Juanita H. J. Vernooy

Breath condenser coatings affect measurement of biomarkers in exhaled breath condensate

Exhaled breath condensate collection is not yet standardised and biomarker measurements are often close to lower detection limits. In the current study, it was hypothesised that adhesive properties of different condenser coatings interfere with measurements of eicosanoids and proteins in breath condensate. In vitro, condensate was derived from a collection model using two test solutions (8-isoprostane and albumin) and five condenser coatings (silicone, glass, aluminium, polypropylene and Teflon). In vivo, condensate was collected using these five coatings and the EcoScreen® condenser to measure 8-isoprostane, and three coatings (silicone, glass, EcoScreen®) to measure albumin. In vitro, silicone and glass coatings had significantly higher albumin recovery compared with the other coatings. A similar trend was observed for 8-isoprostane recovery. In vivo, median (interquartile range) 8-isoprostane concentrations were significantly higher using silicone (9.2 (18.8) pg·mL-1) or glass (3.0 (4.5) pg·mL-1) coating, compared with aluminium (0.5 (2.4) pg·mL-1), polypropylene (0.5 (0.5) pg·mL-1), Teflon (0.5 (0.0) pg·mL-1), and EcoScreen® (0.5 (2.0) pg·mL-1). Albumin in vivo was mainly detectable using glass coating. In conclusion, a condenser with silicone or glass coating is more efficient for measurement of 8-isoprostane or albumin in exhaled breath condensate, than EcoScreen®, aluminium, polypropylene or Teflon. Guidelines for exhaled breath condensate standardisation should include the most valid condenser coating to measure a specific biomarker.

Systemic and local inflammation in asthma and chronic obstructive pulmonary disease: is there a connection?

Increasing evidence indicates that chronic obstructive pulmonary disease (COPD) and probably asthma are associated with low-grade systemic inflammatory changes. In patients with COPD, systemic inflammation is considered a key factor in the pathogenesis of the multicomponent disease manifestations. Spillover of inflammatory mediators into the circulation is generally considered to be the source of this systemic inflammation. Despite this attractive hypothesis, the nature of systemic inflammation in COPD and asthma remains unclear. Available scientific data challenge the spill-over hypothesis. Interventions with biologicals such as TNF-alpha do not modify local or systemic inflammation in these inflammatory respiratory diseases. Adipose tissue-mediated inflammation is discussed as a connecting link of systemic inflammation in asthma and COPD.

Enhanced levels of hyaluronan in lungs of patients with COPD: relationship with lung function and local inflammation

Background: Chronic inflammation and airway remodelling are characteristics of chronic obstructive pulmonary disease (COPD). Hyaluronan (HA) is an extracellular matrix compound with proinflammatory activity. HA levels in induced sputum from patients with COPD were measured and related to local inflammation. The expression of hyaluronan synthase 2 (HAS2) and hyaluronidase 2 (HYAL2) was analysed in lung tissue. Methods: Sputum was obtained from 18 patients with COPD (forced expiratory volume in 1 second (FEV1) 62% predicted (range 20–76)) and 14 healthy smokers. HA and inflammatory markers were measured using ELISA assays. Lung sections were obtained from five patients with severe COPD (FEV1 <30%) and from five smokers, and mRNA levels of HAS2 and HYAL2 were analysed by polymerase chain reaction. Results: HA levels were significantly higher in the sputum from patients with COPD than controls. The COPD population appeared to consist of two subpopulations with either high or moderate HA levels. The subgroup of patients with high HA levels had lower FEV1 than the moderate HA group. In addition, neutrophil influx and levels of interleukin-8, and the soluble tumour necrosis factor receptors R55 and R75 were significantly higher in patients with high HA levels than in those with moderate HA levels and controls. Semiquantitative analysis revealed enhanced expression of HYAL2 in lung tissue of patients with severe COPD compared with control subjects. Conclusion: These data indicate a relationship between HA levels, local inflammation and severity of disease, and suggest enhanced breakdown of HA in the lungs of patients with COPD.

Enhanced pulmonary leptin expression in patients with severe COPD and asymptomatic smokers

Background: Chronic obstructive pulmonary disease (COPD) is characterised by an abnormal inflammatory reaction of the lungs involving activation of epithelial cells. Leptin is a pleiotropic cytokine important in the regulation of immune responses via its functional receptor Ob-Rb. This study was undertaken to test the hypothesis that severe COPD is associated with increased leptin expression in epithelial cells. Methods: Immunohistochemistry for leptin was performed on peripheral lung specimens from 20 patients with COPD (GOLD stage 4), 14 asymptomatic ex-smokers and 13 never smokers. Leptin and Ob-Rb mRNA expression were determined by rtPCR in cultured primary bronchial epithelial cells and primary type II pneumocytes. NCI-H292 and A549 cell lines were used to study functional activation of leptin signalling. Results: Leptin immunoreactivity in lung tissue was observed in bronchial epithelial cells, type II pneumocytes, macrophages (tissue/alveolar) and interstitial lymphocytic infiltrates. rtPCR analysis confirmed pulmonary leptin and Ob-Rb mRNA expression in primary bronchial epithelial cells and pneumocytes. Leptin-expressing bronchial epithelial cells and alveolar macrophages were markedly higher in patients with severe COPD and ex-smokers than in never smokers (p<0.02). Exposure of cultured primary bronchial epithelial cells to smoke resulted in increased expression of both leptin and Ob-Rb (p<0.05). Leptin induced phosphorylation of STAT3 in both NCI-H292 and A549 cells. Conclusions: Leptin expression is increased in bronchial epithelial cells and alveolar macrophages of ex-smokers with or without severe COPD compared with never smokers. A functional leptin signalling pathway is present in lung epithelial cells.

