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.

Expression of ADAMs ("a disintegrin and metalloprotease") in the human lung.

In view of the associations of "a disintegrin and metalloprotease" (ADAM) with respiratory diseases, we assessed the expression of various ADAMs in human lung tissue. Lung tissue was obtained from nine individuals who underwent surgery for lung cancer or underwent lung transplantation for emphysema. Also, 16HBE 14o- (human bronchial epithelial) and A549 (alveolar type II epithelium-like) cell lines were used. Immunohistochemistry was performed with antibodies recognizing different ADAM domains. The ADAMs were typically distributed over the bronchial epithelium. ADAM8 and ADAM10 were expressed diffusely in all layers of the epithelium. ADAM9, ADAM17, and ADAM19 were predominantly expressed in the apical part of the epithelium, and ADAM33 was predominantly and strongly expressed in basal epithelial cells. In smooth muscle, ADAM19 and ADAM17 were strongly expressed, as was ADAM33, though this expression was weaker. ADAM33 was strongly expressed in vascular endothelium. All ADAMs were generally expressed in inflammatory cells. The typical distribution of ADAMs in the lung, especially in the epithelium, is interesting and suggests a localized function. As most ADAMs are involved in release of (pro-) inflammatory mediators and growth factors, they may play an important role in the first line of defense and in initiation of repair events in the airways.

Anti-inflammatory effects of combined budesonide/formoterol in COPD exacerbations.

Systemic corticosteroids and additional short-acting beta2-agonists are commonly used in exacerbations of chronic obstructive pulmonary disease (COPD). In this double-blind study, the combination of a high-dose inhaled corticosteroid with a rapid-onset long-acting beta2-agonist was evaluated in the treatment of out-patient COPD exacerbations. The primary aim was to compare 14-day treatment effects of budesonide/formoterol to placebo on sputum eosinophils and, secondarily, on other indices of inflammation, forced expiratory flow in one second (FEV(1)), symptoms, health status, and adverse events. Forty-five patients not using steroids (37 male, 21/24 current/ex smoker, median packyears 38, age 65 years, FEV(1) 61% predicted), experiencing a COPD exacerbation, were treated at home with budesonide/formoterol (320/9 microg 4 times daily), prednisolone (30 mg daily), or placebo for 14 days. Sputum eosinophils were significantly reduced by budesonide/formoterol (-57%) compared to placebo (+24%) (p = 0.01). Budesonide/formoterol reduced total symptom scores significantly (p = 0.01) compared to placebo. The increase in FEV(1) by 2 weeks of treatment with budesonide/formoterol (125 ml) was not significantly different from that of placebo (43 ml) (p = 0.07). Budesonide/ formoterol treatment did not suppress morning serum cortisol compared to placebo (-16%; p = 0.50). In conclusion, budesonide/formoterol reduces sputum eosinophils and improves symptoms in the treatment of out-patient COPD exacerbations.

Carbon monoxide blocks oxidative stress-induced hepatocyte apoptosis via inhibition of the p54 JNK isoform.

Most chronic liver diseases are accompanied by oxidative stress, which may induce apoptosis in hepatocytes and liver injury. Oxidative stress induces heme oxygenase-1 (HO-1) expression. This stress-responsive cytoprotective protein is responsible for heme degradation into carbon monoxide (CO), free iron, and biliverdin. CO is an important intracellular messenger; however, the exact mechanisms responsible for its cytoprotective effect are not yet elucidated. Thus, we investigated whether HO-1 and CO protect primary hepatocytes against oxidative-stress-induced apoptosis. In vivo, bile duct ligation was used as model of chronic liver disease. In vitro, primary hepatocytes were exposed to the superoxide anion donor menadione in a normal and in a CO-- containing atmosphere. Apoptosis was determined by measuring caspase-9, -6, -3 activity and poly(ADP-ribose) polymerase cleavage, and necrosis was determined by Sytox green staining. The results showed that (1) HO-1 is induced in chronic cholestatic liver disease, (2) superoxide anions time- and dose-dependently induce HO-1 activity, (3) HO-1 overexpression inhibits superoxide-anions-induced apoptosis, and (4) CO blocks superoxide-anions-induced JNK phosphorylation and caspase-9, -6, -3 activation and abolishes apoptosis but does not increase necrosis. We conclude that HO-1 and CO protect primary hepatocytes against superoxide-anions-induced apoptosis partially via inhibition of JNK activity. CO could represent an important candidate for the treatment of liver diseases.

An integrated high-performance liquid chromatography-mass spectrometry system for the activity-dependent analysis of matrix metalloproteases.

Matrix metalloproteases (MMPs) comprise a family of enzymes that play important roles in mediating angiogenesis, the remodelling of tissues and in cancer metastasis. Consequently, they are attractive targets for therapeutic intervention in chronic inflammation, cancer and neurological disorders. In order to study MMPs in body fluids in an activity-dependent manner, we have developed an automated, integrated system comprising an immobilized inhibitor cartridge for activity-dependent enrichment, an immobilized trypsin reactor for rapid on-line proteolysis and a capillary or nanoLC-MS system for separation and identification of the obtained peptide fragments. This targeted proteomics system was optimized with respect to recovery and evaluated through the analysis of urine samples that were spiked with recombinant MMP-12. MMP-12 specific peptide fragments were easily detected in a nanoLC-MS analysis of 500 microL crude urine spiked at a level of 8 nM. These results show the feasibility of selective, activity-dependent enrichment of MMPs from a non-treated biofluid at low nM concentrations.

Effect of ciclesonide treatment on allergen-induced changes in T cell regulation in asthma.

BACKGROUND: The allergen-induced release of CCL17/thymus and activation-regulated chemokine (TARC) may be crucial in asthmatic airway inflammation by recruitment of Th2 cells. In addition, it might lead to aberrant Th2 cell activity through impairment of beta2-adrenergic receptor (beta2-AR) control. We questioned how chemokine patterns change upon allergen challenge and whether treatment with the inhaled steroid ciclesonide can reduce chemokine release and subsequently prevent allergen-induced changes in Th2 cell regulation and migration. METHODS: Asthma patients were double-blindly treated with placebo or 80 microg ciclesonide for 7 days. We studied allergen-induced changes in sputum chemokines, migration of peripheral blood T cells and control of beta2-agonist fenoterol over T cell migration and alpha-CD3/alpha-CD28-induced cytokine production. RESULTS: Treatment with 80 microg ciclesonide significantly diminished the late asthmatic response. The late asthmatic response was associated with increased sputum levels of CCL17 and CCL4 (but none of the other chemokines measured) and loss of beta2-AR control over T cell migration and Th2-type cytokine production. Although ciclesonide treatment did not prevent chemokine release nor altered beta2-AR function in circulating T cells, it exerted an inhibitory effect on TARC-induced T cell migration and alpha-CD3/alpha-CD28-induced cytokine production. CONCLUSION: Our data support the hypothesis that CCL17 is involved in allergen-induced dysregulation of Th2 cell migration and cytokine production. Ciclesonide treatment inhibits T cell migration and cytokine production upon allergen inhalation, which is regulated independently from reducing CCL17 release, but may contribute to beneficial effects of ciclesonide on Th2-mediated airway inflammation.

Cigarette smoke irreversibly modifies glutathione in airway epithelial cells.

In patients with chronic obstructive pulmonary disease (COPD), an imbalance between oxidants and antioxidants is acknowledged to result in disease development and progression. Cigarette smoke (CS) is known to deplete total glutathione (GSH + GSSG) in the airways. We hypothesized that components in the gaseous phase of CS may irreversibly react with GSH to form GSH derivatives that cannot be reduced (GSX), thereby causing this depletion. To understand this phenomenon, we investigated the effect of CS on GSH metabolism and identified the actual GSX compounds. CS and H(2)O(2) (control) deplete reduced GSH in solution [Delta = -54.1 +/- 1.7 microM (P < 0.01) and -39.8 +/- 0.9 microM (P < 0.01), respectively]. However, a significant decrease of GSH + GSSG was observed after CS (Delta = -75.1 +/- 7.6 microM, P < 0.01), but not after H(2)O(2). Exposure of A549 cells and primary bronchial epithelial cells to CS decreased free sulfhydryl (-SH) groups (Delta = -64.2 +/- 14.6 microM/mg protein, P < 0.05) and irreversibly modified GSH + GSSG (Delta = -17.7 +/- 1.9 microM, P < 0.01) compared with nonexposed cells or H(2)O(2) control. Mass spectrometry (MS) showed that GSH was modified to glutathione-aldehyde derivatives. Further MS identification showed that GSH was bound to acrolein and crotonaldehyde and another, yet to be identified, structure. Our data show that CS does not oxidize GSH to GSSG but, rather, reacts to nonreducible glutathione-aldehyde derivatives, thereby depleting the total available GSH pool.

Down-regulation of E-cadherin in human bronchial epithelial cells leads to epidermal growth factor receptor-dependent Th2 cell-promoting activity.

Airway epithelial cells are well-known producers of thymus- and activation-regulated chemokine (TARC), a Th2 cell-attracting chemokine that may play an important role in the development of allergic airway inflammation. However, the mechanism responsible for up-regulation of TARC in allergy is still unknown. In the asthmatic airways, loss of expression of the cell-cell contact molecule E-cadherin and reduced epithelial barrier function has been observed, which may be the result of an inadequate repair response. Because E-cadherin also suppressed multiple signaling pathways, we studied whether disruption of E-cadherin-mediated cell contact may contribute to increased proallergic activity of epithelial cells, e.g., production of the chemokine TARC. We down-regulated E-cadherin in bronchial epithelial cells by small interference RNA and studied effects on electrical resistance, signaling pathways, and TARC expression (by electric cell-substrate impedance sensing, immunodetection, immunofluorescent staining, and real-time PCR). Small interference RNA silencing of E-cadherin resulted in loss of E-cadherin-mediated junctions, enhanced phosphorylation of epidermal growth factor receptor (EGFR), and the downstream targets MEK/ERK-1/2 and p38 MAPK, finally resulting in up-regulation of TARC as well as thymic stromal lymphopoietin expression. The use of specific inhibitors revealed that the effect on TARC is mediated by EGFR-dependent activation of the MAPK pathways. In contrast to TARC, expression of the Th1/Treg cell-attracting chemokine RANTES was unaffected by E-cadherin down-regulation. In summary, we show that loss of E-cadherin-mediated epithelial cell-cell contact by damaging stimuli, e.g., allergens, may result in reduced suppression of EGFR-dependent signaling pathways and subsequent induction of Th2 cell-attracting molecule TARC. Thus, disruption of intercellular epithelial contacts may specifically promote Th2 cell recruitment in allergic asthma.

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 (DeltaPsim) and the production of reactive oxygen species. Fluorescent probes were used to assess DeltaPsim (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 DeltaPsim, 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.

Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis.

Increased lung cell apoptosis and necrosis occur in patients with chronic obstructive pulmonary disease (COPD). Mitochondria are crucially involved in the regulation of these cell death processes. Cigarette smoke is the main risk factor for development of COPD. We hypothesized that cigarette smoke disturbs mitochondrial function, thereby decreasing the capacity of mitochondria for ATP synthesis, leading to cellular necrosis. This hypothesis was tested in both human bronchial epithelial cells and isolated mitochondria. Cigarette smoke extract exposure resulted in a dose-dependent inhibition of complex I and II activities. This inhibition was accompanied by decreases in mitochondrial membrane potential, mitochondrial oxygen consumption, and production of ATP. Cigarette smoke extract abolished the staurosporin-induced caspase-3 and -7 activities and induced a switch from epithelial cell apoptosis into necrosis. Cigarette smoke induced mitochondrial dysfunction, with compounds of cigarette smoke acting as blocking agents of the mitochondrial respiratory chain; loss of ATP generation leading to cellular necrosis instead of apoptosis is a new pathophysiological concept of COPD development.

Der p, IL-4, and TGF-beta cooperatively induce EGFR-dependent TARC expression in airway epithelium.

Thymus and Activation-Regulated Chemokine (TARC) may be critical in Th2 cell recruitment in allergic inflammation; however, the mechanisms of allergen-induced TARC release are unclear. Since airway epithelium is the first line of defense to inhaled allergens, we questioned whether house dust mite allergen (Der p) can induce TARC expression in bronchial epithelial cells, how this is regulated at the molecular level, and if micro-environmental cytokines augment this effect. We examined the effects of Der p and the cytokines IL-4 and TGF-beta on TARC expression in 16HBE cells and primary bronchial asthma epithelium. Real-time PCR and immunofluorescence demonstrated that Der p induces TARC expression in bronchial epithelium. Supernatants from Der p-stimulated 16HBE cells were able to induce TARC-dependent T cell trafficking. IL-4 and TGF-beta cooperatively enhanced Der p-induced TARC expression in 16HBE cells. Specific inhibitors, immunodetection, and gel-shifts revealed that these effects are mediated by phosphorylation of the epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK) signaling and subsequent nuclear factor (NF)-kappaB activation. A Disintegrin And Metalloproteinase (ADAM), a family of proteins involved in shedding of various growth factors, was shown to be responsible for EGFR activation. The increase in TARC production by direct interaction of Der p with the bronchial epithelium may be an important initial step in the generation of allergic inflammation, which is further potentiated by micro-environmental cytokines. Interference with ADAM or EGFR activity may be a novel promising target to prevent TARC release and subsequent allergic inflammation.

Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death.

Cigarette smoke-induced apoptosis and necrosis contribute to the pathogenesis of chronic obstructive pulmonary disease. The induction of heme oxygenase-1 provides cytoprotection against oxidative stress, and may protect in smoking-related disease. Since mitochondria regulate cellular death, we examined the functional expression and mitochondrial localization of heme oxygenase-1 in pulmonary epithelial cells exposed to cigarette smoke extract (CSE), and its role in modulating cell death. Heme oxygenase-1 expression increased dramatically in cytosolic and mitochondrial fractions of human alveolar (A549), or bronchial epithelial cells (Beas-2b) exposed to either hemin, lipopolysaccharide, or CSE. Mitochondrial localization of heme oxygenase-1 was also observed in a primary culture of human small airway epithelial cells. Furthermore, heme oxygenase activity increased dramatically in mitochondrial fractions, and in whole cell extracts of Beas-2b after exposure to hemin and CSE. The mitochondrial localization of heme oxygenase-1 in Beas-2b was confirmed using immunogold-electron microscopy and immunofluorescence labeling on confocal laser microscopy. CSE caused loss of cellular ATP and rapid depolarization of mitochondrial membrane potential. Apoptosis occurred in Beas-2b at low concentrations of cigarette smoke extract, whereas necrosis occurred at high concentrations. Overexpression of heme oxygenase-1 inhibited CSE-induced Beas-2b cell death and preserved cellular ATP levels. Finally, heme oxygenase-1 mRNA expression was elevated in the lungs of mice chronically exposed to cigarette smoke. We demonstrate the functional compartmentalization of heme oxygenase-1 in the mitochondria of lung epithelial cells, and its potential role in defense against mitochondria-mediated cell death during CSE exposure.

Airway inflammation contributes to health status in COPD: a cross-sectional study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation and airway inflammation, accompanied by decreased health status. It is still unknown which factors are responsible for the impaired health status in COPD. We postulated that airway inflammation negatively contributes to health status in COPD. METHODS: In 114 COPD patients (99 male, age: 62 +/- 8 yr, 41 [31-55] pack-years, no inhaled or oral corticosteroids, postbronchodilator FEV1: 63 +/- 9% pred, FEV1/IVC: 48 +/- 9%) we obtained induced sputum and measured health status (St. George's respiratory questionnaire (SGRQ)), postbronchodilator FEV1, hyperinflation (RV/TLC), and airway hyperresponsiveness to methacholine (PC20). Sputum was induced by hypertonic saline and differential cell counts were obtained in 102 patients. RESULTS: Univariate analysis showed that SGRQ total and symptom score were positively associated with % sputum macrophages (r = 0.20, p = 0.05; and r = 0.20, p = 0.04, respectively). Multiple regression analysis confirmed these relationships, providing significant contributions of % sputum macrophages (B = 0.25, p = 0.021) and RV/TLC (B = 0.60, p = 0.002) to SGRQ total score. Furthermore, SGRQ symptom score was associated with % sputum macrophages (B = 0.30, p = 0.03) and RV/TLC (B = 0.48, p = 0.044), whilst SGRQ activity score was associated with % sputum macrophages (B = 0.46, p = 0.002), RV/TLC (B = 0.61, p = 0.015), and PC20 (B = -9.3, p = 0.024). Current smoking and FEV1 were not significantly associated with health status in the multiple regression analysis. CONCLUSION: We conclude that worse health status in COPD patients is associated with higher inflammatory cell counts in induced sputum. Our findings suggest that airway inflammation and hyperinflation independently contribute to impaired health status in COPD. This may provide a rationale for anti-inflammatory therapy in this disease.

Repeated sputum inductions induce a transient neutrophilic and eosinophilic response.

INTRODUCTION: Sputum induction is a tool to monitor airway inflammation, yet it may induce by itself a neutrophilic response when repeated within 24 to 48 h. This limits its repeated use in clinical trials. OBJECTIVE: We aimed to investigate the induction and resolution of inflammation generated by repeated sputum inductions. SUBJECTS AND DESIGN: Sixteen healthy intermittent smokers participated in a study on the short-term effects of smoking. The nonsmoking arm consisted of seven successive sputum inductions with increasing time intervals (3, 6, 12, 24, 48, and 96 h). Inflammatory cellular characteristics and different soluble mediators were investigated. MEASUREMENTS AND RESULTS: The median percentage of sputum neutrophils increased significantly from baseline to 6 h (58.9% [range, 31.8 to 94.2%] to 83.2% [range, 26.7 to 98.3%], respectively). Surprisingly, the percentage of eosinophils also increased significantly from baseline to 6, 12, 24, and 48 h, as follows: 0.3% (range, 0.0 to 1.2%) to 1.7% (range, 0.0 to 15.5%), 2.2% (range, 0.5 to 12.5%), 1.2% (range, 0.0 to 4.8%), and 0.8% (range, 0.0 to 2.8%), respectively. Interleukin-8 increased significantly from baseline to 24 h (1,553 pg/mL [range, 462 to 8,192 pg/mL] to 2,178 pg/mL [range, 666 to 128,544 pg/mL]). CONCLUSIONS: Repeated sputum inductions should preferably be avoided within 48 h. It induces not only a short-lived neutrophilic response but also a prolonged eosinophilic inflammatory response in healthy subjects, possibly by local changes in osmolarity, and subsequent epithelial and/or mast cell activation.

Solid-phase synthesis of succinylhydroxamate peptides: functionalized matrix metalloproteinase inhibitors.

[reaction: see text] A novel solid-phase synthesis strategy toward succinylhydroxamate peptides, using an appropriately protected hydroxamate building block, is described. Rapid and efficient access is gained to amine-functionalized peptides, which can be decorated with, for instance, a fluorescent label. In addition, we demonstrate an on-resin synthesis of a biotinylated photoactivatable hydroxamate peptide, which can be used as an activity-based probe for matrix metalloproteinases and ADAMs.

Innate immune responses to environmental allergens.

Aero-allergens, including plant pollens, house dust mite particles, fungal spores, and mycelium fragments, are continuously inhaled and deposited on the airway mucosa. These particles and their soluble components actively interact with innate recognition systems present in the mucosal layer (e.g., surfactant proteins) and with various receptors present on a diversity of cells in the airways. Deposited particles are first removed by active transportation, the rate of which is either enhanced or inhibited by components present in the inhaled substances. Cleaning further depends on innate recognition, beginning with (a) soluble factors released into the mucosal surface layer that bind different bio-organic components; (b) innate receptors on phagocytic cells, alveolar macrophages, and dendritic monocytes; and (c) innate receptors on airway epithelial cells. Different innate receptor families (Toll-like receptors [TLRs], nucleotide-binding oligomerization domain receptors, and protein-activated receptors [PARs]) have been demonstrated on airway cells, including alveolar macrophages, monocytes, dendritic cells, and airway tissue cells (e.g., epithelial cells and mast cells). However, although the functional role of these receptors has been studied for infectious diseases, the functional role for reaction of airways to inhaled bio-organic substances, including allergens, is largely unexplored. Indirect evidence for functional interactions has come from in vivo animal studies, as well as in vitro studies with monocytes, macrophages, and epithelial cells, which have demonstrated release of cytokines and chemokines after contact with allergens such as house dust mite, cat, pollen, and fungi. Most allergens show direct activation of airway epithelial cells, suggesting a role for the innate receptors. However, the role of TLRs, PARs, and other receptors was studied for only a limited number of allergens. Current studies indicate synergistic interactions between members of the same receptor family (TLRs) as well as synergistic interactions between members of different families (TLRs, PARs, and nucleotide-binding oligomerization domain receptors), modulating responses into feed-forward or inhibitory actions. Study of these synergistic interactions and their genetic variations will provide insight regarding how the innate immune system determines the inflammatory reactions of the airways and the outcome of the T-helper-1- and T-helper-2-type responses to inhaled allergens.

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms.

House dust mite allergens (HDM) cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1) and unknown enzymatic activity (Der p 2, Der p 5) induce biological responses in a human airway-derived epithelial cell line (A549), and if so, to elucidate the underlying mechanism(s) of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR)1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

Interaction of airway epithelial cells (A549) with spores and mycelium of Aspergillus fumigatus.

OBJECTIVE: To study the interaction of airway epithelial cell line A549 with fragments of mycelium, spores of Aspergillus fumigatus in vitro and to determine if toll-like receptors (TLRs) are involved in the process. METHODS: A549 cells were exposed to fragments of A. fumigatus mycelium, zymosan and inactivated A. fumigatus spores. Interleukin 6 (IL-6) and IL-8 released by A549 cells to the culture supernatant were measured by ELISA. Presence of TLR2 and TLR4 on A549 cells were studied by immuno-histochemistry. RESULTS: Mycelium fragments of A. fumigatus showed strong binding to epithelial cells but had limited effects on the release of IL-6 and IL-8 by A549 cells. Irradiated A. fumigatus spores were partly internalised by A549 cells and inhibited A549 cells to produce IL-6. TNF-alpha pre-incubated A549 cells produced increased IL-6 after exposure to zymosan and WIAF. Immuno-histochemistry showed a negative staining for TLR2 and TLR4. CONCLUSIONS: The low levels of cytokines produced by A549 cells after the firm binding of either mycelium or spores of A. fumigatus may lead to insufficient recruitment of inflammatory cells to the infected site, which may result in the escape of detection by the immune defence system. TLR2 and TLR4 are probably not or only in part involved in the above process, although very low expression cannot be excluded.

First study of infliximab treatment in patients with chronic obstructive pulmonary disease.

RATIONALE: Tumor necrosis factor-alpha is believed to be important in the induction and maintenance of airway inflammation in chronic obstructive pulmonary disease. OBJECTIVES: We aimed to evaluate the effect of the anti-tumor necrosis factor-alpha drug infliximab in patients with chronic obstructive pulmonary disease, with percentage of sputum neutrophils as the primary endpoint. METHODS: We performed an exploratory single-center, double-blind, placebo-controlled, randomized, phase 2 trial in which 22 current smokers with mild-to-moderate chronic obstructive pulmonary disease participated. Fourteen patients received three infusions of infliximab (5 mg/kg) at Weeks 0, 2, and 6, and eight patients received placebo infusions. Sputum samples, respiratory symptoms, quality of life, exhaled nitric oxide, lung function parameters, bronchial hyperresponsiveness, resting energy expenditure, and side effects were evaluated. MEASUREMENTS AND MAIN RESULTS: This study did not show a positive short-term effect of infliximab on airway inflammation, lung function, resting energy expenditure, or quality of life. Exhaled nitric oxide increased significantly at Day 2, Week 6, and Week 8 in patients receiving infliximab compared with those receiving placebo. Eight patients in the infliximab group (vs. none in the placebo group) reported increased coughing, but no serious adverse events or increase in respiratory infections were reported during 9 weeks of follow-up. CONCLUSIONS: In this short-term study, no clinically beneficial effects of infliximab were observed, and there were no significant safety issues. Definite conclusions concerning the effectiveness of infliximab treatment in chronic obstructive pulmonary disease await additional studies, including those with a larger number of patients with more advanced disease.