Maurizio Marini

Expression of the potent inflammatory cytokines, granulocyte-macrophage-colony-stimulating factor and interleukin-6 and interleukin-8, in bronchial epithelial cells of patients with asthma

We have previously demonstrated that cultured human bronchial epithelial cells produce cytokines with potent proinflammatory properties on exposure to several stimuli in vitro, and we have hypothesized that these epithelial cell-derived factors may contribute to the pathogenesis of some inflammatory diseases of the bronchial mucosa, particularly asthma, by promoting the infiltration of granulocytes and T cells and their local activation. We provide, in this study, direct evidence of an increased expression of granulocyte-macrophage-colony-stimulating factor, interleukin-6, and interleukin-8 genes and proteins in bronchial epithelium from patients with symptomatic asthma. The up regulation of the production of these cytokines in bronchial epithelial cells of patients with asthma could be abolished in vitro by corticosteroids (hydrocortisone, 10(-7) mol/L), but the up regulation also spontaneously disappeared during a period of 6 days after the removal of the cells from the diseased tissue.

Levels of endothelin in the bronchoalveolar lavage fluid of patients with symptomatic asthma and reversible airflow obstruction

We have previously demonstrated that human bronchial smooth muscle cells possess a single class of specific binding sites for the potent bronchoconstrictive peptide, endothelin 1, and that human bronchial epithelial cells constitutively release an endothelin-like material in culture, which binds to smooth muscle cell receptors with a kinetic analogous to that observed with the authentic peptide. To evaluate the potential role of endothelin in the pathogenesis of asthma, we examined in this study the release of endothelin in the airways of six patients with asthma, both at the time when they were symptomatic and had reversible airflow obstruction and during the remission phase of the disease induced by treatment. Five normal volunteers and five patients with chronic bronchitis and airflow obstruction unaffected by bronchodilators were tested as control subjects. The release of endothelin in airway mucosa was assessed by RIA with the bronchoalveolar lavage fluid recovered during bronchoscopy. Patients with asthma had increased amounts of immunoreactive endothelin in bronchoalveolar lavage fluid than normal control subjects or subjects with chronic bronchitis (p less than 0.05) in absence of any significant alteration in the levels of circulating peptide. Treatment of patients with asthma with oral corticosteroids and inhaled beta-agonists for 15 days resulted in improvement of airflow obstruction and in more than threefold reduction in the contents of endothelin in lavage fluid. Our findings indicate that the potent bronchoconstrictive substance, endothelin, may contribute to the pathogenesis of airflow obstruction in asthma.

BRONCHIAL EPITHELIAL CELLS OF PATIENTS WITH ASTHMA RELEASE CHEMOATTRACTANT FACTORS FOR T LYMPHOCYTES

BACKGROUND T lymphocytes may orchestrate the inflammatory response in atopic asthma, but the mechanisms that promote T-cell accumulation in asthmatic airways are still unclear. In this study, we tested the hypothesis that bronchial epithelial cells of patients with atopic asthma release chemoattractant factors for T lymphocytes. METHODS Sixteen patients with atopic asthma and eight healthy control subjects were selected for this study. Bronchial epithelial cells were isolated from biopsy specimens obtained by means of bronchoscopy and cultured for 48 hours in serum- and hormone-free medium, with or without 10(-6) mol/L histamine. RESULTS Only the supernatants of cells from donors with asthma showed chemotactic activity for T lymphocytes, and this was significantly increased (p < 0.025) by exposure to histamine. Chemotactic activity was in part mediated by interleukin-8 (IL-8), because an antibody against human IL-8 significantly reduced it (p < 0.05) and the cell supernatants contained appreciable amounts of immunoreactive IL-8 (0.89 +/- 0.39 ng/ml). Both the residual chemotactic activity of unstimulated epithelial cells and the increased activity caused by histamine were mediated by a single protease-sensitive substance with an apparent molecular weight of 56,000 d and an estimated isoelectric point of 8.8 to 9.1. The partially purified chemoattractant specifically enhanced the migration of CD4+ T lymphocytes, and its activity was inhibited by the univalent Fab fragment of a monoclonal antibody against CD4. CONCLUSION These results extend our previous observations, indicating an important effector role of bronchial epithelium in asthma.

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.

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.

IL-33 promotes the migration and proliferation of circulating fibrocytes from patients with allergen-exacerbated asthma

The release of IL-33 increases in the bronchial mucosa of asthmatic patients in relation to disease severity and several studies have demonstrated that IL-33 may enhance airway inflammation in asthma. This study tested the hypothesis that IL-33 may also contribute to the development of irreversible structural changes in asthma by favoring the airway recruitment and profibrotic function of circulating fibrocytes during episodes of allergen-induced asthma exacerbation. The circulating fibrocytes from patients with allergen-exacerbated asthma (PwAA) showed increased expression of the specific IL-33 receptor component ST2L in comparison with the cells from non-asthmatic individuals (NAI). Recombinant IL-33 induced the migration of circulating fibrocytes from PwAA at clinically relevant concentrations and stimulated their proliferation in a concentration-dependent manner between 0.1 and 10 ng/ml, without affecting the constitutive release of type I collagen. The recombinant protein did not induce similar responses in circulating fibrocytes from NAI. This study uncovers an important mechanism through which fibrocytes may accumulate in the airways of allergic asthmatics when their disease is not adequately controlled by current treatment and provides novel information on the function of IL-33 in asthma.

