Marco van der Toorn

Lipid-soluble components in cigarette smoke induce mitochondrial production of reactive oxygen species in lung epithelial cells

Reactive oxygen species (ROS) present in cigarette smoke (CS) are thought to contribute to the development of COPD. Although CS-ROS can hardly enter airway epithelial cells, and certainly not the circulation, systemic levels of ROS have been found to be elevated in COPD patients. We hypothesize that lipophilic components present in CS can enter airway epithelial cells and increase intracellular ROS production by disturbing mitochondrial function. Different airway epithelial cells were exposed to CS extract (CSE), hexane-treated CSE (CSE without lipophilic components), gaseous-phase CS, and water-filtered CS (gaseous-phase CS without ROS). Mitochondrial membrane potential (Deltapsi(m)) and ATP levels were assessed using the bronchial epithelial cell line Beas-2b. ROS generation measured directly by DCF fluorescence and indirectly by measuring free thiol groups (-SH) upon exposure to CS was assessed using lung alveolar epithelial cells devoid of functional mitochondria (A549-rho0), with normal A549 cells serving as controls. In Beas-2b cells, CSE (4 h) caused a dose-dependent decrease in Deltapsi(m) and ATP levels, whereas hexane-treated CSE did not. DCF fluorescence in A549 cells increased in response to CSE, whereas this was not the case in A549-rho0 cells. Exposure of A549 cells to CS resulted in a rapid decrease in free -SH, whereas exposure to ROS-depleted CS only resulted in a delayed decrease. This delayed decrease was less pronounced in A549-rho0 cells. Lipophilic components in CS disturb mitochondrial function, which contributes to increased intracellular generation of ROS. Our results are of importance in understanding the systemic effects of smoking observed in patients with COPD.

Change in inflammation in out-patient COPD patients from stable phase to a subsequent exacerbation

Background Inflammation increases during exacerbations of COPD, but only a few studies systematically assessed these changes. Better identification of these changes will increase our knowledge and potentially guide therapy, for instance by helping with quicker distinction of bacterially induced exacerbations from other causes. Aim To identify which inflammatory parameters increase during COPD exacerbations compared to stable disease, and to compare bacterial and non-bacterial exacerbations. Methods In 45 COPD patients (37 male/8 female, 21 current smokers, mean age 65, FEV1 52% predicted, pack years 38) sputum was collected during a stable phase and subsequently during an exacerbation. Results Sputum total cell counts (9.0 versus 7.9 × 106/mL), eosinophils (0.3 versus 0.2 × 106/mL), neutrophils (6.1 versus 5.8 × 106/mL), and lymphocytes (0.07 versus 0.02 × 106/mL) increased significantly during an exacerbation compared to stable disease. A bacterial infection was demonstrated by culture in 8 sputum samples obtained during an exacerbation. These exacerbations had significantly increased sputum total cell and neutrophil counts, leukotriene-B4, myeloperoxidase, interleukin-8 and interleukin-6, and tumor necrosis factor-α (TNF-α) levels, and were also associated with more systemic inflammation compared to exacerbations without a bacterial infection. Sputum TNF-α level during an exacerbation had the best test characteristics to predict a bacterial infection. Conclusion Sputum eosinophil, neutrophil, and lymphocyte counts increase during COPD exacerbations. The increase in systemic inflammation during exacerbations seems to be limited to exacerbations caused by bacterial infections of the lower airways. Sputum TNF-α is a candidate marker for predicting airway bacterial infection.

Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype

BackgroundA major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.MethodsTo this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.ResultsBoth CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.ConclusionsDirect exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.

Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells

•  What is the central question of this study? The endoplasmic reticulum stress response caused by cigarette smoke may lead to excessive apoptosis with disruption of the epithelial barrier, thus contributing to chronic obstructive pulmonary disease. One way of promoting cell survival is to facilitate degradation of cigarette smoke‐induced protein damage through the ubiquitin–proteasome pathway. Direct effects of gas‐phase cigarette smoke on proteasomal activities have not been demonstrated previously. •  What is the main finding and what is its importance? We show that cigarette smoke induces protein damage and triggers the endoplasmic reticulum stress response in human alveolar epithelial cells. A significant reduction of all three proteasomal activities was found. Ineffective degradation of damaged proteins could lead to a sustained epithelial stress response and development of chronic obstructive pulmonary disease.