Myeloperoxidase Deficiency Attenuates Lipopolysaccharide-Induced Acute Lung Inflammation and Subsequent Cytokine and Chemokine Production

Lung neutrophilia is common to a variety of lung diseases. The production of reactive oxygen and nitrogen species during neutrophil oxidative burst has been associated with protein and DNA damage. Myeloperoxidase (MPO) is an enzyme stored in the azurophilic granula of neutrophils. It is important in host defense because it generates the reactive oxidant hypochlorous acid and has been described to play a role in the activation of neutrophils during extravasation. We hypothesized that MPO contributes directly to the development of acute lung neutrophilia via stimulation of neutrophil extravasation and indirectly to the subsequent production of cytokines and chemokines in the lung. To test this hypothesis, wild-type (WT) and Mpo−/− mice were given a single LPS instillation, after which the development of neutrophil-dominated lung inflammation, oxidative stress, and cytokine and chemokine levels were examined. Mpo−/− mice demonstrated a decreased lung neutrophilia that peaked earlier than neutrophilia in WT mice, which can be explained by decreased neutrophil chemoattractant levels in LPS-exposed Mpo−/− compared with WT mice. However, oxidative stress levels were not different in LPS-exposed WT and Mpo−/− mice. Furthermore, in vivo findings were confirmed by in vitro studies, using isolated neutrophils. These results indicate that MPO promotes the development of lung neutrophilia and indirectly influences subsequent chemokine and cytokine production by other cell types in the lung.

Obesity Is Associated with Neutrophil Dysfunction and Attenuation of Murine Acute Lung Injury

Although obesity is implicated in numerous health complications leading to increased mortality, the relationship between obesity and outcomes for critically ill patients appears paradoxical. Recent studies have reported better outcomes and lower levels of inflammatory cytokines in obese patients with acute lung injury (ALI)/acute respiratory distress syndrome, suggesting that obesity may ameliorate the effects of this disease. We investigated the effects of obesity in leptin-resistant db/db obese and diet-induced obese mice using an inhaled LPS model of ALI. Obesity-associated effects on neutrophil chemoattractant response were examined in bone marrow neutrophils using chemotaxis and adoptive transfer; neutrophil surface levels of chemokine receptor CXCR2 were determined by flow cytometry. Airspace neutrophilia, capillary leak, and plasma IL-6 were all decreased in obese relative to lean mice in established lung injury (24 h). No difference in airspace inflammatory cytokine levels was found between obese and lean mice in both obesity models during the early phase of neutrophil recruitment (2-6 h), but early airspace neutrophilia was reduced in db/db obese mice. Neutrophils from uninjured obese mice demonstrated diminished chemotaxis to the chemokine keratinocyte cytokine compared with lean control mice, and adoptive transfer of obese mouse neutrophils into injured lean mice revealed a defect in airspace migration of these cells. Possibly contributing to this defect, neutrophil CXCR2 expression was significantly lower in obese db/db mice, and a similar but nonsignificant decrease was seen in diet-induced obese mice. ALI is attenuated in obese mice, and this blunted response is in part attributable to an obesity-associated abnormal neutrophil chemoattractant response.

Inhibition of LPS-induced pulmonary inflammation by specific flavonoids.

In the present study, the anti-inflammatory effects of the flavonoids flavone, fisetin and tricetin were evaluated in a mouse model of LPS-induced acute pulmonary inflammation. The flavonoid fisetin significantly reduced lung myeloperoxidase-levels and gene-expression of inflammatory mediators such as IL-6, TNF-alpha, IL-1beta, MIP-1alpha and MIP-2. The LPS-induced gene transcription of HO-1 and SOD2 was also significantly reduced by fisetin. Overall, the anti-inflammatory effects of fisetin in this in vivo model were much more pronounced as compared to the observed effects of flavone or tricetin and the anti-inflammatory glucocorticoid dexamethasone. The results of this study indicate that flavonoids such as fisetin might be potential candidates as pharmaceuticals or nutraceuticals in the treatment of pulmonary inflammatory diseases.

NF-κB Activation Is Required for the Transition of Pulmonary Inflammation to Muscle Atrophy

Disease exacerbations and muscle wasting comprise negative prognostic factors of chronic obstructive pulmonary disease (COPD). Transient systemic inflammation and malnutrition have been implicated in skeletal muscle wasting after acute exacerbations of COPD. However, the interactions between systemic inflammation and malnutrition in their contributions to muscle atrophy, as well as the molecular basis underlying the transition of systemic inflammation to muscle atrophy, remain unresolved. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS to model acute disease exacerbation. Systemic inflammation, nutritional intake, and body and muscle weights were determined. Muscle inflammatory signaling and atrophy signaling were examined, and the effect of the muscle-specific inactivation of NF-κB on muscle atrophy was assessed in genetically modified mice. The intratracheal LPS instillation was followed by markedly elevated circulating cytokine concentrations and NF-κB activation in extrapulmonary tissues, including skeletal muscle. The administration of intratracheal LPS increased the expression of muscle E3 ubiquitin ligases, which govern muscle proteolysis, in particular MuRF1, and caused a rapid loss of muscle mass. Reduced food intake only partly accounted for the observed muscle atrophy, and did not activate NF-κB in muscle. Rather, plasma transfer experiments revealed the presence of NF-κB-signaling and atrophy-signaling properties in the circulation of intratracheal LPS-treated mice. The genetic inhibition of muscle NF-κB activity suppressed intratracheal LPS-induced MuRF1 expression and resulted in a significant sparing of muscle tissue. Systemic inflammation and malnutrition contribute to the muscle wasting induced by acute pulmonary inflammation via distinct mechanisms, and muscle NF-κB activation is required for the transition from inflammatory to muscle atrophy signaling.

Leptin Modulates Innate and Adaptive Immune Cell Recruitment after Cigarette Smoke Exposure in Mice

Leptin, a pleiotropic type I cytokine, was recently demonstrated to be expressed by resident lung cells in chronic obstructive pulmonary disease patients and asymptomatic smokers. To elucidate the functional role of leptin in the onset of chronic obstructive pulmonary disease, we tested leptin-deficient ob/ob mice (C57BL/6), leptin receptor-deficient db/db mice (C57BKS), and littermates in a model of cigarette smoke (CS)-induced pulmonary inflammation. Wild-type (WT) C57BL/6 mice were exposed for 4 or 24 wk to control air or CS. Pulmonary leptin expression was analyzed by immunohistochemistry and real-time PCR. Pulmonary inflammation upon 4 wk CS exposure was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue of WT, ob/ob, and db/db mice. Immunohistochemical analysis revealed leptin expression in bronchial epithelial cells, pneumocytes, alveolar macrophages, and bronchial/vascular smooth muscle cells. The 4 and 24 wk CS exposure increased leptin expression in bronchial epithelial cells and pneumocytes versus air-exposed WT mice (p < 0.05). The 4 wk CS exposure resulted in increased accumulation of neutrophils, dendritic cells, macrophages, and lymphocytes in BALF and lung tissue of WT, ob/ob, and db/db mice. CS-exposed ob/ob and db/db mice showed in general higher numbers of neutrophils and lower numbers of CD4+, CD8+, and dendritic cells versus CS-exposed WT mice. Consistently, CXCL1 levels were enhanced in BALF of CS-exposed ob/ob and db/db mice versus WT mice (p < 0.05). Exogenous leptin administration completely restored the skewed inflammatory profile in ob/ob mice. These data reveal an important role of leptin in modulating innate and adaptive immunity after CS inhalation in mice.

Enhanced deposition of low-molecular-weight hyaluronan in lungs of cigarette smoke-exposed mice

Chronic obstructive pulmonary disease (COPD) is characterized by infiltration of inflammatory cells, destruction of lung parenchyma, and airway wall remodeling. Hyaluronan (HA) is a component of the extracellular matrix, and low-molecular-weight (LMW) HA fragments have proinflammatory capacities. We evaluated the presence of HA in alveolar and airway walls of C57BL/6 mice that were exposed to air or cigarette smoke (CS) for 4 weeks (subacute) or 24 weeks (chronic). We measured deposition of the extracellular matrix proteins collagen and fibronectin in airway walls and determined the molecular weight of HA purified from lung tissue. In addition, we studied the expression of HA-modulating genes by RT-PCR. HA staining in alveolar walls was significantly enhanced upon chronic CS exposure, whereas HA levels in the airway walls were already significantly higher upon subacute CS exposure and remained elevated upon chronic CS exposure. This differed from the deposition of collagen and fibronectin, which are only elevated at the chronic time point. In lungs of CS-exposed mice, the molecular weight of HA clearly shifted toward more LMW HA fragments. CS exposure significantly increased the mRNA expression of the HA synthase gene Has3 in total lung tissue, whereas the expression of Has1 was decreased. These in vivo studies in an experimental model of COPD show that CS exposure leads to enhanced deposition of (mostly LMW) HA in alveolar and bronchial walls by altering the expression of HA-modulating enzymes. This may contribute to airway wall remodeling and pulmonary inflammation in COPD.