Francesca Cagnoni

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down‐regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV‐infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti‐p70, anti‐p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV‐infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV‐infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer‐cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV‐infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA‐A and HLA‐B and did not modify the expression of HLA‐C, but also that NK cell clones expressing the “activating” form of the anti HLA‐C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV‐infected autologous target cells. J. Med. Virol. 62:354–363, 2000.

CD40 on Adult Human Airway Epithelial Cells: Expression and Proinflammatory Effects

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-β and IFN-γ, 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-κB 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.

Novel Anti-inflammatory Effects of the Inhaled Corticosteroid Fluticasone Propionate During Lung Myofibroblastic Differentiation

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-β-induced expression of α-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-κB 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.

Quantitative flow cytometric analysis of the effects of cetirizine on the expression of ICAM-1/CD54 on primary cultured nasal cells

An in vitro flow cytometric model has been developed to evaluate the effects of antiallergic drugs such as cetirizine (CTZ) on the expression of surface molecules on primary cultured normal cells. Quantitative analysis demonstrated that HLA class I and ICAM‐1/CD54 molecules are present on both epithelial and stromal cells, and that their expression is strongly enhanced by treatment with interferon‐gamma (IFN‐γ). Nevertheless, the IFN‐γ‐mediated upregulation of ICAM‐1/CD54 was inhibited by treatment with CTZ, demonstrating a direct effect on both cell types. This finding is particularly interesting because ICAM‐1/CD54 is the main rhinovirus receptor, and rhinoviruses are the principal cause of asthma exacerbation in children. Thus, according to data derived from this in vitro model, CTZ should have an important role in the reduction of infectious exacerbation of asthma in atopic patients.

Effects of Triamcinolone Acetonide on Adult Human Lung Fibroblasts: Decrease in Proliferation, Surface Molecule Expression and Mediator Release

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.

Low concentrations of cytokines produced by allergen-stimulated peripheral blood mononuclear cells have potent effects on nasal polyp-derived fibroblasts

Accumulating data show that fibroblasts are important regulators in the development and maintenance of allergic airway inflammation. However, most studies so far have used individual recombinant cytokines in high concentrations, unlikely to be found in vivo. We aimed to investigate how cytokines produced by peripheral blood mononuclear cells (PBMC) affect fibroblast functions. Primary airway fibroblasts where incubated with allergen‐stimulated or non‐stimulated PBMC supernatants from allergic patients. The levels of cytokines in PBMC supernatants were measured and the expression of CD54, CD40 and CD106 as well as the production of eotaxin, interleukin (IL)‐6 and IL‐8 were assessed in fibroblasts. Although the levels of single cytokines measured in PBMC supernatants were low, a significant up‐regulation of the surface molecules as well as of IL‐6 and IL‐8 production was found in fibroblasts cultured with allergen‐stimulated PBMC supernatants as compared to non‐stimulated, while the increase in eotaxin production was not significant. The evaluation of correlations between cytokines produced by PBMC and effects seen on fibroblasts did not indicate a crucial role for any single cytokine. Furthermore, the addition of comparably low concentrations of recombinant interferon (rIFN)‐γ or recombinant tumour necrosis factor (rTNF)‐α did not induce the same effects as PBMC supernatants, the only exception being TNF‐α as a direct inducer of CD54 expression. Our results show that synergistic mechanisms has a more important role than single mediators, highlighting important differences between in vitro experiments, where effects of individual mediators are studied, versus the actual situation in vivo.

Effects of Mizolastine in vitro on Human Immunocompetent and Airway Cells: Evidence for Safety and Additional Property

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.

Cetirizine-induced downregulation of airway fibroblast proliferation and function: a rationale for a different approach to allergy treatment?

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.