Allergic airway inflammation in mice deficient for the antigen-processing protease cathepsin E.

BACKGROUND: Allergic asthma is a Th2-type chronic inflammatory disease of the lung. It is characterized by infiltration of eosinophils, neutrophils, mast cells and T lymphocytes into the airways. Th2 cytokines like interleukin (IL)-4, IL-5 and chemokines like eotaxin are increased in the asthmatic response. The processing and presentation of exogenous antigens is important in the sensitization to an allergen. Cathepsin E (Ctse) is an intracellular aspartic endoprotease which is expressed in immune cells like dendritic cells (DCs). It was found to play an essential role in the processing and presentation of ovalbumin (OVA). The aim of the present study was to investigate the inhibition of Ctse in two different experimental models of allergic airway inflammation. METHODS: Ctse wild-type (Ctse(+/+)) and Ctse-deficient (Ctse(-/-)) bone marrow-derived DCs (BMDCs) were pulsed with OVA/OVA peptide and cocultured with OVA transgenic T II (OT II) cells whose proliferation was subsequently analyzed. Two different in vivo asthma models with Ctse(+/+) and Ctse(-/-) mice were performed: an acute OVA-induced and a subchronic Phleum pratense-induced airway inflammation. RESULTS: Proliferation of OT II cells was decreased when cocultured with BMDCs of Ctse(-/-) mice as compared to cells cocultured with BMDCs of Ctse(+/+) mice. In vivo, Ctse deficiency led to reduced lymphocyte influx after allergen sensitization and challenge in both investigated airway inflammation models, compared to their control groups. CONCLUSION: Ctse deficiency leads to a reduced antigen presentation in vitro. This is followed by a distinct effect on lymphocyte influx in states of allergic airway inflammation in vivo.

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.

Expression, localisation and functional implications of the transporter protein PEPT2 in the upper respiratory tract.

Infectious diseases of the upper respiratory tract are widespread and may initiate exacerbations of chronic respiratory diseases such as bronchial asthma or chronic obstructive pulmonary disease. Nebulisation of antibiotics as a topic treatment may lead to high local drug concentrations. To provide data on drug transport, the present study analysed a specific drug transport system on the molecular and functional level. Messenger RNA of the proton-coupled transporter PEPT2 that mediates physiological transport of oligopeptides as well as peptidomimetics like beta-lactams and aminolevulinic acid was discovered in rat nasal mucosa by RT-PCR. Real-time PCR studies indicated a lower expression level than control kidney samples. PEPT2 immunoreactivity was identified in nasal mucosa tissue. The protein was expressed in epithelial cells, but goblet cells did not exhibit PEPT2 expression. Functional studies with rat preparations led to uptake of a fluorophore-conjugated substrate into epithelial cells of nasal mucosa. Goblet cells did not exhibit uptake activity. The uptake was competitively inhibited by dipeptides demonstrating similar substrate specificity as reported for PEPT2. Together, these data suggest that PEPT2 is likely to play an important role in mucosal peptide metabolism and may represent a novel target for therapeutic efforts in upper airway diseases.

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.

LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing.

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1ß) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1ß. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1ß levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs.