Sabina Lantero

Downregulation of the expression of intercellular adhesion molecule (ICAM)-1 on bronchial epithelial cells by fenoterol, a β2-adrenoceptor agonist

Inflammatory airway disorders, such as asthma and chronic bronchitis, are characterized by overexpression of adhesion molecules on airway epithelial and endothelial cells. This phenomenon is associated with increased adherence and activation of polymorphonuclear leukocytes (PMNs). With the knowledge that beta2-adrenoceptor agonists demonstrate some anti-inflammatory activity in vitro, the present study was designed to evaluate whether fenoterol could interfere with adhesion molecule expression on airway epithelium. Human bronchial epithelial cells (HBECs), obtained by protease digestion from surgically resected bronchi, were stimulated with human recombinant interferon-gamma (rh IFN-gamma) in the presence of (a) fenoterol (10(-12)-10(-5) M); (b) dexamethasone (10(-12)-10(-5) M); and (c) fenoterol and dexamethasone. Because desensitization after high-dose exposure to agonists has been described for many membrane-associated receptors, in additional sets of experiments HBECs were preexposed to fenoterol and, as control, to dexamethasone for 8 hr, then washed and stimulated with rh IFN-gamma in the presence of fresh drugs. The cells were harvested after 24-hr culture and stained by specific monoclonal antibodies. The intensity of intercellular adhesion molecule-1 (ICAM-1) expression was then measured by flow cytometry analysis and expressed as mean fluorescence channel (mfc). The significant increase in ICAM-1 expression on HBECs induced by rh IFN-gamma was inhibited, in a dose-dependent manner, by the two drugs, but fenoterol was more efficient than dexamethasone at all of the concentrations tested (p < 0.05, all comparisons). In addition, the inhibitory activity of fenoterol was not enhanced by the simultaneous presence of dexamethasone in rh IFN-gamma-stimulated HBEC cultures (p > 0.05, all comparisons). Finally, preexposure to fenoterol or to dexamethasone did not induce any modification of the inhibitory effect of the two drugs on ICAM-1 expression (p > 0.05, all comparisons). These results suggest that clinical efficacy of fenoterol in patients with obstructive lung disease may include downregulation of adhesion molecule expression on airway epithelial cells.

An improved method to obtain highly differentiated monolayers of human bronchial epithelial cells.

SummaryElectrophysiological studies of human bronchial epithelial cells in vitro are limited by the scarcity of biological material available for primary culture. To overcome this problem, we set up a protocol in which the cell number is first enlarged in LHC9/RPMI 1640 serum-free medium for up to six passages, each passage giving a four- to eightfold amplification. The cells are then plated at high density on permeable supports. Cell differentiation, monitored by measuring transepithelial potential difference (PD) and electrical resistance (R), is induced with a medium containing serum and a cocktail of different supplements and hormones. Maximal values of PD and R, obtained after 4–7 d of culture on permeable supports, are around −50 mV and 3000–4000 ω/cm2, respectively. Ussing chamber experiments show that basal short-circuit current (ISC) is partially inhibited by the epithelial Na+ channel blocker amiloride. Stimulation with a cAMP-elevating agent induces a ISC increase that is inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Our culture protocol provides a large number of differentiated bronchial epithelial cell monolayers starting from a low amount of material. This characteristic is useful for in vitro studies of ion transport in airway epithelium.

Stimulation of Blood Mononuclear Cells of Atopic Children with the Relevant Allergen Induces the Release of Eosinophil Chemotaxins Such as IL-3, IL-5, and GM-CSF

Peripheral blood mononuclear cells (PBMC) from 10 atopic asthmatic children (atopics), sensitized to Dermatophagoides pteronyssinus (Dp), and from 5 nonatopic healthy children (controls) were stimulated with Dp extract or with birch extract (Be). After 6 days we tested the supernatants (Sn) chemotactic activity toward purified blood eosinbnophils and T-lymphocyte proliferation. Dp induced a statistically significant T-cell proliferation from atopics as compared to controls (p < 0.05), which correlated with the levels of eosinophil chemotactic activity in the Sn (r = 0.713; p < 0.05). Measurable levels of IL-3, IL-5, and GM-CSF were demonstrated in the Sn of Dp-stimulated PBMC from atopics, while eosinophil locomotion toward different concentrations of recombinant human (rh) IL-3, rhIL-5, and rhGM-CSF confirmed that these cytokines were able to stimulate eosinophil chemotaxis in a close concentration range. Preincubation of different concentrations of the same Sn with blocking antisera demonstrated that anti-human (ah) IL-3, ahIL-5, and ahGM-CSF effectively decreased eosinophil chemotaxis (p < 0.05; each comparison). Thus PBMC activation with the relevant allergen induces the release by T cells with a Th2 phenotype of chemotactic factors for eosinophils.

Nasal brushing: a clinically useful procedure in pediatric patients with rhinosinusitis?