Protective effect of nedocromil sodium on the IL1-induced release of GM-CSF from cultured human bronchial epithelial cells.

Cultured human bronchial epithelial cells constitutively produce granulocyte-macrophage colony-stimulating factor (GM-CSF). The synthesis and release of GM-CSF is upregulated in bronchial epithelium of patients with symptomatic asthma and this may contribute to the local activation of inflammatory cells in their bronchial mucosa. The cause of this upregulation of GM-CSF expression is unknown, but an increased release of interleukin-1 (IL1) from other airway resident cells might be involved, as an increase in GM-CSF production can be induced in vitro in normal bronchial epithelial cells by IL1 and the airway secretions of asthmatics contain high amounts of this cytokine. In the present study, we have evaluated the effect of the anti-inflammatory and antiasthmatic drug, nedocromil sodium, on the spontaneous and IL1-induced expression of GM-CSF in cultured bronchial epithelial cells. This compound, at the concentration of 10(-5) M, reduced the IL1-induced increase in GM-CSF release from epithelial cells by more than 40%, but it did not affect the constitutive production of GM-CSF.

The protective effect of frusemide on the generation of superoxide anions by human bronchial epithelial cells and pulmonary macrophages in vitro

Inhaled frusemide has been shown to inhibit bronchoconstriction induced by immunological and nonimmunological stimuli in asthmatic patients. The mechanisms by which this compound exerts its effect in asthmatic airways are unknown, but an inhibitory action on the activation of inflammatory cells or on the responsiveness of sensory epithelial nerves may be involved. In this study, we give evidence that frusemide prevents in part the activation of bronchial epithelial cells and pulmonary macrophages, as it reduces the rate of superoxide anion generation induced by IgE receptor cross-linking and by phorbol myristate acetate by 40-60%. The effect was not specific since we used stimuli which activate different signal transduction pathways for NADPH oxidase stimulation and frusemide was equally effective.

Involvement of fibrocytes in allergen-induced T cell responses and rhinovirus infections in asthma

Allergen exposure and rhinovirus infections that propagate from the upper to the lower airways are the most frequent causes of asthma exacerbation. In patients at increased risk of disease exacerbations, chronic airway inflammation is associated with the airway recruitment of circulating fibrocytes, bone marrow-derived CD34(+)CD45RO(+)CD11b(+)CD13(+)HLA-DR(+) progenitors that have antigen-presenting function and fibroblast-like properties. This study demonstrates that allergen-pulsed circulating fibrocytes from patients with allergic asthma are potent inducer of the predominant release of the T helper type (Th)2 cytokines IL-4 and IL-5 from autologous naïve and memory CD4(+) T cells. This study also provides evidence that circulating fibrocytes from allergic asthmatics are susceptible to rhinovirus infection. Infected cells release high amounts of pro-inflammatory cytokines with minimal production of IFN-α/β. Moreover, allergen-pulsed fibrocytes support prolonged rhinovirus replication and release larger quantities of pro-inflammatory cytokines upon rhinovirus infection than unpulsed fibrocytes. Thus, fibrocytes may amplify allergen-induced, Th2 cell-driven inflammatory responses and promote further inflammation by functioning as a reservoir for rhinovirus replication in asthmatic airways. Through these mechanisms, fibrocytes may play an important role in the provocation of disease exacerbations.

Enumeration of circulating fibrocytes for clinical use in asthma by an optimized single-platform flow cytometry assay.

Background Elevated numbers of circulating fibrocytes are associated with inadequately controlled asthma, poor response to available therapies, and increased risk of adverse outcomes. The lack of reliable and clinically-applicable assays precludes a proper evaluation of blood fibrocyte count as a prognostic biomarker in asthma. This report concerns the use of a multiparameter flow cytometry assay for the enumeration of fibrocytes in the whole blood. Methods Consenting fibrocyte donors were 19 patients with asthma well controlled by current treatment, 16 patients with treatment-resistant asthma, 9 patients with transiently uncontrolled asthma and 14 age-matched normal individuals. Blood sampling was performed once in patients with transiently uncontrolled asthma and twice, at an interval of one week, in the other subjects. The assay was performed in 100 μl of whole blood and involved a sequential gating strategy and absolute fibrocyte counting with a single instrument (single-platform assay). Results The quantification of circulating fibrocytes by this assay was analytically and clinically valid. In individuals with stable clinical conditions, the repeatability of blood fibrocyte counts over one week was good. The intraclass correlation coefficient was 0.939 and 96.88% of the total variability reflected on-average differences among the tested subjects. Stabilized blood samples could be stored at 4 °C for up to 96 h before processing. Conclusions The novel assay for the enumeration of fibrocytes in the whole blood is reliable and clinically applicable. General significance This report demonstrates the validity and reliability of the first optimized assay for the enumeration of circulating fibrocytes in multicenter clinical trials.