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Background Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects’ symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. Methods Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-β1 (TGF-β1). Results The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-β1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. Conclusion and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

Aim: This study aimed to characterize DNA methylation (DNA-me) in promoter region of IL33, IL1RL1 and CCL26 in asthma and their impacts on transcriptional activity in bronchial epithelial cells (BECs). Patients & methods: We performed bis-pyrosequencing, quantitative real-time PCR and sequencing in BECs from ten asthmatic and ten control individuals. Results: We detected lower DNA-me levels of IL33 and CCL26 in asthmatic than control BECs. No correlation was found between methylation and expression levels. Interestingly, carriers of a mutative allele in a haplotype within the promoter of IL33 had a lower IL33 DNA-me level and CCL26 gene expression correlated with eosinophil count. Conclusion: These findings highlight the importance of investigating both epigenetic and genetic mechanisms in understanding the epithelial immune response in asthma.

In an effort to reduce smoking-related diseases, alternative products such as e-cigarettes have been proposed. However, despite their growing popularity, the potential toxicity of e-cigarettes remains largely unknown. In this study, human gingival fibroblasts were repeatedly exposed to cigarette smoke condensate (CSC) and to nicotine-rich (NR) or nicotine-free (NF) e-vapor condensates for 60 min once a day for various time periods. They were then used to perform different analyses. Results indicate that cells exposed to CSC or NR condensates showed an altered morphology and a reduced proliferation rate, as ascertained by MTT and BrdU assays. Fibroblast cultures exposed to either CSC or e-vapor condensates also showed increased levels of TUNEL-positive apoptotic cells, compared to that recorded in the control. Furthermore, the cell scratch test revealed that repeated exposures to CSC or to e-vapor condensates delayed both fibroblast migration and wound healing. It should be noted that CSC was much more damageable to gingival fibroblasts than were the NR and NF e-vapor condensates. The representative chain of damage thus translates to CSC > NR e-vapor condensate > NF e-vapor condensate.

Background Bronchial fibroblasts play a key role in airway remodelling in asthma. They regulate epithelial cell functions such as proliferation through growth factors, cytokines, chemokines and exosomes. The role of exosomes in the communication between epithelial cells and fibroblasts by vehiculing these mediators in asthma remains to be determined. Objective To evaluate the role of exosomes released by bronchial fibroblasts on epithelial cell proliferation in severe asthma. Methods Exosomes were obtained from culture media of primary bronchial fibroblasts and characterized using Western blot, electron microscopy and flow cytometry. Uptake profile of fluorescent-labelled exosomes in epithelial cells was assessed by flow cytometry. Exosome cytokine content was analysed by Cytokine Arrays. Bronchial epithelial cell proliferation was evaluated by BrdU incorporation test. Exosome biogenesis/release was blocked using sphingomyelinase inhibitor. Plasmid transfection was used to modulate transforming growth factor beta 2 (TGF-β2) gene expression. Results We showed that bronchial fibroblasts secreted exosomes, which were internalized by bronchial epithelial cells. Exosomes of severe asthmatic subjects' fibroblasts showed a lower level of TGF-β2 and significantly increased the epithelial cell proliferation of both healthy and severe asthmatic subjects compared to healthy controls' exosomes. Overexpression of TGF-β2 in severe asthmatics' fibroblasts induced enhanced TGF-β2 in exosomes leading to a reduced proliferation of epithelial cells, whereas knockdown of TGF-β2 enhanced epithelial cell proliferation. Conclusion Our study shows that exosomes are involved in fine-tuning intercellular communication in asthma. Exosomes of severe eosinophilic asthmatics' fibroblasts can contribute to airway remodelling, at least in part, by modulating epithelial cell proliferation observed in severe asthma.

The molecular basis for the propensity of a small number of environmental proteins to provoke allergic responses is largely unknown. Herein, we report that mite group 13 allergens of the fatty acid-binding protein (FABP) family are sensed by an evolutionarily conserved acute-phase protein, serum amyloid A1 (SAA1), that promotes pulmonary type 2 immunity. Mechanistically, SAA1 interacted directly with allergenic mite FABPs (Der p 13 and Blo t 13). The interaction between mite FABPs and SAA1 activated the SAA1-binding receptor, formyl peptide receptor 2 (FPR2), which drove the epithelial release of the type-2-promoting cytokine interleukin (IL)-33 in a SAA1-dependent manner. Importantly, the SAA1–FPR2–IL-33 axis was upregulated in nasal epithelial cells from patients with chronic rhinosinusitis. These findings identify an unrecognized role for SAA1 as a soluble pattern recognition receptor for conserved FABPs found in common mite allergens that initiate type 2 immunity at mucosal surfaces. Smole and colleagues show that the soluble pattern recognition receptor serum amyloid A (SAA) recognizes several mite allergenic proteins, including Der p 13 and Blo t 13, which are conserved fatty acid-binding proteins. Such FABP–SAA1 binding triggers epithelial cell release of the type-2-promoting cytokine IL-33, which in turn drives IL-13 production and allergic syptoms.

Airway smooth muscle (ASM) hyperplasia is a key feature of airway remodeling in development of lung diseases such as asthma. Anomalous proliferation of ASM cells directly contributes to ASM hyperplasia. However, the molecular mechanisms controlling ASM cell proliferation are not completely understood. Semaphorins are versatile regulators of various cellular processes including cell growth and proliferation. The role of semaphorins in ASM cell proliferation has remained to be addressed. Here, we report that semaphorin 3A (Sema3A) receptor, neuropilin 1 (Nrp1), is expressed on human ASM cells (HASMC) isolated from healthy and asthmatic donors and treatment of these cells with exogenous Sema3A inhibits growth factor-induced proliferation. Sema3A inhibitory effect on HASMC proliferation is associated with decreased tyrosine phosphorylation of PDGFR, downregulation of Rac1 activation, STAT3 and GSK-3β phosphorylation. Bronchial sections from severe asthmatics displayed immunoreactivity of Nrp1, suggestive of functional contribution of Sema3A-Nrp1 axis in airway remodeling. Together, our data suggest Sema3A-Nrp1 signaling as a novel regulatory pathway of ASM hyperplasia.

