Surfactant Protein D modulates allergen particle uptake and inflammatory response in a human epithelial airway model.

BACKGROUND: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated. METHODS: SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array. RESULTS: SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and led to a decreased secretion of pro-inflammatory cytokines. CONCLUSION: These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen.

Allergen particle binding by human primary bronchial epithelial cells is modulated by surfactant protein D.

BACKGROUND: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. Our previous work demonstrated that SP-D increases the uptake of SPP by alveolar macrophages. In the present study, we investigated the uptake of SPP in human primary epithelial cells and the potential modulation by SP-D. The patho-physiological consequence was evaluated by measurement of pro-inflammatory mediators. METHODS: SPP were isolated from timothy grass and subsequently fluorescently labelled. Human primary bronchial epithelial cells were incubated with SPP or polystyrene particles (PP) in the presence and absence of surfactant protein D. In addition, different sizes and surface charges of the PP were studied. Particle uptake was evaluated by flow cytometry and confocal microscopy. Soluble mediators were measured by enzyme linked immunosorbent assay or bead array. RESULTS: SPP were taken up by primary epithelial cells in a dose dependent manner. This uptake was coincided with secretion of Interleukin (IL)-8. SP-D increased the fraction of bronchial epithelial cells that bound SPP but not the fraction of cells that internalized SPP. SPP-induced secretion of IL-8 was further increased by SP-D. PP were bound and internalized by epithelial cells but this was not modulated by SP-D. CONCLUSIONS: Epithelial cells bind and internalize SPP and PP which leads to increased IL-8 secretion. SP-D promotes attachment of SPP to epithelial cells and may thus be involved in the inflammatory response to inhaled allergen.

A Toll-like receptor 2/6 agonist reduces allergic airway inflammation in chronic respiratory sensitisation to Timothy grass pollen antigens.

BACKGROUND: The hygiene hypothesis negatively correlates the microbial burden of the environment with the prevalence of T helper type 2 (Th2)-related disorders, e.g. allergy and asthma. This is explained by Th1 triggering through pathogen-associated molecular patterns via Toll-like receptors (TLRs). In this study, the biological effects of a TLR2/6 agonist as a potential treatment of allergic inflammation are explored. METHODS: In a model of chronic allergic airway inflammation induced by intranasal administration of Timothy grass pollen allergen extract, early TLR agonism and/or interferon (IFN)-gamma administration was compared to the therapeutic and immune-modulating effects of dexamethasone with regard to the cellular inflammation and cytokine profiles. RESULTS: Eosinophilic inflammation was clearly reduced by TLR2/6 agonism. This effect was also seen without simultaneous administration of IFN-gamma. However, lymphocyte counts were not affected among the different treatment groups. More precise determination of the lymphocyte-mediated immune reaction showed that TLR2/6 agonism induced neither CD4+foxp3+ regulatory T cells in draining lymph nodes nor a pronounced Th1 immune response. In contrast, dexamethasone reduced both sensitisation as well as allergic inflammation and, in addition, CD11c+ antigen-presenting cells in lymph nodes. Our data clearly point to the potential to rebalance Th2-skewed allergic immune responses by therapeutic TLR2/6 agonist administration. CONCLUSION: The use of the TLR2/6 agonist is a promising therapeutic approach in diseases with an imbalance in T cell responses, such as allergy and asthma.

The effect of titanium dioxide nanoparticles on pulmonary surfactant function and ultrastructure.

BACKGROUND: Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO(2)) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling in vitro. In addition, TiO(2) effects on surfactant ultrastructure were visualized. METHODS: A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 microg/ml) of TiO(2) NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope. RESULTS: TiO(2) NSP, but not MSP, induced a surfactant dysfunction. For TiO(2) NSP, adsorption surface tension (gammaads) increased in a dose-dependent manner from 28.2 + or - 2.3 mN/m to 33.2 + or - 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (gammamin) slightly increased from 4.8 + or - 0.5 mN/m up to 8.4 + or - 1.3 mN/m (p < 0.01) at high TiO(2) NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both gammaads (63.6 + or - 0.4 mN/m) and gammamin (21.1 + or - 0.4 mN/m). Interestingly, TiO(2) NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae. CONCLUSION: TiO(2) nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.

Low cytotoxicity of solid lipid nanoparticles in in vitro and ex vivo lung models.

The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) for human lung as a suitable drug delivery system (DDS). Therefore we used a human alveolar epithelial cell line (A549) and murine precision-cut lung slices (PCLS) to estimate the tolerable doses of these particles for lung cells. A549 cells (in vitro) and precision-cut lung slices (ex vivo) were incubated with SLN20 (20% phospholipids in the lipid matrix of the particles) and SLN50 (50% phospholipids in the lipid matrix of the particles) in increasing concentrations. The cytotoxic effects of SLN were evaluated in vitro by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vitality of lung slices was controlled by staining with calcein AM/ethidium homodimer 1 using confocal laser scanning microscopy and followed by quantitative image analysis with IMARIS software. A549 cell line revealed a middle effective concentration (EC(50)) for MTT assay for SLN20 of 4080 microg/ml and for SLN50 of 1520 microg/ml. The cytotoxicity in terms of LDH release showed comparable EC(50) values of 3431 microg/ml and 1253 microg/ml for SLN20 and SLN50, respectively. However, in PCLS we determined only SLN50 cytotoxic values with a concentration of 1500 microg/ml. The lung slices seem to be a more sensitive test system. SLN20 showed lower toxic values in all test systems. Therefore we conclude that SLN20 could be used as a suitable DDS for the lung, from a toxicological point of view.