Protein Levels In Sputum Research Articles

Exogenous IL-25 ameliorates airway neutrophilia via suppressing macrophage M1 polarization and the expression of IL-12 and IL-23 in asthma

BackgroundSevere asthma is associated with substantial mortality and has unmet therapeutic need. A subset of severe asthma is characterized by neutrophilic airway inflammation. Classically activated (or M1) macrophages which express IL-12 and IL-23 are associated with airway neutrophilia in asthma. Exogenous IL-25 was reported to suppress intestinal inflammation in animal models of inflammatory bowel diseases via suppressing IL-12 and IL-23 production. We hypothesize that IL-25 ameliorates airway neutrophilia via inhibiting macrophage M1 polarization and the expression of IL-12 and IL-23 in asthma.MethodsIn a mouse model of neutrophil-dominant allergic airway inflammation, the effect of mouse recombinant IL-25 on airway inflammation were assessed by H&E staining and bronchoalveolar lavage (BAL) cell counting. The percentage of M1 macrophages in lung tissue and BAL cells were analyzed by flow cytometry. Quantitative PCR and immunostaining were performed to measure the expression of Il12, Il23, and inflammatory cytokines. Mechanistic experiments were performed in primary culture of macrophages from mouse lungs. The expression of IL-12, IL-23 and IL-25 in sputum was analyzed in a cohort of severe asthma and subjects with eosinophilic or non-eosinophilic asthma.ResultsIntranasal administration of IL-25 markedly decreased the number of neutrophils in BAL cells in a murine model of neutrophil-dominant allergic airway inflammation. Moreover, exogenous IL-25 decreased the number of M1 macrophages, and reduced the expression of IL-12, IL-23 in the lungs of the mouse model. Exogenous IL-25 also inhibited the expression of inflammatory cytokines IL-1β, IFN-γ, TNF-α and IL-17 A. In vitro, IL-25 suppressed IL-12 and IL-23 expression in lipopolysaccharide (LPS)-stimulated primary culture of mouse pulmonary macrophages. Mechanistically, IL-25 inhibited LPS-induced c-Rel translocation to nucleus via STAT3-dependent signaling. In a cohort of severe asthma, IL-25 protein levels in sputum were significantly lower than control subjects. The transcript levels of IL-12 and IL-23 were increased whereas IL-25 transcripts were decreased in sputum cells from subjects with non-eosinophilic asthma compared to eosinophilic asthma.ConclusionsIL-25 expression is downregulated in subjects with severe or non-eosinophilic asthma. Exogenous IL-25 ameliorates airway neutrophilia, at least in part, via inhibiting macrophage M1 polarization and the expression of IL-12 and IL-23.

Cilia proteins CFAP36 and sentan in induced sputum as possible new markers of epithelial damage in obstructive lung diseases: A preliminary study

Background: Asthma and chronic obstructive pulmonary disease (COPD) are the most common chronic respiratory diseases characterized by inflammation in the lower airways and epithelium remodeling. Dysfunction of cilium is related to severe asthma and COPD, the role of cilium proteins in obstructive lung diseases is not known. The aim of the study was to evaluate the concentration of cilia associated proteins: sentan and CFAP36 in induced sputum (IS) of asthma and COPD patients. Materials/Methods: The study involved 15 patients with asthma, 12 patients with COPD and 17 control subjects (9 non-smoking, 8 smoking) who underwent lung function tests and sputum induction. Sentan, CFAP36, IL-6, IL-8, concentrations were measured in induced sputum supernatants by ELISA. Results: The level of CFAP36 in induced sputum was elevated in asthma patients and subjects with atopy. Cilium protein levels in sputum were not related to spirometric tests results. Both CFAP36 and sentan concentrations were positively correlated with age. The level of sentan was associated with airway neutrophilic inflammation and active smoking status. CFAP36 concentration was negatively related to cell viability, whereas sentan level was positively related, but only in COPD patients. Conclusions: The results of our study revealed CFAP36 and sentan as possible new markers of epithelial damage of different origin in obstructive lung diseases.

The SNP rs13147758 in the HHIP Gene Is Associated With COPD Susceptibility, Serum, and Sputum Protein Levels in Smokers.

BackgroundGenetic association studies have identified single nucleotide polymorphisms (SNPs) related to chronic obstructive pulmonary disease (COPD) susceptibility. The aim of this study was to identify HHIP genetic variants associated with COPD, pulmonary function, and serum and sputum HHIP protein levels in Mexican mestizo smokers.Materials and MethodsAssociation analysis was performed by carrying out a case–control study in Mexican mestizo smokers comprised of two groups: tobacco-smoking subjects with COPD (COPD-TS, n = 222) and smokers without COPD (SWOC, n = 333). We evaluated three SNPs (rs13147758, rs1828591, and rs13118928) in the HHIP gene. Allele discrimination was accomplished by qPCR using TaqMan probes, and determination of protein levels in the serum and sputum supernatants (SS) was performed using ELISA.ResultsStatistically significant differences were observed in the rs13147758 GG genotype (adjusted p = 0.014, OR = 1.95) and the rs13147758-rs1828591 GA haplotype (p = 6.6E-06, OR = 2.65) in the case–control comparison. HHIP protein levels were elevated in SS samples from the COPD-TS group compared to those from the SWOC group (p = 0.03). Based on genotype analysis, HHIP protein levels were lower in the serum samples of rs13147758 GG genotype carriers in the COPD-TS group than in the serum samples of rs13147758 GG genotype carriers from the SWOC group (p < 0.05), but there were no differences in the sputum samples.ConclusionThe rs13147758 GG genotype and the rs13147758-rs1828591 GA haplotype are associated with susceptibility to COPD. Furthermore, an association in protein levels was observed between the HHIP rs13147758 genotype and COPD in Mexican mestizo smokers.

Sputum biomarkers in IPF: Evidence for raised gene expression and protein level of IGFBP-2, IL-8 and MMP-7.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a rare lung disease of unknown origin leading rapidly to death. This paper addresses the issue of whether sputum induction is a suitable tool to study respiratory tract inflammation and potential biomarkers in IPF compared to COPD, a fibrosing airway wall disease.MethodsIn a cross-sectional analysis, 15 IPF patients, 32 COPD and 30 healthy subjects underwent sputum induction. Total sputum cell counts and the amount of TGF- β, IGF-1, IGF-2, IGFBP-1, IGFBP-2, IGFBP-3, IL-8, IL-13, MMP-7, MMP-9, YKL-40, TNF-α and KL-6 in sputum supernatant were analysed. We also profiled gene expression of cells in the induced sputum for TGF-β, MMP-7, YKL-40, IGFBP-2, IL-6, IL-8 and TNF-α.ResultsIPF patients, like COPD, had increased sputum absolute number of neutrophils, eosinophils, macrophages and epithelial cells compared to HS. IPF sputum supernatants had increased concentrations of IGFBP-2, IL-8, TGF-β, MMP-7, MMP-9 and KL-6 (p<0.05, p<0.0001, p<0.05, p<0.05, p<0.0001, p<0.05 respectively) when compared to healthy subjects where COPD had higher IL-6 and TNF-α levels than IPF (p<0.05 and p<0.05 respectively) and HS (p<0.0001 and p<0.001 respectively) and higher IL-8 and MMP-9 than HS (p<0.0001 and p<0.001 respectively). Conversely to IL-6 and TNF-α, MMP-7 was increased in IPF compared to COPD (p<0.05). The KL-6 and MMP-7 protein levels in sputum were inversely correlated with total lung capacity (TLC, % of predicted) in IPF patients (r = -0.73 and r = -0.53 respectively). Sputum gene expression analysis identified a significant increase for IGFBP-2, IL-6, IL-8 and MMP-7 in IPF compared to HS (p<0.05, p<0.01, p<0.05 and p<0.0001 respectively) and for IGFBP-2, YKL-40, IL-6, IL-8 and MMP-7 compared to COPD (p<0.01, p<0.01, p<0.05, p<0.01 and p<0.0001 respectively). Furthermore, gene expression of TGF-β was increased in IPF compared to COPD (p<0.001) but not to HS.ConclusionOur data show clear increase in expression and production of IGFBP-2, IL-8 and MMP-7 in sputum from patients with IPF that may contribute to the disease.

Pulmonary Biomarkers Based on Alterations in Protein Expression after Exposure to Arsenic

ObjectiveEnvironmental exposure to arsenic results in multiple adverse effects in the lung. Our objective was to identify potential pulmonary protein biomarkers in the lung-lining fluid of mice chronically exposed to low-dose As and to validate these protein changes in human populations exposed to As.MethodsMice were administered 10 or 50 ppb As (sodium arsenite) in their drinking water for 4 weeks. Proteins in the lung-lining fluid were identified using two-dimensional gel electrophoresis (n = 3) or multidimensional protein identification technology (MUDPIT) (n = 2) coupled with mass spectrometry. Lung-induced sputum samples were collected from 57 individuals (tap water As ranged from ~ 5 to 20 ppb). Protein levels in sputum were determined by ELISA, and As species were analyzed in first morning void urine.ResultsProteins in mouse lung-lining fluid whose expression was consistently altered by As included glutathione-S-transferase (GST)-omega-1, contraspin, apolipoprotein A-I and A-IV, enolase-1, peroxiredoxin-6, and receptor for advanced glycation end products (RAGE). Validation of the putative biomarkers was carried out by evaluating As-induced alterations in RAGE in humans. Regression analysis demonstrated a significant negative correlation (p = 0.016) between sputum levels of RAGE and total urinary inorganic As, similar to results seen in our animal model.ConclusionCombinations of proteomic analyses of animal models followed by specific analysis of human samples provide an unbiased determination of important, previously unidentified putative biomarkers that may be related to human disease.

