patients with AA. Furthermore, the percentage of peripheral blood apoptotic eosinophils significantly correlated with eosinophil count in sputum (Rs = -0.53, P = 0.02), and ROS production correlated with the serum IL-5 levels (Rs = 0.71, P = 0.01). CONCLUSION: During allergen-induced late-phase airway inflammation, peripheral blood eosinophils demonstrated further alterations of their functional activity manifested by enhanced spontaneous ROS production, increased chemotaxis, and diminished apoptosis in patients with AA.

Peripheral blood Th9 cells and eosinophil apoptosis in asthma patients.

BACKGROUND AND OBJECTIVE: Th9 cells producing interleukin (IL) 9 are novel subset of CD4+ T helper cells, which might contribute to airway inflammation in asthma. Moreover, the effect of IL-9 on eosinophils is still not fully understood. Study aim was to evaluate peripheral blood Th9 cells and eosinophil apoptosis in allergic asthma patients. MATERIALS AND METHODS: Eighteen patients with allergic asthma and fourteen patients with allergic rhinitis were examined. Control group included sixteen healthy subjects. Allergic asthma and rhinitis patients did not use corticosteroids and antihistamines at least for 1 week. Peripheral blood eosinophils and CD4(+) cells were isolated by high density gradient centrifugation and magnetic separation. Th9 cells and apoptotic eosinophils were estimated by flow cytometer. Serum IL-9 and IL-5 concentration were determined by ELISA. RESULTS: Peripheral blood Th9 cells percentage was increased in allergic asthma group compared with allergic rhinitis and control group (0.74%±0.32% vs. 0.19%±0.10% and 0.15%±0.08%, respectively, P<0.05). The same tendency was observed for IL-9 (P<0.01). Percentage of peripheral blood apoptotic eosinophils was decreased in allergic asthma and allergic rhinitis groups compared with control group (P<0.05). IL-9 concentration correlated with percentage of Th9 cells (r=0.64, P<0.05) and negatively with percentage of apoptotic eosinophils in allergic asthma group (r=-0.58, P<0.05). Negative correlation was found between apoptotic eosinophils count and IL-5 concentration in allergic asthma group (r=-0.76, P<0.05). CONCLUSIONS: Patients with allergic asthma demonstrate increased peripheral blood Th9 cells count and serum IL-9, while eosinophil apoptosis is inversely related to IL-9 concentration.

Evaluation of vitamin D levels in allergic and non-allergic asthma.

BACKGROUND AND OBJECTIVE: Some researches show that low vitamin D may play a role in asthma pathogenesis. The aim of this study was to evaluate the serum vitamin D level in asthmatics with different phenotypes and to determine its associations with lung function, IgE, eosinophil count and body mass index (BMI). MATERIALS AND METHODS: The study population comprised 85 patients with asthma and 73 healthy persons. Patients with asthma were divided into groups according to phenotypes. Allergy was assessed using a skin prick test and measuring eosinophil count in peripheral blood and total IgE in serum. Lung function was evaluated by spirometry. Concentration of vitamin D (25(OH)D3) was measured using a commercial ELISA kit. Smoking history was assessed and BMI was calculated for all individuals. RESULTS: The vitamin D level was lower in asthmatics than in the control group (14.36±0.57 vs. 22.13±0.84 ng/mL, P<0.01). There were no significant differences in the vitamin D level between the groups with allergic and non-allergic asthma (14.36±0.77 vs. 14.35±0.74 ng/mL). The low vitamin D level increased the risk of asthma 1.2 times (OR, 1.194; 95% CI, 1.109-1.286, P<0.01). The vitamin D level did not correlate with lung function and markers of allergy in asthmatic patients. The vitamin D level correlated with FEV1/FVC (rs=0.72, P<0.05) in smoking patients with asthma. Correlation between the vitamin D level and BMI was found in all studied subjects (rs=-0.18, P<0.05). CONCLUSIONS: The vitamin D level was lower in asthmatic patients than in healthy individuals despite their hypersensitivity and increase risk of asthma. There was no relation between the vitamin D level and lung function, eosinophil count and total IgE level, whereas the lower vitamin D level was associated with higher BMI.

