Involvement of fibrocytes in asthma and clinical implications.

Bloodborne fibrocytes are bone marrow-derived cells that participate in immune responses and exhibit pro-inflammatory and matrix remodelling properties. In patients with asthma receiving an adequate treatment, the blood fibrocyte count is very low and comparable to that obtained in healthy individuals. In these patients, a transient increase in fibrocyte numbers in the peripheral blood and in the airways occurs in concomitance with increased bronchial inflammation and reflects disease worsening and the need for more intensive treatment. Persistently elevated numbers of fibrocytes in the peripheral blood and in the bronchial mucosa are observed in chronically undertreated or corticosteroid-resistant asthma and are associated with persistent airway inflammation and ongoing remodelling of the bronchial wall. The asthmatic bronchial epithelium is the main source of fibrocyte chemoattractants in asthma and contributes with T helper type 2 lymphocytes and eosinophils to promote the proliferation and pro-remodelling function of recruited fibrocytes. The presence of elevated numbers of fibrocytes in the bronchial mucosa of allergic patients with undertreated or treatment-resistant asthma may also increase the risk of acute exacerbations because these cells can amplify T helper type 2 lymphocyte-driven inflammation on every exposure to the clinically relevant allergen and can promote further inflammation on rhinovirus infections by allowing viral replication and releasing additional pro-inflammatory factors. Improved methods for the isolation and functional analysis of pure populations of viable circulating fibrocytes have allowed a better understanding of the effector role of these cells. A reliable and clinically applicable assay has been developed to measure blood fibrocyte counts as outcome measure in future clinical trials. New therapeutic agents are needed to block both persistent inflammation and fibrocytosis in corticosteroid-resistant asthma.

The C-C motif chemokine ligands CCL5, CCL11, and CCL24 induce the migration of circulating fibrocytes from patients with severe asthma.

The C-C motif chemokine ligand 5 (CCL5), CCL11, and CCL24 are involved in the pathogenesis of asthma, and their function is mainly associated with the airway recruitment of eosinophils. This study tested their ability to induce the migration of circulating fibrocytes, which may contribute to the development of irreversible airflow obstruction in severe asthma. The sputum fluid phase (SFP) from patients with severe/treatment-refractory asthma (PwSA) contained elevated concentrations of CCL5, CCL11, and CCL24 in comparison with the SFP from patients with non-severe/treatment-responsive asthma (PwNSA). The circulating fibrocytes from PwSA expressed the receptors for these chemokines at increased levels and migrated in response to recombinant CCL5, CCL11, and CCL24. The SFP from PwSA induced the migration of autologous fibrocytes, and its activity was significantly attenuated by neutralization of endogenous CCL5, CCL11, and CCL24. These findings suggest that CCL5, CCL11, and CCL24 may contribute to the airway recruitment of fibrocytes in severe asthma.

Interleukin (IL)-4, IL-13, and IL-17A differentially affect the profibrotic and proinflammatory functions of fibrocytes from asthmatic patients.

Fibrocytes contribute to the fibrotic changes most frequently observed in forms of asthma where inflammation is driven by T helper type 2 (Th2) cells. The mechanisms that regulate the profibrotic function of asthmatic fibrocytes are largely unknown. We isolated circulating fibrocytes from patients with allergen-exacerbated asthma, who showed the presence of fibrocytes, together with elevated concentrations of interleukin (IL)-4 and IL-13 and slightly increased concentrations of the Th17 cell-derived IL-17A, in induced sputum. Fibrocytes stimulated with IL-4 and IL-13 produced high levels of collagenous and non-collagenous matrix components and low levels of proinflammatory cytokines. Conversely, fibrocytes stimulated with IL-17A proliferated and released proinflammatory factors that may promote neutrophil recruitment and airway hyperresponsiveness. IL-17A also indirectly increased α-smooth muscle actin but not collagen expression in fibrocytes. Thus, fibrocytes may proliferate and express a predominant profibrotic or proinflammatory phenotype in asthmatic airways depending on the local concentrations of Th2- and Th17-derived cytokines.

Allergen-induced generation of mediators in the mucosa.