Objective Upon use, e-cigarette aerosol comes in contact with various mucosal tissues, including the nasal epithelium, which may lead to nasal pathologies. We therefore assessed the effect of e-cigarettes on nasal epithelial cell and tissue behaviours. Methods Human primary nasal epithelial cells and engineered 3D nasal mucosa tissues were exposed or not to either e-cigarette aerosol or standard cigarette smoke. We then evaluated cell viability and lactate dehydrogenase (LDH) activity. With the tissues analysed tissue structure, the expression of Ki67 proliferating marker, and the secretion of pro-inflammatory cytokines by the engineered nasal mucosa. Results The nasal epithelial cells exposed to e-cigarettes displayed a larger cell size and a faint nucleus following exposure to e-cigarettes. This is supported by the increased levels of LDH activity following exposure to e-cigarettes, compared to that observed in the control. Tissues exposed to e-cigarette aerosol displayed a structural deregulation, with more large-sized cells, fewer Ki67-positive cells, and a reduced proliferation rate, compared to that observed in the non-exposed tissues. Cytokine measurements showed high levels of IL-6, IL-8, TNFα, and MCP-1, demonstrating that e-cigarettes activated pro-inflammatory cytokine responses. Conclusion E-cigarette aerosol showed adverse effects on nasal epithelial cells and nasal engineered mucosa tissue. These findings indicate that e-cigarettes could be a threat to nasal tissues and may impair the innate immune function of nasal epithelial cells.

The key factors underlying the development of allergic diseases-the propensity for a minority of individuals to develop dysfunctional responses to harmless environmental molecules-remain undefined. We report a pathway of immune counter-regulation that suppresses the development of aeroallergy and shrimp-induced anaphylaxis. In mice, signaling through epithelially expressed dectin-1 suppresses the development of type 2 immune responses through inhibition of interleukin-33 (IL-33) secretion and the subsequent recruitment of IL-13-producing innate lymphoid cells. Although this homeostatic pathway is functional in respiratory epithelial cells from healthy humans, it is dramatically impaired in epithelial cells from asthmatic and chronic rhinosinusitis patients, resulting in elevated IL-33 production. Moreover, we identify an association between a single-nucleotide polymorphism (SNP) in the dectin-1 gene loci and reduced pulmonary function in two cohorts of asthmatics. This intronic SNP is a predicted eQTL (expression quantitative trait locus) that is associated with reduced dectin-1 expression in human tissue. We identify invertebrate tropomyosin, a ubiquitous arthropod-derived molecule, as an immunobiologically relevant dectin-1 ligand that normally serves to restrain IL-33 release and dampen type 2 immunity in healthy individuals. However, invertebrate tropomyosin presented in the context of impaired dectin-1 function, as observed in allergic individuals, leads to unrestrained IL-33 secretion and skewing of immune responses toward type 2 immunity. Collectively, we uncover a previously unrecognized mechanism of protection against allergy to a conserved recognition element omnipresent in our environment.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

Background Bronchial fibroblasts are the main structural cells responsible for extracellular matrix production and turnover in lung tissue. They play a key role in airway remodelling in asthma through different cytokines including interleukin (IL-6). Objective To decipher IL-6 signalling in bronchial fibroblasts obtained from severe eosinophilic asthmatics compared to mild asthmatics and healthy controls. Methods Human bronchial fibroblasts were isolated from bronchial biopsies of mild and severe eosinophilic asthmatics and non-atopic healthy controls. IL-6 was assessed by qRT-PCR and ELISA. Phosphorylated STAT3, SHP2 and p38/MAPK were evaluated by Western blot. Chemical inhibitors for SHP2 and p38 were used. Fibroblast proliferation was evaluated by BrdU incorporation test. Results IL-6 release was significantly increased in fibroblasts from mild and severe asthmatics compared to healthy controls. Fibroblasts from severe asthmatics showed a reduced STAT3 activation compared to mild asthmatics and healthy controls. Constitutive activation of phosphatase SHP2 was found to negatively regulate IL-6 induced STAT3 phosphorylation in fibroblasts from severe asthmatics. This effect was accompanied by a decrease in fibroblast proliferation rate due to the activated p38/mitogen-activated protein kinase. SHP2 and p38/MAPK specific inhibitors (PHPS1 and SB212190) significantly induce a restoration of STAT3 phosphorylation, IL-6 target gene expression and cell proliferation. Conclusion These data show dysregulated IL-6 signalling in bronchial fibroblasts derived from severe eosinophilic asthmatic subjects involving the protein tyrosine phosphatase SHP2 and p38MAPK. Collectively, our data provides new insights into the mechanisms by which bronchial fibroblasts regulate airway remodelling in severe asthma.

Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2β1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2β1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2β1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2β1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2β1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor–related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2β1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.

Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc.

Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 μM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO.

Asthma is characterized by airway remodeling associated with an increase in the deposition of ECM proteins such as type I collagen. These components are mainly produced by fibroblasts. Inhaled corticosteroids are considered the cornerstone of asthma therapy. Despite substantial evidence as to the anti-inflammatory action of corticosteroids, their effect on controlling ECM protein deposition in the airways is not completely understood. This study determined the effect of dexamethasone (Dex) on collagen production by bronchial fibroblasts derived from asthmatic and healthy subjects. Expression of procollagen mRNA in fibroblasts from asthmatics and normal controls was determined by quantitative PCR. Regulation of the procollagen-α1I promoter was evaluated by transient transfections. Transforming growth factor-β (TGF-β) protein expression was determined by ELISA. Protein expression of glucocorticoid receptor (GR) and interaction with activator protein-1 (AP-1), a collagen regulatory transcription factor, was assessed by Western blots, coimmunoprecipitations, and EMSA. AP-1 overexpression was performed by transient transfection using c-Fos/c-Jun expression plasmids. Dex significantly downregulated procollagen production and promoter activity in normal fibroblasts but had no effect on asthmatic fibroblasts. AP-1 and GR interaction increased after Dex stimulation in asthmatic fibroblasts. AP-1 overexpression in control fibroblasts abrogated collagen gene response to Dex. These results show that Dex failed to reduce collagen production in fibroblasts from asthmatic subjects. This impaired response may be related to AP-1 overexpression in these cells.