Sputum neutrophil count after bronchial allergen challenge is related to peripheral blood neutrophil chemotaxis in asthma patients.

OBJECTIVE: The aim of this study was to estimate relations between sputum neutrophilia and the chemotactic activity of peripheral blood neutrophils after the bronchial allergen challenge in asthma patients. MATERIALS AND METHODS: Fifteen patients with allergic asthma (AA), 13 patients with allergic rhinitis (AR), all sensitized to Dermatophagoides pteronyssinus, and 8 healthy subjects (HS) underwent bronchial challenge with D. pteronyssinus. Sputum and peripheral blood collection were performed 24 h before, 7 and 24 h after the bronchial challenge. Cell counts were determined by the May-Grünwald-Giemsa method. Neutrophil chemotaxis was analyzed by a flow cytometer; IL-8 levels were measured by ELISA. RESULTS: Sputum neutrophil count and peripheral blood neutrophil chemotaxis of patients with AA were greater 7 and 24 h after the challenge compared with the baseline values and patients with AR and HS (P < 0.05). Moreover, a significant correlation was found between the neutrophil count in sputum and IL-8 levels, and the chemotactic activity of peripheral blood neutrophils 24 h after the bronchial challenge only the patients with AA (P < 0.05). CONCLUSIONS: Increased sputum neutrophil count was found to be associated with an enhanced chemotactic activity of peripheral blood neutrophils during allergen-induced late-phase airway inflammation in patients with allergic asthma.

Th17 response to Dermatophagoides pteronyssinus is related to late-phase airway and systemic inflammation in allergic asthma.

BACKGROUND: Th17 cells may play a role in the development of late-phase allergen-induced airway and systemic inflammation in allergic asthma, although the mechanisms involved remain to be elucidated. METHODS: A total of 36 subjects were enrolled into the study: 15 allergic asthma patients with early asthmatic reaction (n=7) or dual asthmatic reaction (n=8) developed to inhaled D. pteronyssinus, 13 patients with allergic rhinitis, and 8 healthy subjects. Peripheral blood and induced sputum were collected 24h before as well as 7h and 24h after a bronchial challenge with D. pteronyssinus. Th17 cells were analyzed by FACS; IL-17 levels were determined by ELISA. RESULTS: At baseline, the percentage of peripheral blood Th17 cells and serum and sputum IL-17 levels were significantly higher in all groups of studied patients compared with those of healthy subjects. After the bronchial challenge, there was a significant increase in the percentage of peripheral blood Th17 cells and in serum and sputum IL-17 levels in rhinitis and asthma patients compared with their baseline values, particularly in allergic asthma patients with the dual asthmatic reaction. Positive correlations were found between the percentage of Th17 cells and IL-17 levels in serum (Rs=0.649; P=0.009) as well in sputum (Rs=0.583; P=0.022) in allergic asthma patients 24h after the bronchial challenge. CONCLUSIONS: The Th17 response is associated with the development of late-phase airway and systemic inflammation after the inhalation of D. pteronyssinus in patients with allergic asthma.

Local and systemic neutrophilic inflammation in patients with lung cancer and chronic obstructive pulmonary disease.