The inhalation of antigens does not normally lead to allergic inflammation, but airway resident cells and their products may affect the outcome of antigen exposure. It is therefore important to elucidate how potential allergens interact with airway epithelial cells and other cells located within and below the epithelium. Some studies have indicated that certain antigens, particularly the major house dust mite antigen Der p1, penetrate the airway epithelium by intracellular transportation or paracellular passage, depending on their concentrations, time of exposure, and ability of the cells to inactivate them. If an antigen possesses proteolytic activity, such as Der p1, and it reaches high concentrations or the exposure is prolonged, the disruption of the tight junction can also favor the transepithelial passage of other antigens. In this way, the antigens can easily encounter the effector cells located between epithelial cells and below the basement membrane. The magnitude of this phenomenon may be more prominent in the airways of asthmatic patients, as their epithelium is more permeable to Der p1 than the epithelium of nonasthmatic patients and releases cytokines after exposure to very low concentrations of this antigen for brief periods. Epithelial cell activation may facilitate the development of allergic mucosal sensitization to Der p1 and contribute to the antigen-induced inflammatory response by affecting the migration and function of dendritic cells, mast cells, and eosinophils. Also, there might be a secondary release of interleukin-6 and endothelin-1, which can have a detrimental effect on the cardiovascular function.

Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells.

The respiratory epithelium represents the first barrier encountered by airborne Ags. Two major dust mite Ags, Der p3 and Der p9, are serine proteases that may activate lung epithelial cells by interaction with the protease-activated receptor 2 (PAR-2). In this study both Der p3 and Der p9 cleaved the peptide corresponding to the N terminus of PAR-2 at the activation site. Both Ags sequentially stimulated phosphoinositide hydrolysis, transient cytosolic Ca(2+) mobilization, and release of GM-CSF and eotaxin in human pulmonary epithelial cells. These responses were similar to those observed with trypsin and a specific PAR-2 agonist and were related to the serine protease activity of Der p3 and Der p9. Cell exposure to the Ags resulted in a refractory period, indicating that a PAR had been cleaved. Partial desensitization to Der p3 and Der p9 by the PAR-2 agonist suggested that PAR-2 was one target of the Ags. However, PAR-2 was not the only target, because the PAR-2 agonist caused less desensitization to Der p3 and Der p9 than did trypsin. A phospholipase C inhibitor prevented the cytokine-releasing effect of the PAR-2 agonist and abolished or reduced (>70%) the cytokine-releasing effects of Der p3 and Der p9. Our results suggest that Der p 3 and Der p9 may induce a nonallergic inflammatory response in the airways through the release of proinflammatory cytokines from the bronchial epithelium and that this effect is at least in part mediated by PAR-2.

Kinetics of eotaxin expression and its relationship to eosinophil accumulation and activation in bronchial biopsies and bronchoalveolar lavage (BAL) of asthmatic patients after allergen inhalation.

We investigated the kinetics of allergen-induced eotaxin expression and its relationship to eosinophil accumulation and activation in the airways of patients with allergic asthma. Twenty-four patients with allergic asthma and late asthmatic responses to allergen inhalation were randomly allocated into three groups of eight patients each, who received bronchoscopy with bronchial biopsies and BAL at 2, 4 and 24 h, respectively, after the inhalation of the diluent and the allergen. The expression of eotaxin mRNA and protein and eotaxin release were evaluated by in situ hybridization, immunohistochemistry, immunocytochemistry, and radioimmunoassay. Increased transcription from the eotaxin gene preceded the appearance of the late asthmatic response and the influx of activated eosinophils in bronchial tissue and BAL fluid (BALF). This was followed by increased cell expression of eotaxin protein (P<0.001) and increased eotaxin release (P<0.001), which correlated with the numbers of total and activated eosinophils and the level of airflow obstruction at 4 h after allergen exposure (P<0.05 for all correlations). At 24 h after allergen inhalation, enhanced eotaxin expression declined without a similar reduction in the numbers of eosinophils in bronchial biopsies and when there was a further increase in the number of these cells in BALF (P<0.05). These results indicate that eotaxin contributes to the early phase of allergen-induced recruitment of activated eosinophils into the airways of patients with allergic asthma and that other factors are implicated in the persistence of eosinophil infiltration.

Cellular and molecular characteristics of inflammation in chronic bronchitis.

METHODS: To examine the inflammatory process in chronic bronchitis, we evaluated the cell and cytokine profile in bronchoalveolar lavage fluid from 12 chronic bronchitis patients who smoked, six chronic bronchitis patients who did not smoke, 10 subjects control subjects without pulmonary diseases who smoked and eight control subjects who did not smoke. RESULTS: Chronic bronchitis patients who smoked had increased numbers of macrophages, neutrophils, eosinophils, mast cells and activated CD8+ T lymphocytes and predominantly expressed interleukin-8, tumour necrosis factor alpha and interleukin-2 genes and proteins. The number of macrophages and neutrophils and the expression of interleukin 8 were also increased in control subjects who smoked compared with healthy subjects who did not smoke. Chronic bronchitis patients who did not smoke had increased numbers of eosinophils, mast cells and activated CD4+ T lymphocytes and predominantly expressed interleukin-5 and granulocyte-macrophage colony-stimulating factor genes and proteins. CONCLUSION: Thus, the cellular and molecular characteristics of the inflammatory process in chronic bronchitis patients who smoke and do not smoke are different, suggesting a different pathogenesis of the disease.

The role of CD8+ Th2 lymphocytes in the development of smoking-related lung damage.

There is increasing evidence for the existence of different subsets of CD8+ T lymphocytes in humans, according to their cytokine secretion profile. Resistance or susceptibility to infections and the outcome of some inflammatory processes may depend on the lymphokine profile which predominates. We show one consequence of the switching of a host CD8+ T cell response from the Th1 effector function to the Th2 pattern in relation to the exposure to a common toxicant and its pathogenetic implications. Chronic obstructive bronchitis is a pulmonary disease characterized by airway inflammation with predominance of CD8+ T lymphocytes, mucus hypersecretion, repeated airway infections, and decline in lung function. Though smoking-related, it affects only a portion of smokers. The results of this study, comparing the functional characteristics of CD8+ T cell clones from smokers with the disease, unaffected smokers and healthy individuals, indicate that the smokers who have a predominance of CD8+ T lymphocytes of the Th2 phenotype may be predisposed to develop more severe smoking-induced lung damage, with chronic airway inflammation, repeated infections and persistent airflow obstruction.

The allergen Der p1 induces NF-kappaB activation through interference with IkappaB alpha function in asthmatic bronchial epithelial cells.

Asthma is an inflammatory disease of the airways due to an interaction between genetic and environmental factors, and allergy represents the most important predisposing trait. Here, we investigated why and how the allergen most often implicated in the pathogenesis of asthma and other allergic diseases causes the expression of the genes for proinflammatory cytokines in airway epithelium. We found that Der p1 promotes activation of transcriptional factor NF-kappaB by interference with the function of its cytoplasmic inhibitor IkappaB alpha. This is the first report on the effect of an allergen on transcriptional factors. Our results improve the understanding of the mechanisms involved in allergic diseases and suggest potential therapeutic utility of NF-kappaB blockers.

Eotaxin expression and eosinophilic inflammation in asthma.

Asthma is a chronic inflammatory disease of the airways characterized by a marked infiltration of eosinophils in the bronchial mucosa, and the mechanisms that cause the selective recruitment of these cells are areas of active investigation. In this study, we found increased expression of the eosinophil chemoattractant eotaxin in bronchial mucosa of asthmatic patients. The increase in number of cells expressing eotaxin mRNA correlated with the number of eosinophils in the bronchial tissue and with two major clinical and functional indices of disease severity, suggesting that eotaxin is involved in the recruitment of eosinophils and in eosinophil-induced tissue damage in asthma. Cell sources of eotaxin were bronchial epithelial cells, T lymphocytes, macrophages and eosinophils themselves. The use of drugs that interfere with eotaxin synthesis and function may represent a more specific approach in asthma treatment.

Endothelin-1 induces bronchial myofibroblast differentiation.

Endothelin-1 may contribute to bronchial smooth muscle constriction and airway remodelling in asthma, where bronchial epithelial cells represent an important source of this peptide. We report here that asthmatic bronchial epithelial cells exposed to allergens in vitro induce the differentiation of airway fibroblasts into myofibroblasts, and that they do so through a granulocyte/macrophage colony-stimulating factor-mediated upregulation of endothelin-1 production. By this mechanism bronchial epithelial cells may participate in the genesis of bronchial subepithelial fibrosis, a process which contributes to airway narrowing in asthma.

Functional analysis of the preproendothelin-1 gene promoter in pulmonary epithelial cells and monocytes.

At least two human preproendothelin-1 mRNAs are produced by a single gene through the use of different promoters, and the mechanisms controlling the production of these alternative transcripts might be cell-specific. In human lung, endothelin-1 is produced by vascular endothelial cells, bronchial epithelial cells, and pulmonary monocytes/macrophages. Given the important role of endothelin-1 in the pathogenesis of some pulmonary diseases, we carried out a functional analysis of the preproendothelin-1 gene promoter(s) in bronchial epithelial cells and lung monocytes. Two distinct preproendothelin-1 mRNAs were expressed in the two cell populations. Using deletion mutants and enhancer trap transfection experiments, we also identified different regions of the preproendothelin-1 gene necessary for endothelin-1 expression in bronchial epithelial cells and pulmonary monocytes.