Background Allergic asthma is characterized by infiltration of inflammatory cells into the airways. T cell-derived cytokines regulate both airway inflammation and remodelling. In the human airways, T cell–fibroblast interactions may have a role in regulating inflammation and remodelling. Objectives To evaluate the effect of bronchial fibroblast–T cell interaction on profibrogenic cytokine release and determine the nature of the molecules involved in this interaction. Methods Human bronchial fibroblasts obtained from healthy and asthmatic donors were co-cultured with purified T cells derived from peripheral blood of the same subjects. IL-6 mRNA and protein levels were measured by real time PCR and ELISA. CD40, CD40L and α5β1 were evaluated by flow cytometry. Bronchial fibroblasts were stimulated with rsCD40L. Neutralisation was performed using neutralizing antibodies anti-CD40L and anti-α5. Results Contact of T cells with bronchial fibroblasts up-regulated IL-6 at both gene and protein levels. This effect was significantly higher in fibroblasts from asthmatics than those from controls. Blocking CD40L and α5β1 integrin showed a significant inhibition of IL-6 expression in asthmatics but not in healthy controls. Stimulation of fibroblasts with recombinant soluble CD40L up-regulated IL-6 production in asthmatics but not in controls. Adhesion to fibronectin, a α5β1 integrin ligand, is increased in fibroblasts from asthmatics compared to fibroblasts from controls. Conclusion These results showed that interaction of bronchial fibroblasts with T cells increases the production of profibrogenic cytokine IL-6. In asthmatic condition this interaction involves CD40L/α5β1. These results suggest that T cells and structural cells crosstalk in asthma may maintain local mucosal inflammation.

Cysteinyl leukotrienes (CysLTs) contribute to allergic and inflammatory diseases through CysLT 1 -R. We aimed to assess CysLT 1 -R mRNA expression in induced sputum of rhinitics with or without asthma before and following allergen challenges. Both groups underwent nasal and "low dose" lung allergen challenges. Asthmatics also underwent "standard" lung challenge. Sputum was obtained before and at different time-points following the challenges for CysLT 1 -R, 5-lipoxygenase (5-LO), and eotaxin mRNA assessments. At baseline, there was no difference in mediator levels between groups. An increase in CysLT 1 -R mRNA ( p =0.04) and a trend towards an increase in 5-LO and eotaxin ( p =0.06 for both) at 24h post-nasal challenge were observed. Following "low dose" lung allergen challenge, there was a trend towards an increase in CysLT 1 -R ( p =0.07). In conclusion, CysLT 1 -R gene expression changes can be detected in sputum following allergen challenges. No difference was observed between groups, suggesting that changes in CysLT 1 -R expression occur whether or not the subject has concurrent asthma.

Bacteria, moulds, endotoxin and quartz from respirable dust of agricultural and industrial buildings are typically incriminated for the respiratory health decline of exposed workers despite that dust being an undefined mixture and quantification methods of aerosolized bacteria, moulds or endotoxin not being standardized yet. We developed an in vitro alveolar epithelial cell system in which biological activities of peat moss factories' dust might be correlated to bacteria, mould, endotoxin and quartz concentrations of the analyzed samples. Following exposure, interleukin-8 protein secretion, necrosis and apoptosis of the exposed A549 cells were monitored respectively with ELISA on cell supernatants, trypan blue exclusion and DNA fragmentation detection by flow cytometry. Respirable dust was collected with liquid impingers and respirable quartz with 10mm Dorr-Oliver cyclones. We quantified mesophilic bacteria, mesophilic moulds and endotoxins from liquid impinger samples. No correlation was observed between biological activities of dust and bacteria, mould, endotoxin or quartz concentrations under our experimental conditions. Our speculation is that simple measurements, such as dust concentrations, may not be adequate indicators of the human respiratory health hazard for a given environment.

Background House dust mite (HDM) induces allergic asthma in sensitized individuals, although the mechanisms by which HDM is sensed and recognized by the airway mucosa, leading to dendritic cell (DC) recruitment, activation, and subsequent T H 2-mediated responses, are unknown. Objective We sought to define the pathways by which HDM activates respiratory epithelium to induce allergic airway responses. Methods Using a human airway epithelial cell line (16HBE14o-), we studied secretion of the DC chemokine CCL20 after exposure to HDM or other allergens, investigated components of the HDM responsible for the induction of chemokine release, and examined activation of signaling pathways. Central findings were also confirmed in primary human bronchial cells. Results We demonstrate that exposure of airway epithelium to HDM results in specific and rapid secretion of CCL20, a chemokine attractant for immature DCs. The induction of CCL20 secretion is dose and time dependent and quite specific to HDM because other allergens, such as ragweed pollen and cockroach antigen, fail to significantly induce CCL20 secretion. Induction of CCL20 secretion is not protease or Toll-like receptor 2/4 dependent but, interestingly, relies on β-glucan moieties within the HDM extract, as evidenced by the ability of other β-glucans to competitively inhibit its secretion and by the fact that disruption of these structures by treatment of HDM with β-glucanase significantly reduces subsequent chemokine secretion. Conclusion Taken together, our results describe a novel mechanism for specific pattern recognition of HDM-derived β-glucan moieties, which initiates allergic airway inflammation and, through recruitment of DCs, might link innate pattern recognition at the airway surface with adaptive immune responses.

Background: Mechanical strain and cytokine stimulation are two important mechanisms leading to airway remodeling in asthma. The effect of mechanical strain on cytokine secretion in airway fibroblasts is not known. The aim of this study was to determine whether bronchial and nasal fibroblasts differentially alter cytokine secretion in response to mechanical strain. Methods: We measured secretion of the pro-fibrotic cytokine, interleukin-6 (IL-6), and the pro-inflammatory cytokines, IL-8 and monocyte chemotactic protein 1, before and after mechanical strain in bronchial fibroblasts obtained from asthmatic patients [asthmatic bronchial fibroblasts (BAF)] and normal volunteers [normal bronchial fibroblasts (BNF)], and in nasal fibroblasts (NF) obtained from nasal polyps. Cells were grown on flexible membranes and a mechanical strain of 30% amplitude, 1 Hz frequency was applied for 3, 6 and 24 h. Control cells were unstrained. IL-6, IL-8 and monocyte chemotactic protein 1 was measured after 24 h strain using enzyme-linked immunoassay; mRNA was measured by real time polymerase chain reaction. We also measured mRNA for versican, a matrix proteoglycan, known to be upregulated in the asthmatic airway wall. Results: In unstrained conditions, no differences in cytokine secretion were observed. After 24 h strain, BAF secreted more IL-6 than BNF. Mechanical strain increased IL-8 mRNA in BAF, BNF and NF; and IL-6 and versican mRNA, in BAF, only. Conclusions: Cytokine responses to mechanical strain varied in different airway fibroblast populations, and depended on the site of origin, and the underlying inflammatory state. Strain resulted in IL-6 upregulation and increased message for extracellular matrix protein in bronchial fibroblasts from asthmatic patients only, and may reflect these patients’ propensity for airway remodeling.

