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Background: Lung epithelial lining fluid (ELF)-sampled through sputum induction-is a medium rich in cells, proteins and lipids. However, despite its key role in maintaining lung function, homeostasis and defences, the composition and biology of ELF, especially in respect of lipids, remain incompletely understood., Objectives: To characterise the induced sputum lipidome of healthy adult individuals, and to examine associations between different ELF lipid phenotypes and the demographic characteristics within the study cohort., Methods: Induced sputum samples were obtained from 41 healthy non-smoking adults, and their lipid compositions analysed using a combination of untargeted shotgun and liquid chromatography mass spectrometry methods. Topological data analysis (TDA) was used to group subjects with comparable sputum lipidomes in order to identify distinct ELF phenotypes., Results: The induced sputum lipidome was diverse, comprising a range of different molecular classes, including at least 75 glycerophospholipids, 13 sphingolipids, 5 sterol lipids and 12 neutral glycerolipids. TDA identified two distinct phenotypes differentiated by a higher total lipid content and specific enrichments of diacyl-glycerophosphocholines, -inositols and -glycerols in one group, with enrichments of sterols, glycolipids and sphingolipids in the other. Subjects presenting the lipid-rich ELF phenotype also had significantly higher BMI, but did not differ in respect of other demographic characteristics such as age or gender., Conclusions: We provide the first evidence that the ELF lipidome varies significantly between healthy individuals and propose that such differences are related to weight status, highlighting the potential impact of (over)nutrition on lung lipid metabolism.

The adverse effects of petrodiesel exhaust exposure on the cardiovascular and respiratory systems are well recognized. While biofuels such as rapeseed methyl ester (RME) biodiesel may have ecological advantages, the exhaust generated may cause adverse health effects. In the current study, we investigated the responses of bioactive lipid mediators in human airways after biodiesel exhaust exposure using lipidomic profiling methods. Lipid mediator levels in lung lavage were assessed following 1-h biodiesel exhaust (average particulate matter concentration, 159 μg/m 3 ) or filtered air exposure in 15 healthy individuals in a double-blinded, randomized, controlled, crossover study design. Bronchoscopy was performed 6 h post exposure and lung lavage fluids, i.e., bronchial wash (BW) and bronchoalveolar lavage (BAL), were sequentially collected. Mass spectrometry methods were used to detect a wide array of oxylipins (including eicosanoids), endocannabinoids, N-acylethanolamines, and related lipid metabolites in the collected BW and BAL samples. Six lipids in the human lung lavage samples were altered following biodiesel exhaust exposure, three from BAL samples and three from BW samples. Of these, elevated levels of PGE 2 , 12,13-DiHOME, and 13-HODE, all of which were found in BAL samples, reached Bonferroni-corrected significance. This is the first study in humans reporting responses of bioactive lipids following biodiesel exhaust exposure and the most pronounced responses were seen in the more peripheral and alveolar lung compartments, reflected by BAL collection. Since the responsiveness and diagnostic value of a subset of the studied lipid metabolites were established in lavage fluids, we conclude that our mass spectrometry profiling method is useful to assess effects of human exposure to vehicle exhaust.

Metabolomics protocols are used to comprehensively characterize the metabolite content of biological samples by exploiting cutting-edge analytical platforms, such as gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) assays, as well as nuclear magnetic resonance (NMR) assays. We have developed novel sample preparation procedures combined with GC-MS, LC-MS, and NMR metabolomics profiling for analyzing bronchial wash (BW) and bronchoalveolar lavage (BAL) fluid from 15 healthy volunteers following exposure to biodiesel exhaust and filtered air. Our aim was to investigate the responsiveness of metabolite profiles in the human lung to air pollution exposure derived from combustion of biofuels, such as rapeseed methyl ester biodiesel, which are increasingly being promoted as alternatives to conventional fossil fuels. Our multi-platform approach enabled us to detect the greatest number of unique metabolites yet reported in BW and BAL fluid (82 in total). All of the metabolomics assays indicated that the metabolite profiles of the BW and BAL fluids differed appreciably, with 46 metabolites showing significantly different levels in the corresponding lung compartments. Furthermore, the GC-MS assay revealed an effect of biodiesel exhaust exposure on the levels of 1-monostearylglycerol, sucrose, inosine, nonanoic acid, and ethanolamine (in BAL) and pentadecanoic acid (in BW), whereas the LC-MS assay indicated a shift in the levels of niacinamide (in BAL). The NMR assay only identified lactic acid (in BW) as being responsive to biodiesel exhaust exposure. Our findings demonstrate that the proposed multi-platform approach is useful for wide metabolomics screening of BW and BAL fluids and can facilitate elucidation of metabolites responsive to biodiesel exhaust exposure. Graphical Abstract Graphical abstract illustrating the study workflow. NMR Nuclear Magnetic Resonance, LC-TOFMS Liquid chromatography-Time Of Flight Mass Spectrometry, GC Gas Chromatography-Mass spectrometry.