BACKGROUND: Recent investigations suggest that neutrophils play an important role in the immune response to lung cancer as well as chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate the amount of neutrophils and markers of their activity in lung cancer and COPD and in coexistence of these two diseases. METHODS: In total, 267 persons were included in the study: 139 patients with lung cancer, 55 patients with lung cancer and COPD, 40 patients with COPD, and 33 healthy subjects. Peripheral blood and BAL fluid samples were obtained for cell count analysis and determination of NE, MPO levels and ROS production. NE and MPO levels in the serum and BAL fluid were determined by ELISA. ROS production was analyzed by flow cytometer. RESULTS: The percentage, cell count of neutrophils and neutrophil to lymphocyte ratio in the peripheral blood were significantly higher in lung cancer patients with or without COPD compared to COPD patients or healthy individuals (P < 0.05). The percentage and cell count of neutrophils in BAL fluid were significantly lower in patients with lung cancer with or without COPD than in patients with COPD (P < 0.05). However, BAL fluid and serum levels of both NE and MPO were significantly higher in patients with lung cancer than COPD patients or healthy individuals (P < 0.05). Neutrophils produced higher amounts of ROS in patients with lung cancer with or without COPD compared with COPD patients or healthy individuals (P < 0.05). CONCLUSIONS: The results from this study demonstrate higher degree of local and systemic neutrophilic inflammation in patients with lung cancer (with or without COPD) than in patients with COPD.

Reactive oxygen species in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbation of chronic obstructive pulmonary disease.

Chronic airway inflammation can be mediated by an enhanced neutrophil oxidative burst. However, the role of bacteria in the pathogenesis of chronic obstructive pulmonary disease (COPD) exacerbations is highly controversial. The aim of this study was to evaluate the production of reactive oxygen species (ROS) in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbations of COPD (AECOPD). A total of 40 patients with AECOPD, 10 healthy nonsmokers, and 10 "healthy" smokers were enrolled into the study. Peripheral blood and sputum samples were obtained during exacerbation and after recovery. Neutrophils were isolated by high-density gradient centrifugation and magnetic separation. ROS production by neutrophils was investigated after stimulation with phorbol-myristate-acetate and Staphylococcus aureus bacteria. ROS production by neutrophils was assessed as the mean fluorescent intensity using a flow cytometer. IL-8 levels in serum and induced sputum were determinant by ELISA. Spontaneous ROS production was significantly higher in neutrophils from the patients with bacterial AECOPD as compared with nonbacterial AECOPD and stable COPD (P <0.05). ROS production stimulated with PMA and with Staphylococcus aureus was significantly higher in neutrophils isolated from the patients with bacterial AECOPD as compared with nonbacterial and stable COPD (P <0.05). The serum and induced sputum IL-8 levels were significantly increased in the patients with bacterial AECOPD than nonbacterial AECOPD, stable COPS, and "healthy" smokers and nonsmokers (P <0.05) and higher in the induced sputum as the compared with serum in all studied groups (P <0.05). Enlarge CRP level was documented during AECOPD than in all other groups (P <0.05). A markedly increased ROS production in sputum neutrophils during bacterial AECOPD shows an inflammatory response reflecting enhanced local inflammation, which can be mediated by bacterial colonization.

Peripheral blood Th17 cells and neutrophils in Dermatophagoides pteronyssinus-induced early- and late-phase asthmatic response.

BACKGROUND AND OBJECTIVE: Biphasic cellular immune reactions, which follow allergen inhalation, are a specific feature of inflammation in allergic asthma. The aim of this study was to determine the changes in the percentage of peripheral blood Th17 cells and neutrophil functions after Dermatophagoides pteronyssinus-induced early- and late-phase asthmatic response in patients with allergic asthma. MATERIAL AND METHODS: A total of 19 patients with allergic asthma were examined. Eleven patients developed an isolated early-phase asthmatic response (EAR), whereas 8 developed both early- and late-phase (dual) asthmatic responses (DAR) after the bronchial challenge with Dermatophagoides pteronyssinus. The control group included 15 healthy subjects. Peripheral blood collection was performed 24 hours before as well as 7 and 24 hours after the bronchial challenge. The percentage of Th17 cells, and chemotaxis and apoptosis of neutrophils were analyzed by flow cytometry. The serum IL-8 and IL-17 levels were determined by ELISA. RESULTS: After the bronchial challenge, the percentage of Th17 and IL-17 levels increased considerably 7 and 24 hours after the challenge in both groups of patients. Moreover, 24 hours after the challenge, the percentage of Th17 cells and IL-17 levels were significantly higher in the patients with the DAR than those with the EAR or healthy controls. Seven and 24 hours after the challenge, neutrophil chemotaxis was greater in the patients with the DAR as compared with those with the EAR and healthy controls as well. The apoptotic activity of neutrophils was lower 24 hours after the challenge in the patients with the DAR than those with the EAR. CONCLUSIONS: Dermatophagoides pteronyssinus-induced early- and late-phase asthmatic response in patients with allergic asthma was found to be accompanied by an increased percentage of peripheral blood Th17 cells and elevated serum IL-17 levels as well as altered neutrophil functions.