Emphysema is mainly known for the complex inflammatory processes associated with its development. In addition to lung inflammation, it is now accepted that increased alveolar cell apoptosis is also part of emphysema pathophysiology. However, little is known about the mechanisms involved in alveolar apoptosis. We postulate that oxidative stress and proinflammatory cytokines could lead to p53 accumulation, Bax/Bcl-x(L) ratio elevation, and higher tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor levels in the emphysematous lung.To evaluate the expression of p53, Bax, Bcl-x(L), TRAIL, and TRAIL receptors in lung parenchyma from nonemphysematous nonsmokers and smokers and emphysematous smokers and ex-smokers and to determine whether H2O2 and/or TNF can modulate the expression of these apoptotic proteins.p53, Bax, Bcl-x(L), and TRAIL receptor protein levels in lung parenchyma were measured by Western blot, and TRAIL mRNA levels were measured by real-time polymerase chain reaction. Changes in TRAIL receptor, Bax, Bcl-x(L), and p53 protein levels after in vitro H2O2 and/or TNF stimulation of A549 cells were also assessed by Western blot.The p53 protein levels, the Bax/Bcl-x(L) ratio, and TRAIL receptors 1, 2, and 3 protein levels were significantly higher in subjects with emphysema. Moreover, they were also increased after H2O2 and TNF treatments of A549 cells.These findings suggest that oxidative stress and proinflammatory cytokines may be involved in the elevation of p53 levels, the Bax/Bcl-x(L) ratio, and TRAIL receptor levels, new mechanisms that may be implicated in the increased alveolar cell apoptosis that occurs in emphysema.

Background Interaction between lymphocytes and structural cells has been proposed as a key factor in regulating inflammation in asthma. Objective This study was designed to investigate the effect of epithelial cells and fibroblasts on T-lymphocyte survival by using a 3-dimensional tissue-engineered model. Methods Engineered human bronchial mucosal tissues were produced by using fibroblasts, epithelial cells, and autologous T cells from asthmatic and healthy donors. T-cell apoptosis and apoptotic marker expression by T cells were evaluated by using the terminal deoxynucleotidyl transferase biotinylated d-UTP nick end-labeling technique and immunofluorescence, respectively. Cytokines implicated in T-cell survival were measured by means of ELISA in culture supernatants. Results We demonstrated histologically that we were able to generate a well-structured engineered bronchial mucosa by using epithelial cells, fibroblasts, and T cells cultured from healthy and asthmatic subjects. Structural cells from asthmatic subjects cultured in this model induced a significant decrease in the ability of T cells to undergo apoptosis represented by a decrease in DNA fragmentation and proapoptotic molecule expression (Bcl-2–associated X protein and Fas ligand). Structural cells from healthy control subjects have no effect. Among cytokines measured in the supernatants, only TGF-β 1 was significantly increased in the model derived from cells of asthmatic subjects. Conclusion These results support the concept that bronchial structural cells might play a critical role in the regulation of inflammation in asthma by increasing the survival of T lymphocytes. The results also further validated the model as a tool for investigating the interaction between inflammatory and structural cells.

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.

BACKGROUND: Bronchial biopsies are widely used for histopathological, primary cell culture and genetic studies, but very few reports have evaluated their quality.OBJECTIVES AND METHODS: The present project evaluated the quality (using a scoring system) and the general morphology of a pool of six bronchial biopsy specimens taken from three different sampling sites (the lobar, segmental and subsegmental carinae) in 27 subjects (13 asthmatic subjects and 14 healthy controls). The present study also assessed quantitative measurements of structural changes related to asthma.RESULTS: In total, 94.4% of the biopsy attempts had enough tissue to be processed. From these, 61.7% were scored with a good to excellent quality, while 76.5% presented smooth muscle bundles and 40.5% had an intact epithelium wall. The data also confirmed the structural changes observed in asthma, such as increased apparent thickening of the basement membrane, reduced amounts of smooth muscle for healthy controls and decreased percentage of intact epithelium for asthmatic subjects.CONCLUSION: A pool of six bronchial biopsy specimens can provide tissue of excellent quality in both asthmatic and healthy subjects and, consequently, a valuable sample for morphological analysis of mucosal structures.

Background ADAM33 , a disintegrin and metalloproteinase 33 gene, has been identified as a risk factor for asthma and bronchial hyperresponsiveness and has been postulated as a gene for airway remodeling. ADAM8 is strongly induced by allergens and T H 2 cytokines in the lung in experimental asthma. Objectives To assess the importance of these genes in asthma pathogenesis and to investigate whether expression relates to disease severity or deterioration in lung function, we measured the mRNA and protein expression of both genes in bronchial biopsies of subjects with asthma and control subjects. Methods RNA was extracted from frozen endobronchial biopsies of mild, moderate, and severe adults with asthma and controls. Subjects with moderate and severe asthma were taking corticosteroids. The mRNA transcript of both genes was measured by real time RT-PCR using specific primers. Protein expression was examined by immunohistochemistry on paraffin sections. Results ADAM33 mRNA expression was significantly higher in both moderate and severe asthma compared with mild asthma ( P ADAM33 was increased in the epithelium, submucosal cells, and smooth muscle in severe asthma compared with mild disease and controls. ADAM8 mRNA expression was significantly increased in all asthma groups compared with controls. Increased inflammatory cells stained positive for ADAM8 in both moderate ( P P Conclusions These results demonstrate increased expression of both ADAM genes as asthma severity increases. Clinical implications These genes may contribute to the remodeling process that occurs with asthma progression and may have implications for future treatment in severe disease.