Introduction: Systemic sclerosis (SSc) is an autoimmune disorder, which frequently affects the lungs, with manifestations of interstitial lung disease (ILD) with lung fibrosis and of pulmonary hypertension. The pathogenesis remains largely unrecognised., Objective: The aim of this study was to elucidate the inflammation in the bronchial mucosa in patients with SSc., Subjects and Methods: Twenty-three subjects diagnosed with SSc participated. Twelve of the SSc patients showed signs of ILD, four were smokers and seven were treated with oral corticosteroids. Seventeen non-smoking, age- and sex-matched healthy subjects served as controls. Bronchoscopy was performed to sample endobronchial mucosal biopsies, which were immunohistochemically stained using a panel of antibodies against inflammatory markers., Results: The number of neutrophils was significantly elevated in the submucosa of SSc patients, regardless of ILD, or whether the subject was smoking or using oral corticosteroids. No up-regulation of neutrophil chemoattractants or cytokines was seen in the bronchial epithelium. The signal transduction pathways and adhesion molecule expression tended to be suppressed or unchanged in SSc patients compared with controls., Conclusion: It is concluded that SSc is associated with a chronic neutrophilic inflammation in the bronchial mucosal, with signs of suppressed signal transduction, regardless of the presence of interstitial lung disease.

Fluoride has been in focus as a possible causal agent for respiratory symptoms amongst aluminium potroom workers for several decades. Previously, using bronchoalveolar lavage (BAL), we demonstrated airway inflammation in healthy volunteers 24 hours after exposure to hydrogen fluoride (HF). The objective of the present study was to examine early lung responses to HF exposure. Bronchoscopy with BAL was performed 2 hours after the end of 1-hour exposure to HE Significant reductions in the total cell number and the number of neutrophils and lymphocytes were observed in bronchoalveolar portion (BAP), whereas there were no significant changes in the bronchial portion (BP). Significantly decreased concentrations of beta2-MG, IL-6 and total protein were found in both BAP and BP. Additionally, IL-8 was significantly reduced in BP, and ICAM-1 and albumin were present in lower concentrations in BAP. Lung function measurements were not affected by HF exposure. These reported effects are presumably transitory, as many were not present in the airways 24 hours after a similar HF exposure.

Extracellular-superoxide dismutase (EC-SOD) exists primarily in the tissue interstitium and the lung contains particularly large amounts of the enzyme. To determine the roles of EC-SOD and extracellularly formed superoxide radicals in the pulmonary response to the common air pollutant ozone, wild-type mice and mice lacking EC-SOD were exposed to 1.5 ppm ozone for 48 h. The exposure resulted in a marked neutrophilic inflammatory reaction observed both in the bronchoalveolar lavage fluid (BALF) and by histopathology of the lungs, which was much stronger in the mice lacking EC-SOD. Unlike the wild-type mice, the null mutants also showed increased levels of interleukin-6 in the BALF. The ozone exposure also resulted in increased airway mucosal permeability and cell damage as indicated by increased protein and lactate dehydrogenase in the BALF. There was, however, no difference between the two groups of mice.The results suggest that extracellular superoxide radicals are important inflammatory mediators in the pulmonary response to ozone, but in the present model, the radical and the infiltrating neutrophils contributed little to the pulmonary injury The data, together with previous findings, support a role for EC-SOD as a modulator of inflammatory reactions.

