Federica Sabatini

Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate along different pathways including chondrogenic, osteogenic and adipogenic lineages. MSCs with a fibroblast-like morphology have been identified in human fetal lung. However, their frequency and characterization in human adult lung have not been yet evaluated. Therefore, we analyzed the mesenchymal phenotype and differentiation ability of cultured human adult bronchial fibroblast-like cells (Br) in comparison with those of mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (−), CD45 (−), CD90/Thy-1 (+), CD73/SH3, SH4 (+), CD105/SH2 (+) and CD166/ALCAM (+) in Br, ICIG7 and BM212 cells. There was one exception, STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments while they were all devoid of epithelial cytokeratins. Additionally, α-smooth muscle actin was also present in microfilaments of a low number of cells. All cell types predominantly produced collagen and fibronectin extracellular matrix as evidenced by staining with the monoclonal antibodies to collagen prolyl 4-hydroxylase and fibronectin isoforms containing the extradomain (ED)-A together with ED-B in ICIG7 cells. Br cells similarly to fetal lung and marrow fibroblasts were able to differentiate along the three adipogenic, osteogenic and chondrogenic mesenchymal pathways when cultured under appropriate inducible conditions. Altogether, these data indicate that MSCs are present in human adult lung. They may be actively involved in lung tissue repair under physiological and pathological circumstances.

Cysteinyl leukotrienes induce human eosinophil locomotion and adhesion molecule expression via a CysLT1 receptor-mediated mechanism

Background The mechanisms involved in eosinophil recruitment by cysteinyl‐leukotrienes (CysLTs) remain to be defined.

Epithelial cells and fibroblasts: structural repair and remodelling in the airways

Extensive lesions and changes in the architecture of the airway walls are commonly described in patients with respiratory infections, asthma, chronic bronchitis and interstitial lung diseases. Current knowledge identifies in airway epithelial cells and in fibroblasts the two cell types mainly involved in tissue repair after injury. During inflammatory respiratory disorders, extensive injury of airway epithelium may occur, with shedding of a large sheet of damaged cells in the bronchial and alveolar lumen but also with activation of the surviving epithelial cells and of the underlying fibroblasts. Indeed, besides acting as a physical and functional barrier to external agents, the epithelial surface of the bronchi has the capability to modulate the repair processes through the secretion of extracellular matrix proteins and the interaction with interstitial fibroblasts. Besides releasing pro-inflammatory cytokines and chemokines, the surviving epithelial cells and the underlying fibroblasts secrete factors contributing to airway repair, including the formation of the provisional extracellular matrix. This is indeed the substrate to which the epithelial cells at the edge of the lesion can attach to migrate in order to reconstitute the surface layer. In these processes airway epithelial cells receive the support of bronchial wall fibroblasts which actively release cytokines stimulating epithelial cell functions.

Bronchoalveolar lavage and esophageal pH monitoring data in children with "difficult to treat" respiratory symptoms.

Gastroesophageal reflux (GER) may be associated with chronic or recurrent asthma‐like symptoms secondary to bronchoconstrictor reflexes and/or inhalation of gastric content. The presence of lipid‐laden alveolar macrophages has been proposed as an index to establish the degree of gastric aspiration. We evaluated 20 children with “difficult to treat” respiratory symptoms and a clinical history suggestive of GER. All children underwent 24‐hr esophageal pH monitoring (pHm) and fiberoptic bronchoscopy with bronchoalveolar lavage (BAL). The amount of lipid per single macrophage was determined by a semiquantitative method, using fluorescence microscopy to detect Nile‐Red‐stained BAL cells and calculating a lipid‐laden macrophage index (LLMI).

Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway.

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-α, IL-4 and IFN-γ to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as GT values; (d) epidermal growth factor receptor (EGFR)-dependent mitogen-activated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-α (47±3%), IL-4 (43±1%) and INF-γ (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. GT values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm2) × 1000 in control cultures and in those exposed to TNF-α, IFN-γ and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.

Association of increased CCL5 and CXCL7 chemokine expression with neutrophil activation in severe stable COPD

Background: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. Objectives: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I–IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. Methods: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). Results: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2–15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. Conclusion: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

Fluticasone and salmeterol downregulate in vitro, fibroblast proliferation and ICAM-1 or H-CAM expression

Beta2-adrenoreceptor agonists have pharmacological properties that may suggest an inhibitory effect on various aspects of the inflammatory and repair processes that characterize asthma. Since fibroblasts express beta2-adrenoreceptors, the effects of different concentrations (0.1-100 nM) of fluticasone propionate (FP), salmeterol (S) and their combination (FP+S) on lung fibroblast proliferation and adhesion molecule expression were evaluated. Stimulation of human foetal lung fibroblasts with a fibrogenic cytokine, basic fibroblast growth factor (bFGF), resulted in a [methyl-3H] thymidine ([3H]TdR) uptake, four-fold higher than that of control cultures (p=0.0001) and was significantly inhibited by S, at all the concentrations tested (0.1-100 nM; p<0.05). No changes in bFGF-induced cell proliferation were observed in the presence of FP (0.1-100 nM; p>0.05, all comparisons). In addition, the association FP+S did not improve the inhibitory activity of S alone (p>0.05, each comparison). An upregulation of intercellular adhesion molecule-1 (ICAM-1) expression was induced by tumour necrosis factor-alpha (TNF-alpha) (p=0.0004), but not by interleukin-4 (IL-4) (p>0.05), while none of the two cytokines were able to increase hyaluronic-cellular adhesion molecule (H-CAM) expression by lung fibroblasts (p>0.05). A significant downregulation of ICAM-1 or H-CAM expression was demonstrated in the presence of FP or S, at all concentrations tested (0.1-100 nM; p<0.01, each comparison). Interestingly, S (10 nM and 100 nM) was able to enhance the inhibitory activity of FP on ICAM-1 expression (p<0.01), but not on H-CAM expression (p>0.1). These results show that in human foetal lung fibroblasts, fluticasone propionate and salmeterol are effective in modulating in vitro, different lung fibroblast biological functions that are likely to be involved in airway remodelling.

Exhaled nitric oxide levels in non-allergic and allergic mono- or polysensitised children with asthma

BACKGROUND Increased fractional exhaled NO concentrations (Feno) and blood/tissue eosinophilia are frequently reported in allergic children with mild asthma and are thought to reflect the intensity of the inflammation characterising the disease. The aim of this study was to investigate possible differences in Feno levels or in the intensity of the blood eosinophilia in allergic and non-allergic asthmatic children. METHODS 112 children with stable, mild, intermittent asthma with a positive bronchial challenge to methacholine were consecutively enrolled in the study; 56 were skin prick test and RAST negative (non-sensitised) while 56 were sensitised to house dust mites (23 only to house dust mites (monosensitised) and 33 were sensitised to mites and at least another class of allergens (pollens, pet danders, or moulds)). Nineteen sex and age matched healthy children formed a control group. RESULTS Compared with non-allergic patients, allergic children had a significantly higher rate of blood eosinophilia (p=0.0001) with no differences between mono- and polysensitised individuals. Forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), forced expiratory flow at 25–75% of vital capacity (FEF25–75%), and the degree of bronchial reactivity to methacholine were similar in non-atopic and atopic children, with no differences between mono- and polysensitised individuals. Feno levels measured by chemiluminescence analyser were higher in asthmatic children (15.9 (14.3) ppb) than in the control group (7.6 (1.6) ppb, p=0.04) and higher in allergic patients (23.9 (2.1) ppb) than in non-allergic patients (7.9 (0.8) ppb, p=0.0001), but there were no differences between mono- and polysensitised individuals (p>0.1). Significant correlations between blood eosinophilia and Feno levels were seen only in allergic (r=0.35, p<0.01) and in polysensitised individuals (r=0.45, p<0.05). CONCLUSIONS In children with mild asthma, a similar degree of functional disease severity may be associated with a higher inflammatory component in allergic than in non-allergic subjects.

Fluticasone Propionate Downregulates Nasal Fibroblast Functions Involved in Airway Inflammation and Remodeling

Background: Besides being highly effective in the treatment of allergic and nonallergic rhinitis with eosinophilia, intranasal corticosteroids appear to be useful in reducing nasal polypoid lesions and the likelihood of polyp recurrence after surgery. We evaluated the ability of fluticasone propionate to downregulate fibroblast functions related to nasal inflammation and remodeling. Methods: Primary nasal polyp tissue-derived fibroblasts were stimulated with tumor necrosis factor (TNF)-α or interleukin (IL)-4 or basic fibroblast growth factor (bFGF) in the presence of fluticasone propionate (0.1–100 nM). Fibroblast proliferation, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression and eotaxin release were then evaluated. Results: As compared with unstimulated cultures, a significant increase in fibroblast proliferation was observed when the cells were stimulated with bFGF (p < 0.05), but not with TNF-α or IL-4 (p > 0.05). TNF-α induced an upregulation of ICAM-1 expression (p < 0.05), which was not seen in fibroblasts cultured in the presence of IL-4 or bFGF. No changes in VCAM-1 expression were induced by TNF-α, IL-4 or bFGF, whereas both TNF-α and IL-4 increased eotaxin release (p < 0.05). Both bFGF-induced fibroblast proliferation and TNF-α-induced ICAM-1 expression were significantly reduced by fluticasone, starting at the dose of 1 and 10 nM, respectively (p < 0.05). Fluticasone at concentrations of 1–100 nM effectively inhibited eotaxin release by TNF-α- or IL-4-stimulated fibroblasts (p < 0.05). Conclusions: The pharmacologic activity of fluticasone in patients with chronic upper airway inflammatory disease may include inhibition of resident fibroblast functions involved in airway inflammation and remodeling.

Total and allergen‐specific IgE levels in serum reflect blood eosinophilia and fractional exhaled nitric oxide concentrations but not pulmonary functions in allergic asthmatic children sensitized to house dust mites

Although elevated levels of serum immunoglobulin E (IgE) are considered the hallmark of atopic diseases, their clinical value in evaluating subjects with allergic disorders is under debate. To evaluate possible relationships between serum IgE levels and a variety of clinical parameters, 83 mild asthmatic children [10.98‐year‐old (2.95)], sensitized to house dust mites (HDM) Dermatophagoides pteronyssinus (Dp) or D. farinae (Df), were enrolled. As compared with normal control reference values detected in our laboratory, children with allergic asthma had higher blood eosinophil counts (expressed both as percentage and as absolute number) and higher fractional exhaled nitric oxide (FeNO) levels but similar values in pulmonary function parameters. In the allergic asthmatic population, serum levels of total, Dp‐specific or Df‐specific IgE correlated positively with eosinophil counts (Rho ≥ 0.30, p < 0.01, each correlation) and FeNO levels (Rho ≥ 0.33, p < 0.01, each correlation) but not with pulmonary function parameters (p > 0.1, each correlation). Finally, significant correlations, although moderate, were found in the allergic asthmatic population between eosinophil counts and FeNO levels (Rho ≥ 0.42, p < 0.001, each correlation). Thus, in atopic children sensitized to HDM with mild intermittent asthma, IgE levels in blood appear to reflect systemic (blood eosinophils) and organ‐specific (FeNO) markers of allergic inflammation but not pulmonary volumes or the degree of airflow limitation.