The aim of bronchial thermoplasty is to improve asthma symptoms by reducing central airway smooth muscle mass. Up to now, the reduction of smooth muscle mass has been documented for only 1 group of 10 patients who had 15% or more of their pretreatment total bronchial biopsy area occupied by smooth muscle.To evaluate the effects of bronchial thermoplasty on airway smooth muscle mass and airway collagen deposition in adult patients with asthma, regardless of pretreatment smooth muscle area.Seventeen patients with asthma underwent bronchial thermoplasty over the course of three visits. At Visit 1, bronchial biopsies were taken from the lower lobe that was not treated during this session. At Visit 2 (3-14 wk after the first visit), all 17 patients underwent biopsy of the lower lobe treated during the first procedure. At Visit 3 (7-22 wk after the first visit), nine patients agreed to undergo biopsy of the same lower lobe. Histological and immunohistochemical analyses were performed on the biopsy specimens.Bronchial thermoplasty decreased airway smooth muscle from 12.9 ± 1.2% of the total biopsy surface at Visit 1 to 4.6 ± 0.8% at Visit 2 (P 0.0001). For the nine patients who underwent a third biopsy, mean airway smooth muscle area was 5.3 ± 1.3% at Visit 3 (P = 0.0008 compared with baseline). Bronchial thermoplasty also decreased Type I collagen deposition underneath the basement membrane from 6.8 ± 0.3 μm at Visit 1 to 4.3 ± 0.2 μm at Visit 2 (P 0.0001) and to 4.4 ± 0.4 μm for nine patients at Visit 3 (P 0.0001 compared with baseline). Over the course of 1 year after treatment, the doses of inhaled corticosteroid, the number of severe exacerbations, and asthma control all improved (P ≤ 0.02).For patients with severe asthma, bronchial thermoplasty reduced the smooth muscle mass of treated airway segments, regardless of the baseline level of muscle mass. Treatment also altered the deposition of collagen. At follow-up, bronchial thermoplasty improved asthma control; however, the limited number of subjects did not allow us to evaluate possible correlations between these improvements and the studied histological parameters. Further studies are needed to confirm these results and evaluate their persistence.

Background: Animal and human studies demonstrated that interleukin (IL)-12, a Th1 cytokine, reduces blood and bronchial eosinophilia, and airway hyperreactivity. According to current concepts, these effects are mediated through the release of cytokines promoting eosinophil recruitment and activation. However, the presence of IL-12 receptors on eosinophils suggests that IL-12 also acts directly on eosinophils. We postulated that IL-12 directly modulates eosinophil functions and has the capacity to regulate eosinophil degranulation, migration and survival, in vitro. Method: Effects of IL- 12 on purified human blood eosinophils were evaluated for peroxidase (EPO) release, eotaxin-induced migration through a model of basement membrane (Matrigel), and survival. Results: IL-12 inhibited 50% of PAF and secretory IgA-induced EPO release (n = 8, p < 0.001). IL-12 also reduced eotaxin-induced migration through Matrigel by 54 ±6% (n = 6, p < 0.01). These effects were not explained by an IL-12-induced impaired viability or apoptosis. Conclusion: Our results demonstrate that IL-12 directly modulates eosinophil functions without promoting apoptosis and explain, at least in part, the effects of IL-12 on eosinophils observed in in vivo studies.

Asthma is characterized by an increase in airway smooth muscle mass and a decreased distance between the smooth muscle layer and the epithelium. Furthermore, there is evidence to indicate that airway smooth muscle cells (ASMC) express a wide variety of receptors involved in the immune response. The aims of this study were to examine the expression of CCR3 on ASMC, to compare this expression between asthmatic and nonasthmatic subjects, and to determine the implications of CCR3 expression in the migration of ASMC. We first demonstrated that ASMC constitutively express CCR3 at both mRNA and protein levels. Interestingly, TNF-α increases ASMC surface expression of CCR3 from 33 to 74%. Furthermore, using FACS analysis, we found that ASMC CCR3 is expressed to a greater degree in asthmatic vs control subjects (95 vs 75%). Functionality of the receptor was demonstrated by calcium assay; the addition of CCR3 ligand eotaxin to ASMC resulted in an increase in intracellular calcium production. Interestingly, ASMC was seen to demonstrate a positive chemotactic response to eotaxin. Indeed, ASMC significantly migrated toward 100 ng/ml eotaxin (2.2-fold increase, compared with control). In conclusion, the expression of CCR3 by ASMC is increased in asthmatics, and our data show that a CCR3 ligand such as eotaxin induces migration of ASMC in vitro. These results may suggest that eotaxin could be involved in the increased smooth muscle mass observed in asthmatics through the activation of CCR3.

Cytokines and chemokines have been implicated in the pathogenesis of asthma. Inhaled corticosteroids have been shown to decrease the number of eosinophils and to downregulate T H 2 cytokines but to increase neutrophils. The effect of corticosteroids on chemokine expression in asthma has not yet been investigated.We sought to investigate the effect of a 2-week course of oral corticosteroid (methylprednisolone, 40 mg/d) on the expression of CXC chemokines (IL-8 and IFN-gamma-inducible protein 10 [IP-10]) and CC chemokines (eotaxin and monocyte chemotactic proteins [MCPs] 1-4) in endoscopic biopsy specimens of 13 patients with moderate-to-severe asthma.CD3, major basic protein, and elastase immunoreactivities were monitored before and after treatment by using immunocytochemistry. Eotaxin, IL-8, IP-10, MCP-1, MCP-2, MCP-3, and MCP-4 mRNA expression in epithelium and submucosa were studied by using in situ hybridization.Corticosteroids reduced the number of CD3-positive T cells and major basic protein-positive eosinophils ( P.05), whereas the number of neutrophils were increased ( P.05). Corticosteroids also reduced the number of eotaxin ( P.05), MCP-3, and MCP-4 mRNA-positive cells ( P.001) in the epithelium and subepithelium. However, corticosteroids caused a significant increase in the epithelial expression of IL-8 ( P.001), IP-10 ( P.05), and MCP-2 mRNAs ( P.01). Corticosteroids had no effects on MCP-1 mRNA expression.Our results demonstrate the dual nature of corticosteroids. Although corticosteroids can downregulate the expression of some asthma-associated chemokines, such as eotaxin, MCP-3, and MCP-4, they can also upregulate the expression of other chemokines, including IL-8, IP-10, and MCP-2. The failure of oral corticosteroids to inhibit IL-8 mRNA expression might contribute to persistent airway neutrophilia observed in patients with moderate-to-severe asthma, despite treatment with corticosteroids.

Summary Background Remodelling of the asthmatic airway includes increased deposition of proteoglycan (PG) molecules. One of the stimuli driving airway remodelling may be excessive mechanical stimulation. Objective We hypothesized that fibroblasts from asthmatic patients would respond to excessive mechanical strain with up-regulation of message for PGs. Methods We obtained fibroblasts from asthmatic patients (AF) and normal volunteers (NF) using endobronchial biopsy. Cells were maintained in culture until the fifth passage and then grown on a flexible collagen-coated membrane. Using the Flexercell device, cells were then subjected to cyclic stretch at 30% amplitude at 1 Hz for 24 h. Control cells were unstrained. Total RNA was extracted from the cell layer and quantitative RT-PCR performed for decorin, lumican and versican mRNA. Results In unstrained cells, the expression of decorin mRNA was greater in AF than NF. With strain, NF showed increased expression of versican mRNA and AF showed increased expression of versican and decorin mRNA. The relative increase in versican mRNA expression with strain was greater in AF than NF. Conclusions These data support the hypothesis that proteoglycan message is increased in asthmatic fibroblasts subject to mechanical strain. This finding has implications for the mechanisms governing airway wall remodelling in asthma.

Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-alpha. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF-alpha in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF-alpha increased MT4-MMP mRNA expression. This effect appeared at 4h and reached a maximum at 8h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF-alpha increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-alpha. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling.

This study aimed to determine whether there is a persistent or different type of airway inflammation in patients with an incomplete reversibility of airflow obstruction (IRAO) despite optimal treatment and if so, whether it is associated with an accelerated decline of pulmonary function. Fifteen asthmatic patients with IRAO, and 23 with complete reversibility of airflow obstruction (CRAO) had a spirometry and an induced-sputum (IS) analysis. Past FEV 1 values were recorded over 2–12 years during periods of stable asthma. Medians (range) for IS cell differentials were: lymphocytes, 0(0–3)/1(0–2)%; neutrophils, 56(13–88)/38(3–84)% and eosinophils, 2.0(0–82)/4.0(0–68)%, (all P >0.05). Among non-smoking patients, those with IRAO had more neutrophils in IS than those with CRAO ( P =0.019). Mean (±sem) yearly fall in FEV 1 in IRAO or CRAO patients was 54±21/84±16 ml/year ( P >0.05, predicted age-related decline ≤26ml/year, P =0.0008). In the whole group of asthmatic patients, decline of FEV 1 /year was inversely correlated with the % neutrophils in sputum ( r s =−0.436, P =0.008) and, in IRAO patients, with the duration of asthma ( r s =−0.559, P =0.037). In conclusion, persistent airway inflammation and increased decline in pulmonary function can be observed in both asthmatic patients with IRAO/CRAO and are of similar magnitude. Non-smoking patients with IRAO had more neutrophils in IS than CRAO.

Asthma is characterized by an airway remodeling process involving altered extracellular matrix deposition such as collagen, fibronectin and proteoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects. Not surprisingly, it has been suggested that proteoglycan deposition may alter airway properties in asthma including airway hyperresponsiveness. In chronically inflamed airway tissues, fibroblasts likely represent an activated fibrotic phenotype that contributes to the excessive deposition of different extracellular matrix components. To investigate whether this was the case for proteoglycans, the production of hyaluronan, perlecan, versican, small heparan sulphate proteoglycans (HSPGs), decorin and biglycan was quantified in the culture medium of primary bronchial fibroblast cultures, established from four normal and six asthmatic subjects. Values were further correlated to the airway responsiveness (PC(20) methacholine) of donor subjects. Fibroblasts from subjects with the most hyperresponsive airways produced up to four times more total proteoglycans than cells from subjects with less hyperresponsive or normoresponsive airways. We observed a significant negative correlation between the PC(20) and perlecan, small HSPGs and biglycan, while such correlation was absent for decorin and close to significant for hyaluronan and versican. Altered proteoglycan metabolism by bronchial fibroblasts may contribute to the increased proteoglycan deposition in the bronchial mucosa and to airway hyperresponsiveness characterizing asthma.

Background: Blood eosinophils have mRNA for FcγRIIIB (CD16) but no or minimal spontaneous CD16 expression. Because IFN-γ and chemotactic factors induce eosinophil CD16 expression in vitro, we postulated that blood eosinophils could express CD16. Objective: Blood of nonallergic controls and subjects with allergic rhinitis, allergic and nonallergic asthma, or hypereosinophilia of various etiologies were analyzed for leukocyte CD16 surface expression. Methods: CD16 + eosinophils were identified on the basis of physico-optic characteristics, major basic protein, CD49b expression, and sorting by flow cytometry and microscope examination. Results: Subjects with allergic rhinitis and subjects with asthma had higher median percentages of CD16 + eosinophils (8.1% [1% to 48.6%] and 7.3% [1.4% to 31.1%], respectively) than nonallergic controls and nonallergic asthmatics (3% [0% to 11%] and 4.6% [2.9% to 5.1%], respectively). In subjects with hypereosinophilia, CD16 + eosinophils were increased only in a case of drug allergy. When subjects with mild allergic asthma were challenged with a relevant aeroallergen, blood CD16 + eosinophils further increased during or after the late-phase response (6 to 48 hours after challenge; mean ± SEM, 9.4% ± 2.5% to 20.0% ± 3.0%). CD16 + eosinophils expressed more IL-5 receptor but less CD11b and IL-12p35 than did CD16 − eosinophils. Conclusion: Upregulation of blood CD16 + eosinophils in allergic conditions and its association with a modified phenotype suggest that CD16 receptor could play a role in eosinophil activation in allergy. (J Allergy Clin Immunol 2002;109:463-9.)

Background Allergic asthma is characterized by inflammation and airway remodeling. Bronchial epithelium is considered a key player in coordinating airway wall remodeling. In mild asthma, the epithelium is damaged and fails to proliferate and to repair, whereas in severe asthma, the epithelium is highly proliferative and thicker. This may be due to different regulatory mechanisms. The purpose of our study was to determine the role of miRNAs in regulating proliferation of bronchial epithelial cells obtained from severe asthmatic subjects in comparison with cells obtained from mild asthmatics and healthy controls. Methods Human bronchial epithelial cells (BEC) were isolated by bronchoscopy from bronchial biopsies of healthy donors and patients with mild and severe asthma. MiRNA expression was evaluated using the TaqMan low-density arrays and qRT-PCR. Transfection studies of bronchial epithelial cells were performed to determine the target genes. Cell proliferation was evaluated by BrdU incorporation test. Results MiR-19a was upregulated in epithelia of severe asthmatic subjects compared with cells from mild asthmatics and healthy controls. Functional studies based on luciferase reporter and Western blot assays suggest that miR-19a enhances cell proliferation of BEC in severe asthma through targeting TGF-β receptor 2 mRNA. Moreover, repressed expression of miR-19a increased SMAD3 phosphorylation through TGF-β receptor 2 signaling and abrogated BEC proliferation. Conclusion Our study uncovers a new regulatory pathway involving miR-19a that is critical to the severe phenotype of asthma and indicates that downregulating miR-19a expression could be explored as a potential new therapy to modulate epithelium repair in asthma.