Short-term exposure to ozone at peak ambient levels induces neutrophil influx and impairs lung function in healthy humans. In order to investigate the mechanisms contributing to neutrophil recruitment and to examine the role of T-cells in the acute inflammatory response, we exposed 12 healthy humans to 0.2 parts per million (ppm) of ozone and filtered air on two separate occasions for 2 h with intermittent periods of rest and exercise (minute ventilation = 30 L x min(-1)). Fibreoptic bronchoscopy was performed 6 h after the end of exposures. Total protein, tryptase, histamine, myeloperoxidase, interleukin (IL)-8 and growth-related oncogene-alpha (Gro-alpha) were measured and total and differential cell counts were performed in bronchoalveolar lavage (BAL) fluid. Flow cytometry was performed on BAL cells to study total T-cells, T-cell receptors (alphabeta and gammadelta), T-cell subsets (CD4+ and CD8+ cells) and activated T-cell subsets (CD25+). Using immunohistochemistry, neutrophils, mast cells, total T-cell numbers, T-cell subsets, CD25+ T-cells and leukocyte endothelial adhesion molecules including P-selectin, E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 were quantified in the bronchial biopsies. Paired samples were available from nine subjects. Following ozone exposure there was a threefold increase in the proportion of polymorphonuclear neutrophils (PMNs) (p=0.07) and epithelial cells (p=0.05) in BAL fluid. This was accompanied by increased concentrations of IL-8 (p=0.01), Gro-alpha (p=0.05) and total protein (p=0.058). A significant positive correlation was demonstrated between the two chemokines and proportion of PMNs in BAL fluid. After ozone exposure there was a significant decrease in the CD4/CD8 ratio (p=0.05) and the proportion of activated CD4+ (p=0.01) and CD8+ T-cells (p=0.04). However, no significant changes were demonstrable in any of the inflammatory markers studied in the biopsies. Short-term exposure of healthy humans to 0.2 ppm ozone induced a neutrophil influx in peripheral airways at 6 h post exposure, but no apparent inflammatory response in proximal airways. This response seems to be mediated at least in part by interleukin-8 and growth-related oncogene-alpha.

Eight healthy volunteers were exposed to purified lipopolysaccharide (LPS) by controlled inhalation. Bronchoalveolar lavage (BAL) 3 hours after exposure revealed a pronounced neutrophilia, increase in lymphocytes, fibronectin concentration, and decrease in alveolar macrophage phagocytosis, as compared to a reference BAL.

Various lines of evidence suggest a respiratory route of transmission of nephropathia epidemica (NE). To study the response of the respiratory tract in NE, fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) was performed in 5 patients in the acute phase of the disease. Compared to a reference group of 15 healthy individuals, BAL fluid of NE patients contained significantly higher total numbers of cells (p < 0.05) and significantly higher numbers of lysozyme-positive macrophages (p < 0.01), CD8+ T cells (p < 0.01), and natural killer (NK) cells (p < 0.01). There was no significant difference in numbers of CD4+ T cells, B cells, or neutrophils. When blood samples of 16 patients were examined at various intervals after onset of NE, a significant decrease in the number of NK cells (p < 0.01) was found in the acute phase of the disease. The findings are compatible with the presence of a local host response in the lower respiratory tract to NE virus infection.

Following the basal descriptive studies of the bronchoalveolar inflammatory cell response induced by single exposure with nitrogen dioxide (NO2) in man, it was considered important to clarify the cell response to repeated exposure with NO2. This investigation was, therefore, undertaken with bronchoalveolar lavage (BAL) 3 weeks before and 24 h after six repeated exposures with 4 ppm NO2 (7 mg.m-3) in ten healthy volunteers. The exposures were performed during 20 min and every second day. Analysis of the recovered BAL fluid demonstrated that repeated exposures to NO2 caused a lung cell response different from that reported after a single exposure. Amounts of lavaged alveolar macrophages, B-cells, and natural killer (NK)-cells were decreased and the T-helper-inducer/cytotoxic-suppressor cell ratio was altered, but there was no lymphocytosis or mastocytosis as after single exposure. Lymphocyte numbers in peripheral blood were reduced after exposure. These results suggest that repeated exposure with NO2 adversely affects the immune defence. This could contribute to the increased susceptibility to airway infections reported to be associated with NO2 exposure.

Bacterial endotoxin has been suggested as responsible for the development of subjective symptoms and transient or chronic lung function impairment seen after exposure to organic dusts in cotton mills, poultry houses, swine confinement buildings and saw mills. Animal experiments have demonstrated bronchoalveolar neutrophilia being the most prominent cell response in animals following bacterial lipopolysaccharide (LPS) inhalation. The present study was conducted to obtain information on some aspects of the early inflammatory response to inhaled LPS in man. Eight healthy nonsmoking subjects, 23-27 yrs old, underwent bronchoalveolar lavage (BAL), 3 h after a provocation test with 100 micrograms LPS from E. coli dissolved in 2 ml isotonic NaCl. The solution was aerosolized with a jet nebulizer and inhaled. The calculated dose delivered to the lung was approximately 25 micrograms, which equals exposure in some occupational settings. The BAL data for each individual subject were compared with data from a control BAL performed at least 6 weeks prior to the LPS challenge. The major cellular response to LPS, reflected in BAL fluid, was an approximately hundredfold increase in neutrophils. The total number of lymphocytes was on average tripled. The alveolar macrophage phagocytosis of opsonized yeast particles in vitro was significantly reduced. A further indicator of an ongoing inflammation was an increase in fibronectin. No changes were seen in the levels of BAL albumin, indicating that the elevated level of fibronectin could not be explained by an increased permeability, but rather by a local production. The results correspond with data from animal studies and further supports the hypothesis that bacterial LPS is important in the pulmonary reaction induced by organic dusts.

Bronchoalveolar lavage (BAL) fluid was analysed from 21 patients with breast cancer, stage T1N0M0, who had undergone tumour resection and post-operative local irradiation (accumulated dose 56 Gy). The lavage was performed two months after radiotherapy, in the anterior part of the lingula (left side) or of the right middle lobe (right side), depending on which side had been exposed to radiation. The patients had significantly increased concentrations of fibronectin (FN) (p less than 0.001), hyaluronan (HA) (p less than 0.01) and albumin (p less than 0.05) in BAL fluid compared with the healthy controls (n = 19). However, when the patients were separated, according to smoking history, it was obvious that the inflammatory reaction occurred entirely in the nonsmoking patient group (n = 10), whilst no difference could be found between the smoking patients (n = 11) and the controls. In the nonsmoking patient group, there was a sevenfold increase in BAL concentrations of FN and a threefold increase in HA. Moreover, four patients had detectable levels of procollagen III peptide in BAL, all were nonsmokers. The smoking habits of the controls had no influence on the BAL measurements. These findings indicate that smoking interferes with the radiation-induced early inflammatory connective tissue reaction of the lung. Finally, the results justify further investigation of interaction of smoking with cancer treatment, both from the view of therapy effectiveness and reduction of adverse effects.

Mechanised production of peat for fuel consumption is associated with high concentrations of organic dust, which is inhaled by the peat workers. In the present study 17 workers at two peat bogs in northern Sweden were examined. Personal sampling of total dust and the respirable fraction was performed during several workshifts. Dynamic spirometry was carried out before and at the end of shifts. Bronchoscopy with bronchoalveolar lavage (BAL) was performed in six subjects at the end of the working season and the results were compared with unexposed reference subjects. Peat workers using modern machines with ventilated cabins containing air filters were found to be exposed to low concentrations of peat dust. The recorded dust concentrations were below the threshold limit value for organic dust (5 mg/m3 air) in all but one worker. The respirable fraction of peat dust recorded in the breathing zone of the workers correlated significantly with a decrease in forced expiratory volume in one second (FEV1). The effect on lung function in non-asthmatic peat workers was, however, small. The concentration of lysozyme positive alveolar macrophages in BAL fluid was significantly lower in the peat workers compared with reference subjects. An inverse correlation was found between the mentioned cells and exposure to the respirable fraction of the peat dust. Furthermore, one particularly dust exposed worker had pronounced increases in alveolar macrophages, fibronectin concentration, and mast cells in BAL fluid.

The combination of environmental chamber exposure and bronchoalveolar lavage (BAL) was used to study the effects of the common air pollutant nitrogen dioxide (NO2). Eighteen healthy nonsmokers were exposed to NO2 during 20 min in an exposure chamber during light bicycle ergometer work. All subjects were examined with BAL at least 3 wks before exposure, as a reference. The subjects were re-examined with BAL, in groups of eight, 24 h after exposure to 4, 7 and 10 mg NO2.m.3 (2.25, 4.0 and 5.5 ppm), respectively. An inflammatory cell response was found after exposure to all concentrations. An increase in the number of lymphocytes in BAL fluid was observed after 7 and 10 mg.m.3 (p less than 0.05 and 0.02, respectively). An increase in the number of mast cells, that appears to be dose-dependent, was found after exposure to all concentrations. The proportion of lysozyme positive alveolar macrophages was elevated after exposure to 7 mg.m.3. The inflammatory mediators fibronectin, hyaluronan, angiotensin converting enzyme (ACE) and beta 2-microglobulin were unchanged by exposure. Due to the findings of inflammatory cell changes far below the peak exposure limits for work places in industrialized countries, 9-18 mg.m.3, the safety of these limits is questioned. Studies are in progress in our laboratory using BAL to evaluate the effects of repeated NO2 exposure.

The combination of environmental chamber exposure and bronchoalveolar lavage (BAL) was used to study the time-course of the cell response in the human lung to nitrogen dioxide (NO2). Healthy subjects were exposed for 20 min to 7 mg NO2.m-3 (4 ppm), a concentration which occurs indoors in industries and is below the peak exposure limit for work places in most countries, 10 mg.m-3 (5.5 ppm). BAL was performed in all subjects several weeks before exposure and 4, 8, 24 and 72 h after exposure, in eight subjects at each time. Mastocytosis and lymphocytosis were found in BAL fluid 4-24 h after exposure, with normalization after 72 h. A mild increase in lysozyme positive macrophages was found 24-72 h after exposure. The time-course of the human pulmonary cell response to NO2, demonstrated in BAL fluid, represents a new and previously not reported finding after exposure to this common air pollutant. Our findings are diverging from results obtained in animal studies, using approximately the same NO2 concentrations, indicating that the results from the animal studies may not be transferable to man.

Environmental chamber exposure and bronchoalveolar lavage (BAL) were used to study the dose-response relationship between short-term exposure to sulfur dioxide (SO2) and inflammatory reactions in the human lung as reflected in BAL fluid. Healthy subjects were exposed to 10, 13, 20, or 30 mg/m3 for 20 min. BAL was performed several weeks preexposure and 24 h postexposure. Mast cells, lymphocytes, lysozyme positive macrophages, and the total number of macrophages were significantly increased after SO2 exposure. A dose-dependent increase in the cell response in BAL fluid was observed after exposure to 10-20 mg/m3, but no further increase was detected after 30 mg/m3. Inflammatory cell response was found in BAL fluid at SO2 levels that occur in industrial indoor environments worldwide, and cell response to SO2 was also seen below the short-term exposure limit of Sweden and many other countries (13 mg/m3).

Bronchoalveolar lavage (BAL) has been performed in 22 healthy nonsmoking male volunteers after exposure to 8 ppm SO2 (20 mg/m3). The exposure level exceeds the US Short-Term Exposure Limit (STEL) of 5 ppm, but occurs as peak exposures in industrial indoor environments. Exposures were made during light work on a bicycle ergometer in an environmental exposure chamber for 20 min. BAL was performed 2 wk or more before exposure and 4, 8, 24, and 72 h after exposure in eight subjects at each time interval. Four hours after exposure significant increases were found in the numbers of lysozyme-positive macrophages, lymphocytes, and mast cells (p less than 0.02 to 0.05). Lymphocytes, lysozyme-positive macrophages, total count of alveolar macrophages, and total cell number increased to peak values 24 h after exposure (p less than 0.02 to 0.05). Seventy-two hours after exposure the cell numbers and distribution had returned to normal. The time course of the cell reactions found in BAL fluid after controlled SO2 exposure represents a new and previously not reported response to a noxious gas.

Bronchoalveolar lavage (BAL) which has not previously been used in investigating the effect of sulphur dioxide (SO2) on the human lung was performed on 12 subjects before and after controlled chamber exposure with SO2 for 20 minutes. BAL fluid 24 hours after exposure with 10 mg SO2/m3 (4 ppm, 10 subjects) showed increased alveolar macrophage activity as judged by an increase in lysozyme positive macrophages. Twenty four hours after 20 mg/m3 (4 subjects) a further increase was seen, which was accompanied by an increase in total numbers of macrophages and lymphocytes. Seventy two hours after exposure (4 subjects) cell numbers had virtually returned to pre-exposure levels. These previously uninvestigated reactions indicate potentially noxious effects of SO2 in the lungs at exposure levels that are regarded as relatively safe.