Sinusitis is a common complication of non-allergic and allergic rhinitis, and can trigger lower respiratory diseases, such as bronchitis and asthma. Standard radiography is unable to give any data about the underlying pathological mechanisms (infectious or allergic) involved and infectious rhinosinusitis is very common in pediatric age, even in allergic patients. We investigated the possibility of obtaining more useful diagnostic information, performing nasal brushing (NB) on 117 children with recurrent respiratory symptoms. The following hypothesis were evaluated: (1) whether NB neutrophil/eosinophil percentages and/or NB culture could predict the radiological evidence of maxillary sinusitis; and (2) whether differences between nonallergic and allergic patients could be detected. In the total patient group and in the nonallergic group, the comparison of NB neutrophil percentages in patients with and without maxillary sinusitis showed a statistically significant difference (median 2 and 18%, respectively; P < 0.001). In the nonallergic group, a NB neutrophil rate > or = 5% was chosen as a cut-off between positive and negative NB diagnosis of rhinosinusitis and NB data were compared with radiological investigations. The results obtained showed that NB was fairly sensitive (91%) and predictive (84%). In allergic patients, neither neutrophil nor eosinophil percentages significantly correlated with the presence of sinusitis. Microbiological studies showed that, even if the presence of bacteria in NB resulted associated with sinusitis, a negative culture was not predictive of the absence of the disease. We therefore suggest that NB describes the present inflammatory status of the upper airways, hence, it is more suitable to describe the inflammation related to ongoing upper respiratory tract infections rather than chronic inflammation due to allergic rhinitis, characterized by relapsing episodes of acute inflammation. In conclusion, we propose to consider NB a reliable tool in the diagnosis of rhinosinusitis, particularly in nonallergic pediatric patients. Compared to standard radiological techniques, NB makes it possible to avoid radiation exposure and gives information about the pathological mechanisms involved in the single patient.

Requirement for Different Presenting Cells and for Different Processing Mechanisms by Human CD4 T Helper Clones Specific for M. tuberculosis Antigens

Human T helper cells specific for mycobacterial antigens have been extensively investigated. Differences have been detected according to antigen specificity and to fine epitope specificity. In this work we have analyzed two additional parameters that allow discrimination among antigen specific T helper cells: requirement for certain types of antigen presenting cells (APC) and requirement for protease-sensitive antigen processing pathways. We used T cell clones from peripheral blood or from pleural exudates, and specific for different antigenic fractions of M. tuberculosis. APC were autologous peripheral blood mononuclear cells, adherent monocytes, adherent pleural monocytes, EBV transformed B lymphocytes and dendritic cells. Seven clones out of twelve were stimulated by all APC irrespective of their specificity, whereas other clones had more selective requirements. When protease inhibitors were used during antigen pulsing of APC, the production of certain epitopes, and thus T cell activation, was impaired with six clones out of sixteen. These results demonstrate that the human T helper repertoire specific for mycobacterial antigens is highly diverse also according to APC populations needed for presentation and to processing mechanisms required for production of the relevant T epitopes.

Insensitivity of volume-sensitive chloride currents to chromones in human airway epithelial cells

1 Chromones (sodium cromoglycate and sodium nedocromil) block cell swelling‐activated Cl− channels in NIH‐3T3 fibroblasts and endothelial cells. This has led to hypothesize that cell volume regulation might be involved in asthma pathogenesis. 2 Using whole‐cell patch‐clamp experiments, we studied the effect of chromones on volume‐sensitive Cl− currents in transformed human tracheal epithelial cells (9HTEo‐) and in primary cultures of human bronchial epithelial cells (BE). 3 Cl− currents activated by hypotonic shock were poorly blocked by extracellular nedocromil or cromoglycate. The block was voltage‐dependent since it was observed only at positive membrane potentials. At the concentration of 5 mm, the current inhibition by both chromones at +80 mV was about 40% for 9HTEo‐ and only 20% for BE. 4 Intracellular application of chromones elicited a voltage‐independent inhibition in 9HTEo‐ cells. Under this condition, volume‐sensitive Cl− currents were reduced at all membrane potentials (60 and 45% inhibition by 2 mm nedocromil and cromoglycate respectively). In contrast intracellular chromones were ineffective in BE cells. 5 The relative refractoriness to chromones, in contrast with the high sensitivity shown by other Cl− channels, suggests that the epithelial volume‐sensitive Cl− channel is not involved in asthma.

Characteristics and clinical significance of the lymphocytic alveolitis in interstitial lung disorders

Although the mechanisms responsible for lung damage and respiratory function deterioration for each type of alveolitis are not entirely known, with the opportunity to study the cells present in the lower respiratory tract, their functions and the mediators released in different conditions, we will be able to better understand the link between the inflammatory process, the acute tissue damage, the progression of the disease and the pulmonary scarring. This knowledge will be helpful in a better management of patients with interstitial lung diseases modulated by immunologic mechanisms.