Cigarette Smoke-Induced Damage-Associated Molecular Pattern Release from Necrotic Neutrophils Triggers Proinflammatory Mediator Release

Cigarette smoking, the major causative factor for the development of chronic obstructive pulmonary disease, is associated with neutrophilic airway inflammation. Cigarette smoke (CS) exposure can induce a switch from apoptotic to necrotic cell death in airway epithelium. Therefore, we hypothesized that CS promotes neutrophil necrosis with subsequent release of damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), alarming the innate immune system. We studied the effect of smoking two cigarettes on sputum neutrophils in healthy individuals and of 5-day CS or air exposure on neutrophil counts, myeloperoxidase, and HMGB1 levels in bronchoalveolar lavage fluid of BALB/c mice. In human peripheral blood neutrophils, mitochondrial membrane potential, apoptosis/necrosis markers, caspase activity, and DAMP release were studied after CS exposure. Finally, we assessed the effect of neutrophil-derived supernatants on the release of chemoattractant CXCL8 in normal human bronchial epithelial cells. Cigarette smoking caused a significant decrease in sputum neutrophil numbers after 3 hours. In mice, neutrophil counts were significantly increased 16 hours after repeated CS exposure but reduced 2 hours after an additional exposure. In vitro, CS induced necrotic neutrophil cell death, as indicated by mitochondrial dysfunction, inhibition of apoptosis, and DAMP release. Supernatants from CS-treated neutrophils significantly increased the release of CXCL8 in normal human bronchial epithelial cells. Together, these observations show, for the first time, that CS exposure induces neutrophil necrosis, leading to DAMP release, which may amplify CS-induced airway inflammation by promoting airway epithelial proinflammatory responses.

Critical role of aldehydes in cigarette smoke-induced acute airway inflammation.

BackgroundCigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation.MethodsBALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs.ResultsCompared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein.ConclusionAldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

Poly(ethylene glycol)-Based Stable Isotope Labeling Reagents for the Quantitative Analysis of Low Molecular Weight Metabolites by LC−MS

Stable isotope labeling (SIL) in combination with liquid chromatography-mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of 13C-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of 13C atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C18 reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities.

Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition.

Cyclosporin A‐induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (ΔΨm) and the production of reactive oxygen species. Fluorescent probes were used to assess ΔΨm (JC‐1, 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzimidazolyl‐carbocyanine iodide), reactive oxygen species [DCF, 5‐ (and 6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo‐3, glycine N‐[4‐[6‐[(acetyloxy)methoxy]‐2,7‐dichloro‐3‐oxo‐3H‐xanthen‐9‐yl]‐2‐[2‐[2‐[bis[2‐[(acetyloxy)methoxy]‐2‐oxyethyl]amino]‐5‐methylphenoxy]ethoxy]phenyl]‐N‐[2‐[(acetyloxy)methoxy]‐2‐oxyethyl]‐(acetyloxy)methyl ester] in human kidney cells (HK‐2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A‐sensitive P‐glycoprotein. We used transfected GLC4 cells expressing P‐glycoprotein as control for GLC4 cells. NIM811 (N‐methyl‐4‐isoleucine‐cyclosporin) and PSC833 (SDZ‐PSC833) were applied as selective mitochondrial permeability transition pore and P‐glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two‐fold increase in JC‐1 fluorescence in HK‐2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC‐1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5‐fold increase in DCF fluorescence (P < 0.05) and a two‐fold increase in Fluo‐3 fluorescence (P < 0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither ΔΨm, ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non‐mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P‐glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A‐induced oxidative stress and disturbed Ca2+ homeostasis.

Metalloproteinase Profiling in Lung Transplant Recipients With Good Outcome and Bronchiolitis Obliterans Syndrome

Background Bronchiolitis obliterans syndrome (BOS), the major cause of death on lung transplantation, is characterized by bronchiolar inflammation and tissue remodeling. Matrix metalloproteinases (MMPs) have been implicated in these processes, although it is still unclear whether MMP activity and binding to their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is abnormal in BOS. Methods We studied total MMP-1,-2,-3,-7,-8,-9,-12,-13 levels, their activity state using activity-based extraction and their binding to TIMP-1, ‐2, ‐3, and ‐4 in bronchoalveolar lavage (BAL) of lung transplant recipients with good outcome and BOS using a multiplex immunoassay. Results The BAL levels of TIMP-1 and ‐2 and MMP-2, ‐3, ‐7, ‐8, and ‐9 were significantly increased in BOS compared to good outcome recipients. Interestingly, activity of MMP-7, but none of the other MMPs, was detected in good outcome recipients, whereas no active MMPs were observed in BOS recipients. However, BAL levels of TIMP-bound MMP-8 and ‐9 were higher in BOS than in good outcome recipients, suggesting activity of these MMPs in an earlier stage. Conclusions We demonstrate that development of BOS is associated with increased levels of TIMP-1 and ‐2 and total MMP-2, ‐3, ‐7, ‐8, and ‐9. Although active MMP-7 was only observed in good outcome recipients, levels of TIMP-bound MMP-8 and ‐9 were higher in BOS. By enabling profiling of active and TIMP-bound MMPs, our novel method may open opportunities for the screening of early predictors for BOS.