Inducibility of RANTES mRNA by IL-1beta in human bronchial epithelial cells is associated with increased NF-kappaB DNA binding activity.

RANTES is a member of a large supergene family of proinflammatory chemokines that seems to play an important role in inflammatory processes. It is produced by many cell types in response to specific stimuli and during inflammatory reactions, but the marked differences in the pattern of induced expression suggest that different control mechanisms regulate transcription of RANTES in various tissue types. This is supported by the presence of a large number of potential binding sites for transcriptional factors in the promoter region of the RANTES gene. Our data indicate that expression of RANTES mRNA induced by IL-1beta in human lung epithelial cells is associated with the activation of the transcriptional factor NF-kappaB.

Endothelin-1 induces increased fibronectin expression in human bronchial epithelial cells.

Endothelin-1 may be involved in the pathogenesis of asthma by causing bronchial smooth muscle constriction and airway remodelling. Bronchial epithelial cells represent an important source of endothelin-1 in this disease, and increased release of epithelial cell-derived endothelin-1 may contribute to the genesis of subepithelial fibrosis by promoting fibroblast proliferation and collagen production. In this study, we demonstrate that endothelin-1 upregulates fibronectin gene expression and fibronectin release in bronchial epithelial cells via an ETA receptor. Fibronectin is an important component of the extracellular matrix which is deposited in excess in the subepithelial area of asthmatic bronchial mucosa, and it represents a potent chemotactic factor for fibroblasts. Thus, endothelin-1 may induce subepithelial fibrosis both directly and by the autocrine mechanism reported here.

Bronchial epithelial cells of atopic patients with asthma lack the ability to inactivate allergens.

The cause of asthma is still unknown. Though most asthmatic patients are atopic, only certain atopic subjects develop this disease, and atopic asthmatics recover from asthma after transplantation of lungs from nonasthmatic donors. Thus, there might be a primary local defect in the bronchial wall which affects the expression of asthma in atopic individuals. We show here that the bronchial epithelial cells from atopic patients with asthma are aberrantly permeable to the airborne allergen most often implicated in the pathogenesis of this disease, indicating how it can have access to the antigen-presenting cells (dendritic cells) below the apical surface of the epithelium in vivo. In addition, allergen exposure induces asthmatic epithelial cells to express granulocyte/macrophage colony-stimulating factor, a cytokine involved in the proliferation and function of dendritic cells.

Constitutive expression of endothelin in bronchial epithelial cells of patients with symptomatic and asymptomatic asthma and modulation by histamine and interleukin-1.

BACKGROUND: An upregulation of endothelin-1 expression occurs in bronchial epithelial cells of asthmatic patients. This peptide may mediate bronchoconstriction in asthma, but the mechanisms that modulate endothelin-I synthesis and release are unknown. OBJECTIVE: This study was done to compare the pattern of endothelin-1 expression in patients with symptomatic and asymptomatic asthma and evaluate the ability of inflammatory factors to upregulate endothelin-1 synthesis and release in the epithelial cells of subjects who are free of symptoms. METHODS: Two groups of 10 asthmatic patients were selected. One group had symptomatic asthma with airflow obstruction and moderately to severely increased airway responsiveness. The second group was free of symptoms: they did not show airflow obstruction, and airway responsiveness was borderline or slightly increased. Bronchial biopsy specimens were obtained by means of bronchoscopy and used for immunohistochemical evaluation, epithelial cell isolation, and stimulation experiments with interleukin-1 and histamine. RESULTS: Endothelin-1 immunoreactivity was detected in vivo in the bronchial epithelial cells of all the patients with symptoms and in only two subjects without current symptoms. Incubation of bronchial epithelial cells from patients with asymptomatic asthma with interleukin-1 or histamine, for 8 to 24 hours, resulted in increased expression of endothelin-1 messenger RNA and release of appreciable amounts of the peptide to the culture medium. Those effects were dose- and time-dependent. Histamine and interleukin-1 were effective at concentrations similar to those detected in the bronchoalveolar lavage of patients with symptomatic asthma. CONCLUSION: Endothelin-expression is upregulated in bronchial epithelial cells of asthmatic patients with symptoms and evidence of functional derangement as compared with patients without symptoms and airflow obstruction. Exposure of cells from patients with asymptomatic asthma to factors that are released during acute exacerbation of the disease induces endothelin synthesis and release.

Increased peak flow variability in children with asymptomatic hyperresponsiveness.

