Claudio Mastruzzo

Effect of immunotherapy on asthma progression, BHR and sputum eosinophils in allergic rhinitis

Background:  Bronchial hyperresponsiveness (BHR) and airway inflammation are frequently associated with allergic rhinitis, and may be important risk factors for the development of asthma. Specific immunotherapy (SIT) reduces symptom in subjects with allergic rhinitis, but the mechanisms are not clear.

Endothelin-1 Induces Proliferation of Human Lung Fibroblasts and IL-11 Secretion Through an ET A Receptor-Dependent Activation of Map Kinases

Endothelin‐1 (ET‐1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET‐1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET‐1 in the presence or absence of an overnight pre‐treatment with either ETA or ETB selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen‐activated protein kinases (MAPK). ET‐1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL‐6/IL‐11 release into cell culture supernatants. All these effects were inhibited by the selective ETA antagonist BQ‐123, but not by the specific ETB antagonist BQ‐788. The stimulatory influence of ET‐1 on IL‐11, but not on IL‐6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET‐1 exerts a profibrogenic action via an ETA receptor‐dependent, MAPK‐mediated induction of IL‐11 release and cell proliferation.

Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

RationaleGap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition.MethodsWe investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF).ResultsHere we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF.ConclusionThese results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

Monitoring of seasonal variability in bronchial hyper-responsiveness and sputum cell counts in non-asthmatic subjects with rhinitis and effect of specific immunotherapy.

Bronchial hyper‐responsiveness (BHR) is documented in a proportion of non‐asthmatic individuals with allergic rhinitis (NAAR) and reflects inflammatory events in the lower airways. Natural exposure to allergens is known to modulate BHR and the level of airway inflammation in asthma, but less consistently in NAAR. Specific immunotherapy (SIT) attenuates symptoms possibly by reducing BHR and airway inflammation. The influence of natural exposure to Parietaria pollen on BHR and sputum cell counts of NAAR was investigated and the effect of Parietaria SIT examined. Thirty NAAR, monosensitized to Parietaria judaica, participated in a randomized, double‐blind, placebo‐controlled, parallel group study of the effects of a Parietaria pollen vaccine on symptoms/medication score, BHR to inhaled methacholine and adenosine 5′‐monophosphate (AMP), and cell counts in the sputum collected out of and during the pollen seasons for 36 months. Seasonal variation in BHR to inhaled methacholine and AMP and changes in sputum cell counts were documented. Changes were consistent for AMP, but not methacholine, and invariably associated with modifications in sputum eosinophils and epithelial cells. The clinical efficacy of Parietaria SIT was associated with a decline in the seasonal deterioration of BHR to AMP, whereas no significant effect was observed on BHR to methacholine or sputum cell differentials. Between‐groups comparison of the seasonal changes in PC15 methacholine values and sputum cell differentials calculated as the AUC were not statistically significant, whereas a significant difference in PC15 AMP was demonstrated throughout the study (P=0.029), the median (inter‐quartile range) AUC values being 2478.5 (1153.3–3600.0) and 1545.5 (755.3–1797.9) for the SIT‐ and placebo‐treated group, respectively. Bronchial airways of NAAR exhibit features of active inflammation that deteriorate during natural allergen exposure, particularly with regard to BHR to AMP. The clinical efficacy of Parietaria SIT was exclusively associated with attenuation in seasonal worsening of PC15 AMP, suggesting that AMP may be useful in monitoring changes in allergic inflammation of the airways.

Intranasal steroid reduces exhaled bronchial cysteinyl leukotrienes in allergic patients

Background Allergic rhinitis (AR) precedes and is often associated with bronchial asthma. Indeed, local and systemic inflammations in both conditions are very similar. Cysteinyl‐leukotrienes (cys‐LTs) are generated during early‐ and late‐phase allergic reactions and induce smooth‐muscle contraction, microvascular leakage, and mucous hypersecretion. Cys‐LTs are detected in exhaled breath condensate (EBC) of asthmatics and regardless of bronchial symptoms, they are also found in EBC of rhinitic patients.