Determinants of the Severity of Exercise‐Induced Bronchoconstriction in Patients with Asthma

Aim. In examining the mechanisms of exercise‐induced bronchoconstriction (EIB), it is important to determine which factors most strongly affect the severity of EIB. We determined such factors in patients with asthma by stepwise multiple‐regression analysis. Methods. Twenty‐three patients with asthma underwent pulmonary function tests, methacholine provocation test, and sputum induction. Eosinophilic inflammatory indices and airway vascular permeability index (ratio of albumin concentrations in induced sputum and serum) were examined in sputum samples, and then an exercise test was performed by all asthmatics. Results. There was a significant correlation between the severity of EIB and degree of eosinophilic inflammation in induced sputum. Moreover, there was a significant correlation between the severity of EIB and airway vascular permeability index. Although we could not find a significant correlation between the severity of EIB and 1‐sec forced expired volume, 20% provocation concentration of (PC20) methacholine tended to be correlated with the severity of EIB. By stepwise multiple‐regression analysis, we also found that airway vascular permeability index, eosinophil cationic protein levels in sputum, and PC20 methacholine are independent predictors of the severity of EIB. Conclusion. We found that airway vascular hyperpermeability, eosinophilic inflammation, and bronchial hyperreactivity are independent factors predicting the severity of EIB.

Increased levels of nitric oxide derivatives in induced sputum in patients with asthma

Background: Nitric oxide (NO) plays an important role as an inflammatory mediator in the airways. However, because direct measurement of endogenous NO has been difficult in vivo, the exact pathologic roles of NO in human airway inflammation have remained unclear. Objective: This study was designed to determine whether NO may be harmful by amplifying allergic inflammation in asthmatic airways. Methods: In this study we examined the concentration of stable end products of NO, nitrite and nitrate, in induced sputum in 18 patients with asthma and 10 normal control subjects and evaluated the relationship between levels of NO derivatives in sputum and cellular and biochemical profiles, the degree of airflow obstruction, and the cytotoxic activities for epithelial cells. Results: The concentration of NO derivatives in induced sputum was significantly higher in patients with asthma than in normal control subjects (1086 ± 325 μmol/L, 577 ± 115 μmol/L; p < 0.05). Percentages of eosinophil counts and eosinophil cationic protein levels in sputum were also significantly higher in patients with asthma. Moreover, percentages of eosinophil counts and eosinophil cationic protein levels in sputum in patients with asthma were significantly correlated with the concentration of NO derivatives in sputum ( r = 0.63, p < 0.01; r = 0.56, p < 0.05, respectively). In addition, we found that the concentration of NO derivatives in sputum in patients with asthma was significantly correlated with the degree of airflow obstruction (FEV 1/forced vital capacity) ( r = –0.62, p < 0.01) and with percentages of shedding epithelial cells ( r = 0.61, p < 0.01). Conclusion: We have shown that a higher concentration of NO derivatives was found in induced sputum of patients with asthma as compared with normal subjects. The clinical implication of our findings is that measurement of NO derivatives in induced sputum may be useful for assessing allergic inflammation in airways. (J Allergy Clin Immunol 1997;99:624-9.)

Analysis of plasma-protein leakage and local secretion in sputum from patients with asthma and chronic obstructive pulmonary disease.

In order to assess the usefulness of sputum analysis in studying plasma-protein exudation and local secretion of proteins in the airways, we measured specific proteins in the sputum sol phase (SSP) and sputum gel phase (SGP) from patients with stable asthma or chronic obstructive pulmonary disease (COPD). Protein levels in SSP showed relatively small variations between two subsequent visits of each patient (n = 22), as also reflected by intraclass correlation coefficients above 0.79. Protein levels differed between SSP and SGP, but inclusion of the SGP data did not affect the variation of protein levels in sputum. The degree of plasma-protein leakage was estimated from the relative coefficients of excretion (RCE) of alpha 2-macroglobulin and albumin (QA2M/QALB), and of alpha 2-macroglobulin and ceruloplasmin (QA2M/QCP), which do not depend on variable dilution of sputum. Despite the heterogeneity of the study group of 26 patients with asthma (atopic [13] smokers [13], including five patients using inhaled steroids), QA2M/QALB and QA2M/QCP correlated both with bronchial hyperreactivity (Spearman rank: r = -0.45 and r = -0.36, p < 0.05) and with blood eosinophil counts (r = 0.37 and 0.56, p < 0.05). We conclude that protein levels in SSP are relatively constant in patients with stable asthma or COPD; in patients with asthma, the plasma-protein leakage, as measured with the RCE in SSP, appears to correlate with indirect indices of airway inflammation.