Response of peripheral blood Th17 cells to inhaled Dermatophagoides pteronyssinus in patients with allergic rhinitis and asthma.

BACKGROUND: Recent studies have shown the importance of Th17 cells in the development of allergic airway diseases. We examined Dermatophagoides pteronyssinus-induced changes in peripheral blood Th17 cells to establish the importance of these cells in late-phase allergic inflammation in patients with allergic rhinitis (AR) and allergic asthma (AA). METHODS: Eighteen patients with mild-to-moderate/severe persistent AR, 14 patients with intermittent- or mild-to-moderate persistent AA, and 15 healthy subjects (HS) were examined. All patients had positive skin test to D. pteronyssinus. Study subjects underwent bronchial challenge with D. pteronyssinus. The peripheral blood Th1, Th2, and Th17 cells were determined by flow cytometry 24 h before and 7 and 24 h after challenge. The serum IL-17 levels were determined by ELISA. RESULTS: The percentage of Th17 cells and IL-17 levels was significantly higher in patients with AR and AA compared with HS before and after challenge. Twenty-four hours after challenge, the percentage of Th17 cells increased significantly in patients with AA compared with baseline values. The IL-17 levels rose markedly in patients with AR and AA after challenge. Moreover, 24 h after challenge, the percentage of Th17 cells and IL-17 levels were significantly higher in patients with AA than those with AR. CONCLUSIONS: Percentages of peripheral blood Th17 cells and serum IL-17 levels were found to be higher in patients with AR and AA. An increase in the percentage of Th17 cells following challenge shows that Th17 cells may have an important role in the development of late-phase allergen-induced inflammation.

Peripheral blood neutrophil activity during Dermatophagoides pteronyssinus-induced late-phase airway inflammation in patients with allergic rhinitis and asthma.

Recent investigations suggest that neutrophils may play an important role in the late-phase allergen-induced inflammation in allergic airway diseases. The aim of this study was to evaluate neutrophil chemotaxis, phagocytic activity, and reactive oxygen species (ROS) production in patients with allergic rhinitis and asthma challenged with inhaled Dermatophagoides pteronyssinus. Eighteen patients with allergic rhinitis and 14 with allergic asthma, all sensitized to D. pteronyssinus, as well as 15 healthy individuals underwent bronchial challenge with D. pteronyssinus. Peripheral blood collection and neutrophil isolation were performed 24 h before as well as 7 and 24 h after bronchial challenge. Neutrophils chemotaxis, phagocytic activity, and ROS production were analyzed by flow cytometer. Neutrophil chemotaxis and ROS production were increased, while phagocytic activity was decreased 24 h before challenge in patient groups compared with healthy individuals. After challenge, neutrophil chemotaxis and phagocytic activity increased after 7 and 24 h, when ROS production, only after 24 h. Bronchial allergen challenge had no influence for neutrophils activity in healthy subjects. Activated chemotaxis, phagocytic activity, and ROS production of peripheral blood neutrophils after challenge with D. pteronyssinus reflect an enhanced systemic inflammation in allergic rhinitis and asthma patients with induced late-phase airway inflammation.