| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 93 === The fungi are regarded as the main source of allergens that can cause IgE-mediated allergic and atopic disease. Penicillium and Aspergillus are the most common indoor fungal species in Taiwan. It has been recognized that inhalation of fungal allergens can produce allergic rhinitis and asthma in susceptible individuals. But the mechanism about how these allergens induce allergic reaction is still not clear. Recent studies have shown the existence of PAR-2 (proteinase-activated receptor 2) which is expressed on the bronchial and respiratory epithelium cells may be activated by proteolytic allergens. Thus we used the allergen Pen c13, the serine protease we had purified from P. citrinum, to investigate the mechanism of allergy.
The coding cDNA of Pen c13 was cloned into vector pQE30 and expressed in E. coli M15 as a His-tag fusion protein and purified by affinity column. The rPen c13 reacted with serum IgE from 25% of allergic patients. Purified nPen c13 elicited dose-dependent basophil and mast cell histamine release in patients allergic to Penicillium.
In the pulmonary epithelial cell line A549, GM-CSF, IL1β, IL-6 and IL-8 mRNA levels were enhanced by nPen c13. Furthermore, ELISA was also used to detect IL-8 and IL-6 in protein level. Both of them showed significant release after the stimulation by nPen c13 in a dose-dependent manner. The maximum concentrations which IL-8 and IL-6 released were observed at 0.1ug/ml and 0.05 ug/ml respectively. From these experiments we conclude that nPen- c13 can induce inflammatory reaction in A549. Serine protease inhibitors, PMSF and AEBSF, were then chosen to inactivate nPen c13 in order to check whether the inflammatory reaction was caused by nPen c13. The result obtained indicated that IL-8 stimulated by nPen c13 was abolished when protease activity was inhibited by PMSF and AEBSF. Furthermore, Western blot analysis showed that COX-2 expression was also enhanced by nPen c13.
Additionally, we tried to understand whether PAR-2 involved in this inflammatory reaction of A549 caused by nPen c13, antibody against the cleavage region of PAR-2 was used in order to block PAR-2 cleavage by nPen c13. The result indicated the expression of IL-8 was reduced in A549. Then cells were treated with Ca2+ free buffer and the production of IL-8 by nPen c13 significantly decreased, so Ca2+ may play an important role in the inflammatory reaction induced by PAR-2.
The present study indicated that nPen c13 can indeed stimulate histamine release and induce inflammatory reaction in respiratory epithelium cells. The latter reaction of nPen c13 involves its own serine protease activity and PAR-2 may play a role in this result. Our findings are similar to previous studies of mite allergen with PAR-2. Advances in understanding the molecular mechanism of atopy may lead to new therapies for allergic diseases in the future.
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