RESUMEN
Epidemiologic studies suggest an inverse correlation between infection and development of allergy. The purpose of this study was to test the hypothesis whether a preexisting T helper 1 (Th1)-type immune response elicited by Mycobacterium bovis-bacillus Calmette-Guerin (BCG) immunization could suppress allergic airway inflammation induced by the mite allergen Dermatophagoides pteronyssinus group 2 (Der p2) in an animal model. C57BL/6 mice were immunized with subcutaneous injection of BCG, then intraperitoneal Der p2 emulsified in alum. Der p2-specific immunoglobulin G1 and cytokine production from splenocytes were measured after Der p2 sensitization, and pulmonary function and airway inflammation were determined after inhalation challenge with Der p2. The intraperitoneal Der p2 with alum injection was able to induce Der p2-specific immunoglobulin G1 production, which could be downregulated by the pretreatment with BCG + Der p2. The inoculation of BCG + Der p2 caused splenocytes to produce more interferon-gamma, and this level was higher than that elicited by Der p2 or buffer alone. The positive interferon-gamma-staining CD4 cells were also increased after activation by phorbol myristate acetate and ionomycin. Lung pathology examination found decreased airway inflammation (associated with the best pulmonary function and least airway desquamation) in the mice inoculated with BCG + Der p2. In this Der p2-induced allergy model, BCG inoculation with Der p2 can cause a Th1-type immune response that hinders Der p2-induced allergic sensitization and the development of airway inflammation.
Asunto(s)
Antígenos Dermatofagoides/inmunología , Vacuna BCG , Hipersensibilidad Respiratoria/prevención & control , Animales , Proteínas de Artrópodos , Asma/inmunología , Asma/prevención & control , Vacuna BCG/inmunología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/prevención & control , Recuento de Linfocito CD4 , Células Cultivadas , Regulación hacia Abajo , Inmunoglobulina G/biosíntesis , Inflamación/patología , Inflamación/prevención & control , Interferón gamma/inmunología , Interleucina-5/biosíntesis , Interleucina-5/metabolismo , Pulmón/inmunología , Pulmón/patología , Pulmón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Pyroglyphidae/inmunología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/patología , Bazo/citología , Bazo/inmunología , Células Th2/inmunologíaRESUMEN
BACKGROUND: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. METHODS: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). RESULTS: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. CONCLUSIONS: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.