High pulmonary eosinophil counts are associated with asthma symptoms and severity. Bronchial epithelial cells (BECs) produce CC chemokines, notably CCL26 (eotaxin-3), which recruits and activates eosinophils from asthmatic patients. This suggests that CCL26 production by BECs might be involved in persistent eosinophilia in patients with severe asthma despite treatment with high corticosteroid doses.We sought to determine whether CCL26 levels correlate with eosinophilia and asthma severity.Human CC chemokine expression was assessed by means of quantitative PCR or a quantitative PCR array in vehicle- or IL-13-treated BECs. CCL26 was quantitated by means of ELISA. Immunohistochemistry analyses of CCL26 and major basic protein were done on bronchial biopsy specimens.IL-13 selectively induced CCL26 expression by BECs. This increase was time-dependent and more prominent in BECs from patients with severe eosinophilic asthma. CCL26 levels measured in supernatants of IL-13-stimulated BECs also increased with asthma severity as follows: patients with severe eosinophilic asthmapatients with mild asthma ≈ healthy subjects. Immunohistochemistry analyses of bronchial biopsy specimens confirmed increased levels of CCL26 in the epithelium of patients with mild and those with severe eosinophilic asthma. Tissue eosinophil counts did not correlate with CCL26 staining. However, sputum CCL26 levels significantly correlated with sputum eosinophil counts (P.0001), suggesting that CCL26 participates in the movement of eosinophils from the tissues to the airway lumen.These results show a relation between CCL26 production by IL-13-stimulated BECs, sputum eosinophil counts, and asthma severity. They also suggest a role for CCL26 in the sustained inflammation observed in patients with severe eosinophilic asthma and reveal CCL26 as a potential target for treating patients with eosinophilic asthma that are refractory to classic therapies.

IL-17 is a cytokine that has been reported to be produced by T lymphocytes. In vitro, IL-17 activates fibro-blasts and macrophages for the secretion of GM-CSF, TNF-alpha, IL-1beta, and IL-6. A number of these cytokines are involved in the airway remodeling that is observed within the lungs of asthmatic individuals.In this study, we investigated the expression of IL-17 in sputum and bronchoalveolar lavage specimens obtained from asthmatic subjects and from nonasthmatic control subjects.IL-17 was detected through use of immunocytochemistry, in situ hybridization, and Western blot. Bronchial fibroblasts were stimulated with IL-17, and cytokine production and chemokine production were detected through use of ELISA and RT-PCR.Using immunocytochemistry, we demonstrated that the numbers of cells positive for IL-17 are significantly increased in sputum and bronchoalveolar lavage fluids of subjects with asthma in comparison with control subjects (P.001 and P.005, respectively). We demonstrated that in addition to T cells, eosinophils in sputum and bronchoalveolar lavage fluids expressed IL-17. Peripheral blood eosinophils were also positive for IL-17, and the level of IL-17 in eosinophils purified from peripheral blood was significantly higher in subjects with asthma than in controls (P.01). To further investigate the mechanism of action of IL-17 in vivo, we examined the effect of this cytokine on fibroblasts isolated from bronchial biopsies of asthmatic and nonasthmatic subjects. IL-17 did enhance the production of pro-fibrotic cytokines (IL-6 and IL-11) by fibroblasts, and this was inhibited by dexamethasone. Similarly, IL-17 increased the level of other fibroblast-derived inflammatory mediators, such as the alpha-chemokines, IL-8, and growth-related oncogene-alpha.Our results, which demonstrate for the first time that eosinophils are a potential source of IL-17 within asthmatic airways, suggest that IL-17 might have the potential to amplify inflammatory responses through the release of proinflammatory mediators such as alpha-chemokines.

Background: Natural exposure to pollen provokes an increase in airway responsiveness in nonasthmatic subjects with seasonal allergic rhinitis. This natural exposure may induce inflammatory cell recruitment and cytokine release, leading to lower airway inflammation. Objective: The aim of this study was to characterize lower airway inflammation in nonasthmatic pollen-sensitive subjects. Methods: We performed immunohistochemical tests on bronchial biopsy specimens from subjects with rhinitis who had no past or current history of asthma to evaluate cytokine expression and inflammatory cell numbers and activation both in and out of the pollen season. Results: The number of CD4 + , CD8 + , and CD45RO + lymphocyte subpopulations were significantly higher during the pollen season compared with the out-of-season period ( P + cells tended to increase after natural pollen exposure ( P = .06). The number of IL-5 + cells increased significantly after natural exposure to pollen compared with out-of-season numbers ( P + , CD4 + , CD45RO + , and EG1 + cells. The numbers of tryptase-positive, IFN-γ + , and IL-4 + cells did not change after natural exposure. Conclusion: This study showed that natural pollen exposure was associated with an increase in lymphocyte numbers, eosinophil recruitment, and IL-5 expression in the bronchial mucosa of nonasthmatic subjects with allergic rhinitis. (J Allergy Clin Immunol 2000;106:904-10.)

Caveolae are plasma membrane microdomains that have been implicated in organizing and concentrating certain signaling molecules. Caveolins, constitute the main structural proteins of caveolae. Caveolae are abundant in terminally differentiated cell types. However, caveolin-1 is down-regulated in transformed cells and may have a potential tumor suppressor activity. In the lung, caveolae are present in the endothelium, smooth muscle cells, fibroblasts as well as in type I pneumocytes. The presence of caveolae and caveolin expression in the bronchial epithelium, although probable, has not been investigated in human. We were interested to see if the bronchial epithelia express caveolins and if this expression was modified in cancer cells. We thus tested for caveolin-1 and -2 expression several bronchial epithelial primary cell lines as well as eight lung cancer cell lines and one larynx tumor cell line. Both caveolin-1 and -2 are expressed in all normal bronchial cell lines. With the exception of Calu-1 cell line, all cancer cell lines showed very low or no expression of caveolin-1 while caveolin-2 expression was similar to the one observed in normal bronchial epithelial cells.

Asthma treatment has moved from bronchodilator therapy to an emphasis on anti-inflammatory therapy. Airway inflammation is believed to induce airway hyper-responsiveness (AHR) through the release of mediators that increase the airway response to agonists. However, the exact contribution of airway inflammation in the physiology of airway hyper-responsiveness remains undefined. Structural modifications in airways resulting from inflammation may contribute to the development and persistence of AHR and the development of asthma. This paper reviews some of the main components of airway inflammation and structural changes in asthma, and discusses how these processes may interact to modify airway function and induce respiratory symptoms.

