Proteolytic activity in cowshed dust extracts induces C5a release in murine bronchoalveolar lavage fluids which may account for its protective properties in allergic airway inflammation.

OBJECTIVE: Intranasal application of cowshed dust extract (CDE) during sensitisation in a murine model of experimental asthma leads to a significant alleviation of the clinical parameters of the allergic immune response. However, neither the immunological mechanisms underlying this protective effect nor all of the protective substances included in CDE have yet been described. Recently, complement factor 5a (C5a) receptor signalling has been identified to play a regulatory role in allergic airway disease. Thus we investigated whether CDE can activate the complement system to release biologically active C5a in the lung. METHODS: Proteins included in CDE were identified by mass spectrometry. Complement cleaving activity of a serine protease identified in CDE was validated with the purified enzyme, and the biological activity of the released C5a was determined. C5a was applied in a murine model of allergy to prove its protective impact on allergic airway disease. RESULTS: CDE induced the release of C5a in murine bronchoalveolar lavages (BAL). We identified a serine protease from the midgut of tenebrio molitor larvae in CDEs which was able to induce the release of biologically active C5a in murine BAL. We applied C5a in different doses to female Balb/c mice during the sensitisation phase and during the first antigen challenge and showed that C5a has the ability to dampen important parameters of allergic airway inflammation, such as infiltration of proinflammatory cells into lung tissue or Th2 cytokine secretion by lung cells. CONCLUSIONS: We conclude that the C5a generating enzyme included in CDE might account for some of the allergy protective effects of CDE by generation of C5a in murine lungs.

Specific Immunotherapy in a Murine Model of Grass Pollen (Phl p5b)-Induced Airway Inflammation.

Background: The use of ovalbumin as a model allergen in murine models of allergic asthma is controversially discussed since it is not an aeroallergen and sensitization can only be achieved by using strong Th2-inducing adjuvants. Therefore, in this study, a murine model of asthma has been established in which sensitization against the major grass pollen allergen Phl p5b was performed without using aluminum hydroxide (alum). We used this model for specific immunotherapy. Methods: Female, 5-6-week-old mice were sensitized by six subcutaneous (s.c.) injections of 20 µg Phl p5b followed by four provocations to induce allergic airway inflammation. For desensitization, 1 mg of Phl p5b was injected subcutaneously during allergen challenge for one to a maximum of four times. Three days after the last challenge, the allergic immune response was analyzed. Results: Sensitized and challenged animals showed a significant infiltration of eosinophils into the airways, and the production of interleukin-5 (IL-5) by in vitro re-stimulated splenocytes could be detected. Furthermore, hyper-responsiveness of the airways was verified by invasive measurement of airway resistance in methacholine-challenged animals. Desensitized animals showed a significant reduction of all parameters. Conclusion: In this study, a murine model of asthma has successfully been established by sensitization against the clinically relevant allergen Phl p5b without using alum. S.c. injection of allergen dose dependently led to desensitization of sensitized mice. We suggest that this model is useful to study adjuvant effects of immune modulatory substances on immunotherapy without the interference of alum.

T-cell polarization depends on concentration of the danger signal used to activate dendritic cells.

Although several studies have focused on allergic sensitization by dendritic cells, to date it is still open under which conditions these antigen-presenting cells are able to induce an allergic immune response. Our study reveals that BMDCs pulsed with LPS-free ovalbumine did not induce allergic disease. However, when BMDCs were activated with low-dose LPS during pulsing with allergen, these cells expressed an inflammatory set of cytokines and co-stimulatory molecules like CD86 and OX40L. Moreover, activated cells were able to prime mice for massive eosinophilic inflammation of the lung, airway hyper-reactivity, IgE production and production of Th2 cytokines by lymphocytes. Blocking experiments showed that expression of OX40L is not involved in induction of Th2 response. Interestingly, BMDCs that were activated with high dose of LPS lose their Th2-sensitizing capacity. Instead these cells induce a Th17 type immune response. We conclude that presentation of allergen by dendritic cells generated with GMCSF is not sufficient to lead to induction of allergic immune response. Further activation of BMDCs is required to prime mice for allergic immune response. In this study, we show that LPS is a suitable stimulus. However, when cells were activated with high dose LPS they tended to induce a Th17 response.

Conference Scene: Summary report from EAACI: London 2010.

Immunotherapy is, to date, the only effective curative method for the treatment of allergic disorders. Great efforts have been made to improve the efficacy, safety and patient compliance with this method. The growing understanding of the immunological mechanisms underlying immunotherapy has led to new approaches for immunotherapy involving the routes of administration and the kinds of molecules used. In addition, new vaccines are being created that combine the advantageous immunological charasteristics of different substances, such as virus-like particles linked to allergens. Many new results from ongoing research into these topics were presented at the 29th congress of the European Academy of Allergy and Clinical Immunology in London.