Chronic bronchitis (CB)ischaracterized byinflammatory changes inthebronchial tissue andbyrecurrent bronchitis exacerbations. Inaddition, defective systemic andlocal immunemechanisms havebeendemonstrated andbiologic response modifiers (BRMs) have beenrecently introduced forclinical useinpatients with CB.Westudied 24patients withCBbybronchoalveolar lavage (BAL), before andafter a4-week treatment protocol withinhaled Immucytal (Pierre-Fabre Pharma Srl, Milan, Italy), aBRM composed ofbacterial ribosomal fractions andmembraneproteoglycanes. Compared withnormal controls (NC), before treatment BALin patients withCB contained increased proportions of neutrophils (NC,0.8 ±0.2percent; CB,3±1percent), of eosinophils (NC,0.1±0.02percent; CB,0.6±0.2percent); andoflymphocytes (NC,6±1percent; CB,13+2 percent; p<0.01 eachcomparison) withhigher percentagesofCD3+andCD8+lymphocytes (p<0.01 each comparison). InBALfrompatients withCBthere were also higher levels ofalbumin andoftheratio IgG/ albumin (p<0.01 andp<0.05, respectively, compared withNC). After Immucytal treatment, theproportions of (hronicbronchitis (CB) ischaracterized clinically

HLA-DR class II molecules are expressed by a variety of nonlymphoid cells, including the respiratory epithelium. However, it is not known if ciliated bronchial epithelial cells express the HLA-DR genes, if the expression of class II molecules on their surface can be modulated by immune mediators and, finally, if these cells, like other HLA-DR-positive epithelial cells, have the potential to serve as antigen-presenting cells. To answer these questions, we collected ciliated bronchial epithelial cells by brushing and by suction during fiberoptic bronchoscopy and by scraping surgically resected bronchi. The number of cells recovered by brushing or suction during fiberoptic bronchoscopy was similar (P greater than 0.2), but lower than that obtained by scraping surgically resected bronchi (P less than 0.01); however, compared with brushing, suction of ciliated bronchial epithelial cells resulted in a better viability (P less than 0.05). HLA-DR antigens on ciliated bronchial epithelial cells were detected by immunofluorescence using the PTF 29.12 and the L243 monoclonal antibodies, both recognizing HLA-DR molecules on the vast majority of ciliated bronchial epithelial cells. Cytoplasmic dot blot analysis demonstrated that ciliated bronchial epithelial cells had mRNA HLA-DR transcripts, and Northern blot hybridizations showed that the size of the HLA-DR messages was the same observed in other HLA-DR-positive cells. Interestingly, ciliated bronchial epithelial cells showed a significant decline of HLA-DR expression after 5 days in culture, but the addition of gamma-interferon to the cell cultures was associated with the persistence of the expression of class II antigens on the cell surface (P less than 0.01 with control cultures at 5 days). Finally, while ciliated bronchial epithelial cells were ineffective in stimulating allogeneic T cell proliferation in a 6-day primary mixed leukocyte reaction (MLR), the addition of phorbol myristate acetate to the MLR was able to induce a significant T cell proliferation (P less than 0.001, all comparisons). Thus, human ciliated bronchial epithelial cells express HLA-DR surface antigens and have mRNA molecules for the HLA-DR genes, and the expression of the surface class II antigens can be modulated in vitro by immune mediators.(ABSTRACT TRUNCATED AT 400 WORDS)

CD40/CD40 ligand interaction is an important pathway for B and T cell cooperation and function; functional CD40 molecules have recently been found on nonhematopoietic cells. We detected CD40 in vivo on normal human respiratory epithelial cells and showed that its expression is increased on inflamed airway epithelium. Subsequently, we analyzed its expression and function on primary cultures of human airway epithelial cells. Our data show that CD40 is up-regulated by IFN-beta and IFN-gamma, its ligation increases the surface expression of CD54 and CD106 and it may stimulate the release of IL-6 and IL-8. The use of Janus kinase 3 (JAK3) and NF-kappaB inhibitors suggests that both basal and CD40-induced release of the two cytokines is JAK3-dependent. Using colocalization techniques, we revealed the existence of CD40/JAK3 and CD40/TNFR-associated factor 2 interplay. The extent of these interactions may be partial (2-40% of the cells) or massive (80-90% of the cells) in cultured cells. Stimulation via CD40 causes a significant increase in the number of cells expressing colocalization only in the cultures displaying low frequency of initial colocalization. Thus, airway epithelial cells, activated by CD40, may behave as effector cells of the inflammation process and should be considered priority targets for anti-inflammatory therapy. This work identifies CD40 and the correlated JAK3 signaling molecule as potential molecular targets to block the inflammatory functions of epithelial cells.