Interaction between human lung fibroblasts and T-lymphocytes prevents activation of CD4+ cells

BackgroundT lymphocytes are demonstrated to play an important role in several chronic pulmonary inflammatory diseases. In this study we provide evidence that human lung fibroblasts are capable of mutually interacting with T-lymphocytes leading to functionally significant responses by T-cells and fibroblasts.MethodsHuman lung fibroblast were co-cultured with PMA-ionomycin activated T-CD4 lymphocytes for 36 hours. Surface as well as intracellular proteins expression, relevant to fibroblasts and lymphocytes activation, were evaluated by means of flow cytometry and RT-PCR. Proliferative responses of T lymphocytes to concanavalin A were evaluated by the MTT assay.ResultsIn lung fibroblasts, activated lymphocytes promote an increase of expression of cyclooxygenase-2 and ICAM-1, expressed as mean fluorescence intensity (MFI), from 5.4 ± 0.9 and 0.7 ± 0.15 to 9.1 ± 1.5 and 38.6 ± 7.8, respectively. Fibroblasts, in turn, induce a significant reduction of transcription and protein expression of CD69, LFA-1 and CD28 in activated lymphocytes and CD3 in resting lymphocytes. In activated T lymphocytes, LFA-1, CD28 and CD69 expression was 16.6 ± 0.7, 18.9 ± 1.9 and 6.6 ± 1.3, respectively, and was significantly reduced by fibroblasts to 9.4 ± 0.7, 9.4 ± 1.4 and 3.5 ± 1.0. CD3 expression in resting lymphocytes was 11.9 ± 1.4 and was significantly reduced by fibroblasts to 6.4 ± 1.1. Intracellular cytokines, TNF-alpha and IL-10, were evaluated in T lymphocytes. Co-incubation with fibroblasts reduced the number of TNF-alpha positive lymphocytes from 54,4% ± 6.12 to 30.8 ± 2.8, while IL-10 positive cells were unaffected. Finally, co-culture with fibroblasts significantly reduced Con A proliferative response of T lymphocytes, measured as MTT absorbance, from 0.24 ± 0.02 nm to 0.16 ± 0.02 nm. Interestingly, while the activation of fibroblasts is mediated by a soluble factor, a cognate interaction ICAM-1 mediated was demonstrated to be responsible for the modulation of LFA-1, CD28 and CD69.ConclusionFindings from this study suggest that fibroblasts play a role in the local regulation of the immune response, being able to modulate effector functions of cells recruited into sites of inflammation.

Inhaled lysine acetylsalicylate (L-ASA) attenuates histamine-induced bronchoconstriction in asthma

When administered by inhalation, histamine provokes dose‐related bronchoconstriction in asthmatic subjects mainly by a direct activation of histamine H1‐receptors on airway smooth muscle. However, little is known of the change in airway responsiveness to histamine after cyclooxygenase blockade. The aim of the study was to investigate the effect of the potent cyclooxygenase inhibitor, lysine acetylsalicylate (L‐ASA), administered by inhalation on histamine‐induced bronchoconstriction in a group of 16 asthmatic subjects. The subjects studied attended the laboratory on four separate occasions to receive nebulized L‐ASA (solution of 90mg/ml) or matched placebo (glycine solution of 30 mg/ml) 15 min before bronchoprovocation tests with histamine and methacholine in a randomized, double‐blind order. Changes in airway caliber were followed as forced expiratory volume in 1 s (FEV1), and agonist responsiveness was expressed as the provocative concentration causing a 20% fall in FEV1 from baseline (PC20).

Acute additive effect of montelukast and beclomethasone on AMP induced bronchoconstriction.

Bronchial hyperresponsiveness to 5-adenosine mono-phosphate (AMP) is a marker of airway inflammation. Inhaled corticosteroids and antileukotrienes are used as anti-inflammatory drugs for the treatment of asthma. To find out if these two drugs exert their protection in an additive fashion, we compared the effects of acute treatment with inhaled beclomethasone (BDP) and montelukast (ML), alone or in combination, on methacholine and AMP induced bronchoconstriction. 15 asthmatic patients undertook methacholine and AMP challenges at baseline and after receiving ML or BDP, alone or in combination, in a randomized, double-blind, double-dummy placebo-controlled, crossover design. BDP pretreatment significantly increased the AMP PC(20) value (68.34+/-15.9mg/mL) as compared to placebo (22.87+/-5.7mg/mL). Combined treatment, BDP plus ML, afforded a further significant increase of AMP PC(20) (154.57+/-55.0mg/mL) as compared to each single treatment. The significant protection exerted by combined treatment as compared to each single active treatment was also demonstrated by the change of AMP PC(20) doubling dose as compared to placebo and each single active treatment. Our findings suggest that these two agents exert their acute additive protection against AMP induced bronchoconstriction acting on distinct inflammatory pathways and their combined use might provide greater protection against inflammatory response elicited by AMP than either drug alone.