The relevance of increased methacholine airway responsiveness detected in children with no current or past symptoms of asthma is not known. We wished to determine whether the presence of airway hyperresponsiveness in asymptomatic children is also associated with abnormal variability of peak expiratory flow (PEF). In 12 asymptomatic children with methacholine hyperresponsiveness, we examined the diurnal variation of peak expiratory flow (PEF) and response to inhaled bronchodilator. Twelve asthmatic children with a comparable range of methacholine hyperresponsiveness, and 12 normal children without methacholine responsiveness, were used as positive and negative controls. The children were aged 11 (range 9-14) yrs. The mean diurnal variation of PEF in those children with asymptomatic hyperresponsiveness was increased at 9.3%, to a degree comparable to the symptomatic asthmatic children (10.7%), and greater than the normal children (5.7%). Methacholine stimulated airway constriction was associated with symptoms in subjects from each group, indicating that the children were capable of perceiving airway constriction. We conclude that asymptomatic children with methacholine airway hyperresponsiveness have other evidence of mild variable airflow obstruction with increased diurnal PEF variability, and can perceive airflow limitation. The lack of symptoms in the children with airway hyperresponsiveness could be due to an insufficient stimulus to cause symptomatic obstruction, or the absence of eosinophilic airway inflammation, which may be a requirement for the development of symptomatic airway hyperresponsiveness.

Mast cell chemotactic activity of RANTES.

RANTES is a cytokine produced by activated T-lymphocytes that has been shown to exert chemotactic activity for memory-type CD4 T-lymphocytes and eosinophils. In this study, RANTES caused directional migration of human mast cells. When compared to other potential chemoattractants of the same cells, RANTES was found to be more potent than fibronectin and the c-kit receptor ligand, on a molar basis. This cytokine may be a common mechanism in allergic reactions which culminate in the selective migration of memory CD4 T-lymphocytes, eosinophils and mast cells at the tissue site. Asthma and allergic rhinitis may represent possible clinical examples.

Detection of cytokines and their cell sources in bronchial biopsy specimens from asthmatic patients. Relationship to atopic status, symptoms, and level of airway hyperresponsiveness.

This study evaluated immunoreactivity for several cytokines in bronchial tissue of asthmatic patients and related this to the clinical and functional characteristics. Patients were allocated into two different groups on the basis of their atopic status (atopic and nonatopic), with two subgroups of symptomatic and asymptomatic subjects in each. Five healthy volunteers were tested as control subjects. After clinical and functional assessment, all of the subjects underwent bronchoscopy. Several biopsy specimens were obtained for immunohistochemical and immunoelectron microscopic evaluation. Symptomatic asthmatic subjects had increased expression of immunoreactive interleukin (IL) 1 beta, IL-2, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF alpha) when compared to the asymptomatic patients or normal control subjects. The cell sources of IL-1 beta were monocytes and dendritic cells in atopic patients and monocytes alone in nonatopic asthmatic subjects. The CD4+ T lymphocytes from atopic asthmatic subjects predominantly expressed IL-3, IL-4, IL-5, and GM-CSF immunoreactivity, whereas CD4+ T cells from nonatopic patients predominantly expressed IL-2, IL-3, and IL-5, and GM-CSF immunoreactivity. Mast cells showed immunoreactivity for TNF alpha, IL-3, IL-5, and GM-CSF. Immunostaining for TNF alpha and GM-CSF was also detected in bronchial epithelial cells and monocytes. Tissue eosinophilia and the level of airway hyperresponsiveness more closely correlated with IL-5 immunoreactivity in atopic asthmatic subjects and with IL-2 and GM-CSF immunoreactivity in nonatopic patients.

Bronchoconstrictive responses to inhaled ultrasonically nebulized distilled water and airway inflammation in asthma.

Twenty-two asthmatic patients with a range of airway hyperresponsiveness to methacholine underwent a bronchial challenge with ultrasonically nebulized distilled water (UNDW). The presence of positive responses to this stimulus was related to the extent of airway inflammation, as assessed by histochemical and immunohistochemical evaluation of bronchial biopsy specimens. Twelve patients had airflow obstruction during distilled water inhalation and they showed more severe disease than subjects with no response, as demonstrated by the higher degree of nonspecific bronchial hyperresponsiveness (p < 0.01), higher variability of peak expiratory flow rates (p < 0.01), symptom scores (p < 0.01), and daily use of bronchodilators (p < 0.01). Those patients also had increased numbers of mast cells and eosinophils (p < 0.01) and increased percentage of bronchial epithelial cells expressing endothelin 1 immunoreactivity (p < 0.01). Thus, positive responses to inhaled UNDW reflect the bronchial hyperresponsiveness consistent with moderate to severe asthma and may be due to the release of mediators with bronchoconstrictive properties from inflammatory cells or activated resident cells or both.