Given the large phenotypic diversity of asthma, our aim was to characterize molecular profiles related to asthma severity using selected remodeling biomarkers in induced sputum.Induced sputum from healthy controls, patients with mild to moderate asthma and severe asthma were collected. Twelve selected biomarkers previously associated to airway remodeling such as connective tissue growth factor (CTGF), fibroblast growth factor (FGF)-2, matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, procollagen type 1 and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in sputum samples using ELISA or Luminex technology. FGF-2 level was also evaluated in bronchial biopsies using immunohistochemistry.Sputum of severe asthma was characterized by reduced percentage of macrophages and increased percentage of neutrophils and eosinophils. FGF-2, MMP-1 and TIMP-1 levels increased with asthma severity. Interestingly, only FGF-2 level inversely correlated with FEV1/FVC ratio. Although percentage of eosinophils correlated with asthma severity, it did not correlate with FGF-2 levels. Increased levels of FGF-2 with asthma severity were confirmed in bronchial biopsies by immunohistochemistry.Level of FGF-2 in induced sputum represents a relevant remodeling biomarker of asthma severity and significantly correlates with pulmonary function. FGF-2 sputum biomarker is proposed to reveal the phenotype of asthma characterized by fixed airflow obstruction.

Background Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-β1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects. Methods Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-β1, IL-22 and TGF-β1+IL-22. Results Primary bronchial epithelial cells stimulated with TGF-β1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-β1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics. Conclusion The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-β1 cooperativity in driving EMT in primary human bronchial epithelial cells.

Background Chronic airway diseases, including asthma, are characterized by increased airway smooth muscle (ASM) mass that is due in part to growth factor-mediated ASM cell proliferation and migration. However, the molecular mechanisms underlying these effects are not completely understood. Semaphorin 3E (Sema3E) has emerged as an essential mediator involved in cell migration, proliferation, and angiogenesis, although its role in ASM cell function is not investigated. Objectives We sought to determine the expression of Sema3E receptor, plexinD1, in human ASM cells (HASMCs); effect of Sema3E on basal and platelet-derived growth factor (PDGF)-induced proliferation and migration; and underlying signaling pathways. Methods Expression of plexinD1 in HASMCs was studied with RT-PCR, immunostaining, and flow cytometry. The effect of Sema3E on HASMC proliferation and migration was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation, cell count, and Boyden chamber assay. Sema3E-mediated intracellular signaling was investigated with fluorescent microscopy, flow cytometry, Rac1 activation, and Western blot analysis. Results HASMCs from healthy persons expressed plexinD1 more than HASMCs from asthmatic patients. Sema3E increased plexinD1 expression in HASMCs from asthmatic patients. Recombinant Sema3E inhibited PDGF-mediated HASMC proliferation and migration, which was associated with F-actin depolymerization, suppression of PDGF-induced Rac1 guanosine triphosphatase activity, and Akt and extracellular signal-regulated kinase 1 and 2 phosphorylation. Bronchial biopsies from patients with mild asthma displayed immunoreactivity of plexinD1, suggesting the potential in vivo role of Sema3E–PlexinD1 axis in HASMC function. Conclusion This study provides the first evidence that Sema3E receptor is expressed and plays functional roles in HASMCs. Our data suggest a regulatory role of Sema3E in PDGF-mediated proliferation and migration, leading to downregulation of ASM remodeling.

Background Factors associated with asthma remission need to be determined, particularly when remission occurs in adulthood. Objective To evaluate airway responsiveness and inflammation in adult patients in asthma remission compared with adults with mild, persistent symptomatic asthma. Methods Adenosine monophosphate and methacholine responsiveness were evaluated in 26 patients in complete remission of asthma, 16 patients in symptomatic remission of asthma, 29 mild asthmatic patients and 15 healthy controls. Blood sampling and induced sputum were also obtained to measure inflammatory parameters. Results Perception of breathlessness at 20% fall in forced expiratory volume in 1 s was similar among groups. In subjects with symptomatic remission of asthma, responsiveness to adenosine monophosphate and methacholine was intermediate between mild asthma and complete asthma remission, with the latter group similar to controls. Asthma remission was associated with a shorter duration of disease. Blood immunoglobulin E levels were significantly increased in the asthma group, and blood eosinophils were significantly elevated in the complete asthma remission, symptomatic remission and asthma groups compared with controls. The suppressive function of regulatory T cells was lower in asthma and remission groups compared with controls. Conclusion A continuum of asthma remission was observed, with patients in complete asthma remission presenting features similar to controls, while patients in symptomatic asthma remission appeared to be in an intermediate state between complete asthma remission and symptomatic asthma. Remission was associated with a shorter disease duration. Despite remission of asthma, a decreased suppressor function of regulatory T cells was observed, which may predispose patients to future recurrence of the disease.

International audience; Background: Airway disorders are common in regular chlorinated swimming pool attendees, particularly competitive athletes, but the impact of intense swimming training on airway function and structure remains unclear.Objective: This study aimed to evaluate airway inflammation and remodeling in elite swimmers.Methods: Twenty-three elite swimmers were tested during off-training season. All had exhaled nitric oxide measurement, methacholine test, eucapnic voluntary hyperpnea challenge, allergy skin prick tests, and bronchoscopy with bronchial biopsies. Clinical data and tissues from 10 age-matched mild-asthmatic and 10 healthy nonallergic subjects were used for comparison.Results: Swimmers had increased airway mucosa eosinophil and mast cell counts than did controls (P < .05). They had more goblet cell hyperplasia and higher mucin expression than did healthy or asthmatic subjects (P < .05). A greater submucosal type I and III collagen expression and tenascin deposition was also observed in swimmers than in controls (P < .05). Neither exhaled nitric oxide nor airway responsiveness to methacholine or eucapnic voluntary hyperpnea challenge correlated with these inflammatory and remodeling changes.Conclusion: Intense, long-term swimming training in indoor chlorinated swimming pools is associated with airway changes similar to those seen in mild asthma, but with higher mucin expression. These changes were independent from airway hyperresponsiveness. The long-term physiological and clinical consequences of these changes remain to be clarified.

Background Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma. Objectives and Methods We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using 3H-thymidine incorporation, cell count and Boyden chamber assays. Results PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-17 (IL-17) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the fibroblast growth factor-2 (FGF-2)-driven HASMC chemotactic activity. Conclusions Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma.