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Functional role of lysosomal proteases in allergic airway inflammation

 
: Pilzner, C.
: Groneberg, D.; Welte, T.; Braun, A.

Hannover, 2011, 98 Bl.
Hannover, Medizinische Hochschule, Diss., 2011
English
Dissertation
Fraunhofer ITEM ()
cathepsin E; allergic airway inflammation; allergic asthma

Abstract
Cathepsin E (Ctse) is an intracellular aspartic endoprotease of the pepsin family. It is expressed in immune cells such as dendritic cells (DC) and macrophages. In in vitro experiments Ctse was found to play an essential role in processing and presentation of the full Ovalbumin (OVA) protein but not of OVA-derived peptides. It was hypothesised that an inhibition of Ctse expression leads to a decrease of allergic airway inflammation in an OVA-induced or in a phleum pratense induced experimental model of asthma. Allergic asthma is a Th2 type chronic inflammatory disease of the lung. It is characterized by a DC dependent infiltration of eosinophils, neutrophils, mast cells and T lymphocytes into the airways. Th2-cytokines such as interleukin (IL) IL-4, IL-5 and chemokines such as eotaxin are increased in the asthmatic response. In this thesis bone marrow-derived dendritic cells (BMDC) from Ctse wildtype (Ctse+/+) and Ctse deficient (Ctse -/-) mice were pulsed with OVA or OVA peptide and cocultured with OVA transgenic T cells II (OT II cells) whose proliferation was analysed subsequently. Following two different in vivo asthma models were performend. In an acute asthma model Ctse+/+ and Ctse-/- mice were sensitized intraperitonally with OVA combined with aluminium hydroxide (Alum) on day 0, 14 and 21. On day 27 and 28 Ctse+/+ and Ctse-/- mice were challenged with an OVA aerosol. In a subchronic model of allergic airway inflammation Ctse+/+ and Ctse-/-mice were repetitive sensitized intranasally with phleum pratense extract over a period of 11 weeks. Investigating the influence of Ctse deficiency in antigen processing in more detail DQTM OVA was used in an in vivo experiment with Ctse+/+ and Ctse-/- mice. Analysing the proliferation of CFSE labelled OT II cells which were injected into Ctse+/+ and Ctse-/- mice was performed to determine the antigen presentation in vivo. Finally real-time PCR experiments and Western blot analyses were conducted to investigate the expression of different Cathepsins in isolated lung DC compared to in vitro generated BMDC. Proliferation of OT II cells was decreased when cocultured with BMDC of Ctse-/-mice as compared to cells cocultured with BMDC of Ctse+/+ mice. In vivo sensitization and provocation in the acute OVA induced as well as in the phleum pratense induced airway inflammation lead to no increase in the amounts of T lymphocytes of sensitized Ctse-/- mice compared to the Ctse+/+ sensitized group. However no quantitative difference in the amount of eosinophilic recruitment between the Ctse+/+ and Ctse-/- mice was detected. There was no difference between Ctse+/+ and Ctse-/- mice in processing of DQTM OVA. No difference could also be detected between the Ctse+/+ and Ctse-/- sensitized mice in the proliferation of CFSE labelled injected OT II cells. Investigating the expression of different Cathepsins in isolated lung DC compared to in vitro generated BMDC showed that Cathepsin B, C and L were 10 times less expressed in lung DC compared to BMDC, that Cathepsin S was 3 times less expressed in lung DC and that Cathepsin K was 20 times more expressed in lung DC compared to BMDC. Cathepsin D, E and H showed no difference in expression in lung DC compared to BMDC. In conclusion the deficiency of Ctse has an effect on the antigen processing and presentation in vitro. Investigating this effect in an acute OVA induced and subchronic phleum pratense induced airway inflammation, showed no lymphocyte infiltration in Ctse-/- mice in both investigated airway inflammations. However a quantitative difference in the amount of eosinophilic recruitment between the Ctse+/+ and Ctse-/- mice was not found in both induced airway inflammations. Mechanistic investigations of processing and presentation of Ovalbumin in vivo showed as well no difference between Ctse+/+ and Ctse-/- mice. Therefore, a distinct role of Ctse in states of allergic airway inflammation, due to no lymphocyte infiltration in Ctse-/- mice in both investigated asthma models, can be assumed.

: http://publica.fraunhofer.de/documents/N-180977.html