Background: Lung fibroblasts may have a pivotal role in airway inflammation as they are involved in continuous cycles of mediator secretion, proliferation, activation and cross-talk with recruited inflammatory cells. The role of fibroblasts as intermediate participants in the inflammatory network suggests that they could represent an important target for drugs commonly used in asthma; thus, we investigated the effects of triamcinolone acetonide (TAA) on primary human lung fibroblasts. Methods: The in vitro activity of increasing concentrations (10–9 to 10–7 M) of TAA in fibroblast cultures was evaluated as regards the following parameters: proliferation, extracellular matrix (ECM) release, cytokine/chemokine secretion and surface antigen expression. Results: All concentrations of TAA decreased fetal calf serum (FCS)-induced fibroblast proliferation, whereas in the presence of FCS plus basic fibroblast growth factor TAA was only effective at 10–8 and 10–7M. TAA failed to decrease ECM, whereas at 10–8 and 10–7M it decreased IL-6 and IL-8 secretion to different extents. In the presence of IFN-γ the drug was able to reduce VCAM-1 expression at all of the tested concentrations; on the other hand, in TGF-β1-driven cultures a decrease in CD54 expression was detected with TAA at 10–8 and 10–7M. Conclusions: TAA acts on some functional properties of human lung fibroblasts that make these cells active participants in the inflammatory network. The ability of TAA to inhibit lung fibroblast proliferation may prevent or even reverse some of the histological changes that characterize airway remodeling in chronic inflammatory diseases; moreover, IL-6, IL-8 and surface molecule decreases by TAA may suggest a direct anti-inflammatory effect of the drug by suppression of resident lung cell function.

We studied whether different bronchial responses to allergen in asthma and rhinitis are associated with different bronchial inflammation and remodeling or airway mechanics. Nine subjects with mild asthma and eight with rhinitis alone underwent methacholine and allergen inhalation challenges. The latter was preceded and followed by bronchoalveolar lavage and bronchial biopsy. The response to methacholine was positive in all asthmatic but in only two rhinitic subjects. The response to allergen was positive in all asthmatic and most, i.e., five, rhinitic subjects. No significant differences between groups were found in airway inflammatory cells or basement membrane thickness either at baseline or after allergen. The ability of deep inhalation to dilate methacholine-constricted airways was greater in rhinitis than in asthma, but it was progressively reduced in rhinitis during allergen challenge. We conclude that 1) rhinitic subjects may develop similar airway inflammation and remodeling as the asthmatic subjects do and 2) the difference in bronchial response to allergen between asthma and rhinitis is associated with different airway mechanics.

Asthma is characterized by an irreversible subepithelial fibrosis with the appearance of myofibroblasts, which can be now considered important early participants in inflammatory responses as well as potential targets for anti-inflammatory drugs. In this study, we show that fluticasone propionate (FP), a powerful inhaled corticosteroid (ICS), displays novel anti-inflammatory effects on human lung fibroblasts during their myofibroblastic differentiation. Indeed, FP inhibits in lung myofibroblasts, at a very early stage of differentiation, the activation of Janus kinase/STAT pathways induced by IL-13 (tyrosine kinase 2, STAT1, STAT3, STAT6, mitogen-activated protein kinase). Contrarily, in mildly or fully differentiated myofibroblastic cultures, FP still displays a potential anti-inflammatory activity even if it only inhibits tyrosine kinase 2 phosphorylation. Moreover, FP inhibits constitutive and TGF-beta -induced expression of alpha -smooth muscle actin, the main marker of myofibroblastic differentiation, both in very early and in mild differentiated myofibroblasts. Finally, FP displays an additional powerful anti-inflammatory effect, decreasing nuclear translocation of NF-kappaB independent of the degree of myofibroblastic differentiation. These data 1) suggest that myofibroblasts are priority targets for ICS, which is able to revert them to a normal phenotype even if they appear to be already engaged in their differentiation, and 2) may help to explain why asthma is improved by an early ICS treatment, whereas advanced asthma is more resistant to these drugs.

OBJECTIVE:To investigate whether the state of activation of circulating T-cells in childhood asthma could be related to serum IgE levels and/or to blood eosinophilia. METHODS:Seventeen atopic asthmatic children, sensitized to Dermatophagoides pteronyssinus (Der p), in stable condition at the time of the study and 15 sex-matched and age-matched controls were studied. The expression of activation surface markers (HLA-DR and CD25) on peripheral blood mononuclear cells (PBMCs) was tested by monoclonal antibodies and FACS analysis, while the PBMC proliferative response to Der p antigens was measured by tritiated thymidine (3HTdR) incorporation. RESULTS:As compared to controls, atopic children showed higher eosinophil counts (P < .01), similar lymphocyte counts (P > .1, each comparison) but higher proportion of HLA-DR+ and CD25+ T-lymphocytes (P < .05, each comparison). A significant Der p allergen-induced PBMC proliferation was observed in atopic children (P < .01) but not in controls (P > .1). Both in controls and in atopic children, no correlations were found between lymphocyte counts and eosinophil counts or total or allergen-specific IgE levels (P > .1, each comparison). In contrast, weak correlations were detected between the degree of allergen-induced PBMC proliferation and: a) allergen-specific IgE levels in serum (P < .05) and b) eosinophil counts (P < .05). CONCLUSION:These data support the concept that the degree of activation of allergen-specific T-lymphocytes in blood may reflect the intensity of allergic sensitization in childhood asthma.

Recently, airway fibroblasts captured the attention of both allergists and basic scientists since they are no longer considered as mere bystanders, as far as allergic airway diseases are concerned. The aim of the present study was to assess the effects of different Cetirizine (Cet) concentrations (0.01, 0.05, 0.1 mg/ml) on human airway fibroblast proliferation and on CD54 expression. By means of flow cytometry analysis, we evaluated CD54 expression by airway fibroblasts in basal conditions or after gammaIFN stimulation in the presence of Cetirizine; we also evaluated the effect of the drug on cell proliferation by a [3H]thymidine incorporation assay. All of the tested doses of Cetirizine were able to significantly reduce CD54 upregulation induced by gammaIFN; concerning the fibroblast proliferation, we observed a dose-dependent inhibition of [3H]thymidine incorporation. These results show that Cetirizine exerts a biologic effect directly on human airway fibroblasts, suggesting a new rationale in the use of this compound.

Background: Mizolastine is a potent, peripherally acting, selective H1-receptor antagonist with potential anti-inflammatory properties. The aim of the study was to evaluate the in vitro effects of mizolastine on the expression of adhesion molecules by primary human airway epithelial and stromal cultures; moreover, the activity of mizolastine on parameters which reflect the immune response efficacy was investigated. Methods: Airway epithelial and stromal cells were collected from hypereosinophilic subjects by enzymatic digestion of polyps or turbinates. Cells were stimulated with interferon (IFN)-γ (500 IU/ml) in the presence of various mizolastine concentrations (6 × 10–8–6 × 10–6 M) for 24 h and the expression of CD106, CD54, CD58 and HLA class I was evaluated. Peripheral blood mononuclear cells from healthy volunteers were incubated with 1% phytohemagglutinin or anti-CD3 monoclonal antibody (20 ng/ml) in the presence of mizolastine, then T lymphocyte proliferation, HLA-DR expression and T cell subpopulations were evaluated. Results: Both in epithelial and stromal cultures, IFN-γ significantly upregulated all of the tested surface molecules (p < 0.05). The highest dose of mizolastine (6 × 10–6 M), corresponding to 10-fold the peak plasma level after a single oral administration of 10 mg, was able to act on fibroblasts, significantly downregulating the expression of CD54 (p < 0.05). Regarding T lymphocyte proliferation, the addition of mizolastine did not induce any significant change; furthermore, mizolastine was ineffective at all of the tested concentrations on both HLA-DR expression and CD4+/CD8+ ratio. Conclusions: This study demonstrated that mizolastine is able to selectively downregulate CD54 expression on stimulated stromal but not epithelial cells without impairing the immune system effectors. The possible clinical significance of these results are an antiallergic property and CD54 modulation on fibroblasts with a good safety profile as far as the lymphocyte response is concerned.

beta(2)-adrenoreceptor agonists have the ability to downregulate in vitro the proliferative response of peripheral blood mononuclear cells (BMCs). This activity could be related to a variety of beta(2)-adrenoreceptor-mediated functions, including induction of cell apoptosis in activated T-cells. To test this hypothesis, BMCs from atopic subjects, sensitized to house dust mites (Dermatophagoides [Der p]) and/or to Parietaria were incubated with fenoterol 10(-8)-10(-5) M) in the presence of (a) purified allergen extracts (Der p [5 mu g/mL] or Parietaria 15 mu g/mL]) or (b) antigens (tetanus toroid [1 mu g/mL] or Candida albicans [5 x 10(5) bodies/ mL]). The BMC proliferation was assessed by [H-3] thymidine incorporation and cell apoptosis was assessed by evaluating DNA fragmentation by a fluorescence technique, using propidium iodide. In cultures stimulated with Der p or with Parietaria, fenoterol induced a dose-dependent inhibition of BMC proliferation, significant also at the lowest concentration tested (10(-8) M) (p < 0.05, each comparison). In contrast, the inhibitory activity of the drug on tetanus-toxoid-stimulated BMCs was significant only at the highest dose tested (10(-5) M) (p < 0.05), whereas no effect was seen when BMCs were stimulated with C, albicans extract (p > 0.05). The different inhibitory efficacy of fenoterol appeared to be related to the degree of activation of beta(2)-adrenoreceptors on the different BMC populations that responded to the different stimuli. Indeed, in the presence of fenoterol (10(-6) and 10(-5) M), a significant increase in cyclic adenosine monophosphate (cAMP) levels was seen in Der p- or Parietaria-stimulated cells (p< 0.05; each comparison), but not in cell cultures stimulated with tetanus toroid or with C. albicans extracts (p > 0.05; each comparison). Finally, the percentage of cells with fragmented DNA was lower in cultures stimulated with Der p or Parietaria than in those stimulated with tetanus toroid or C. albicans, and the presence of fenoterol did not modify cell apoptosis (p > 0.05; each comparison). Thus, the different inhibitory activity of fenoterol on BMCs activated by allergens (Der p or Parietaria) or by antigens (tetanus toroid or C. albicans) seems to be related to differences in beta(2)-adrenoreceptor expression and/or function in the different antigen-specific T-cell subsets, but it is not influenced by changes in cell apoptosis.

Patients with chronic obstructive lung disorders often show increased susceptibility to airway infections. As beta 2-adrenoceptor agonists, in addition to reversing the contractile response of bronchial smooth muscles, may inhibit a variety of inflammatory and immuno-effector cell functions, it is possible that these drugs interfere with host defence mechanisms. The present study was designed to test in vitro whether fenoterol, a short-acting beta 2-adrenoceptor agonist, could modify human blood neutrophil recruitment and antimicrobial activity. Pre-exposure to fenoterol significantly reduced neutrophil migration towards the complement component C5a, at concentrations ranging from 10(-7) M to 10(-5) M, or towards lipopolysaccharide, at a concentration of 10(-5) M (P < 0.05, each comparison). In contrast, the drug (10(-8)-10(-5) M) did not significantly modify the increased expression of lymphocyte function-associated antigen (LFA-1, i.e. CD11a/CD18) the macrophage antigen-1 (Mac-1, i.e. CD11b/CD18) induced by N-formylmethionylleucylphenylalanine (fMLP) (P > 0.05, each comparison). Finally, incubation of neutrophils with fenoterol (10(-8)-10(-5) M) did not significantly influence phagocytosis or intracellular killing of bacteria (Staphylococcus aureus) or H2O2 release induced by tetradecanoyl-phorbol-acetate (P > 0.1 for each comparison). These results suggest that short-acting beta 2-adrenoceptor agonists, such as fenoterol, are able partially to reduce neutrophil recruitment in the airways without interfering with the processes involved in phagocytic activity against bacteria.

Treatment of allergic asthma with inhaled corticosteroids results in local down-regulation of proinflammatory cytokine synthesis and in marked decrease in tissue eosinophilia. Blood concentrations of inhaled corticosteroids, although significantly lower than those measured in the lung, may still have antiinflammatory effects on circulating eosinophils, reducing their ability to migrate. The aim of our study was to evaluate in vitro the activity of budesonide on blood eosinophils by measuring their chemotactic response, eosinophil cationic protein (ECP) release, and hydrogen peroxide (H2O2) production in the presence of different drug concentrations similar to those obtained at airway level (10−8 and 10−7 M) and at blood level (10−10 and 10−9 M). Partially purified blood eosinophils, isolated from 23 asthmatic subjects, were used to evaluate the activity of budesonide on: (1) chemotaxis toward the activated fifth component of complement (C5a, 0.1 μg/ml) or recombinant human (rh) interleukin (IL)-5 (200 pg/ml), (2) ECP release by cells stimulated with tetradecanoylphorbol acetate (TPA) and (3) H2O2 production by TPA-activated cells. The chemotactic response to C5a was down-regulated significantly by budesonide only by the highest concentrations tested (10−8 and 10−7 M); differently, budesonide was effective in inhibiting eosinophil migration toward rhIL-5, at all concentrations tested (p < 0.01, each comparison). By contrast, no drug-induced modifications were observed in ECP release or in H2O2 production (p > 0.05, each comparison). We conclude that concentrations of budesonide similar to those obtained in vivo are effective in inhibiting eosinophil locomotion but not in down-regulating the release of reactive oxygen species and granule-associated proteins.

Background: Alveolar macrophages (AMs) are more efficient antigen-presenting cells in allergic individuals than in nonatopic subjects.Objective: We studied whether this difference may be correlated to increased expression of membrane costimulatory molecules, such as the B7 molecules (CD80 and CD86).Methods: Eleven subjects with allergic asthma sensitized to Dermatoplagoides pteronyssinus and 5 healthy nonatopic volunteers underwent bronchoalveolar lavage, and the costimulatory molecule expression on AMs was evaluated. Peripheral blood T cells, either freshly isolated or as established D pteronyssinus-specific cell lines, were cultured with autologous monocytes or AMs as antigen-presenting cells. In vitro allergen-induced proliferation and cytokine production were evaluated in the presence of B7-blocking reagents.Results: Allergic individuals had a significantly higher proportion of AMs expressing the CD80 molecule than control subjects (28.5% +/- 14.8% vs 1.4% +/- 1.2%; P < .001), whereas no difference was observed in CD86 expression (2.0% +/- 2.3% vs 1.1% +/- 0.6; P > .1). In a large proportion of the asthmatic subjects we studied, AMs were presenting soluble antigens (tetanus toroid and streptolysin-O) to freshly isolated T cells more efficiently than AMs from nonatopic control subjects. Finally, both T-cell proliferation and cytokine production of D pteronyssinus-specific established T-cell lines were inhibited by a CD80-blocking antibody in a dose-dependent manner.Conclusion: Costimulation by means of CD80 expressed by AMs is probably involved in the amplification of the allergen-specific T-lymphocyte response in the airways of asthmatic subjects.

The possibility that salmeterol could interfere with the inhibitory effect of glucocorticosteroids on allergen-induced activation of leukocytes was assessed in cultures of human blood mononuclear cells (BMCs). BMCs, recovered from 27 patients (21 ± 4 years of age) sensitized to Dermatophagoides pteronyssinus (Der p), were incubated with a purified Der p extract (10 µg/ml) for 7 days in the presence of salmeterol (10–8 and 10–7 M) and/or dexamethasone (10–10 and 10–9 M). BMC proliferation and cytokine release were respectively tested by [3H]thymidine incorporation and EASIA. Stimulation with Der p extract induced a significant BMC proliferation (Der p 18.7 ± 1.9 cpm × 103, control 1.8 ± 0.3 cpm × 103, p < 0.01) which was significantly inhibited both by salmeterol and dexamethasone (p < 0.05, each comparison). The inhibition of BMC proliferation induced by dexamethasone (10–9 M) was significantly enhanced by salmeterol 10–7 M (p < 0.05). Evaluation of cytokine levels in BMC supernatants showed that Der p extract significantly increased the release of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-2 (p < 0.001, each comparison). Salmeterol (10–7 M) or dexamethasone (10–9 M) induced a similar, statistically significant, downregulation of GM-CSF release (p < 0.05, each comparison) and induced a slight, not significant, decrease in the production of IFN-γ, TNF-α, IL-1β and IL-2 (p > 0.05, each comparison). Interestingly, salmeterol (10–7 M) significantly enhanced the inhibitory activity of dexamethasone (10–9 M) on the release of GM-CSF, TNF-α, IL-1β and IL-2 (p < 0.05, each comparison) but not of IFN-γ (p > 0.05). Thus β2-adrenoceptors may enhance the inhibitory activity of low doses of corticosteroids on allergen-induced BMC activation.

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.