Efficacy And Mechanism Of Recombined Bla G 2 Immunotherapy In Murine Allergic Asthma

OBJECTIVE:To investigate the therapeutic efficacy and mechanism of recombined Bla g 2 (rBla g 2) allergen in murine allergic asthma by intraperitoneal injection.METHODS:1. Cloning and expressing of rBla g 2 allergen.2. Eighteen BALB/C mice were randomly divided into three groups: normal control group (group A), asthma group (group B) and recombined protein rBla g 2 treatment group (group C). Mice in group B and C were subcutaneously immunized weekly with recombined protein rBla g 2 (50μg) formulated in Al(OH)3 adjuvant for two weeks. Group A received only adjuvant emulsified with normal saline. Two weeks after the last immunization, mice in group C were administered with rBla g 2 (100μg), and at the same time group A and B were given PBS. All the mice received eight treatments at 2-day intervals with the same dose of allergen. One week after the last immunotherapy, mice in group B and C were intranasally challenged with 50μg rBla g 2 daily for seven days, while Group A were treated with PBS. From the 51st day (first challenge) to the 57th, symptoms of all the mice in the animal center were observed, at 4'clock every afternoon. 24 hours after the last challenge, all the mice were measured as the following.3. Tweenty-four hours after the last challenge, Airway hyperresponsiveness (AHR) was measured using unrestained whole-body plethysmography (WBP) with a two chamber system. The mice were subjected to aerosolized PBS, followed by progressively increasing doses of methacholine (1, 3, 10, 30, 100 mg/ml), then to protract breath curve for analyzing Penh value change.4. Mice were bled from the retro-orbital venous plexus and sacrificed, then the lungs were immediately lavaged via the trachea cannula with PBS, total cellular score and cell classification of bronchoaleolar lavage fluid (BALF) were assayed.5. All the mice serum IgE and IgG2a were determined by enzyme-linked immunosorbent assay (ELISA).6. After the lavage, the lungs were immediately removed, and examined their inflammatory changes with general HE staining.7. Bcl-2 expressing of eosinophils (EOS) of lung was assayed with immunohistochemically.8. The spleens were removed, and gene expressing TLR-2, TLR-4, PAR-2 and PAR-4 were assayed.RESULTS:1. Compared with mice in group A, those in group B after challenge showed the following symptoms, such as gasp, deeper and faster breath, and dysphoria. Penh increased (P<0.05). Total number of cells increased, EOS cells account for 90%. Serum specific IgE and IgG2a inceased (P<0.01). Lung was characterized by a dense mixed cellular infiltration, tissue edema and damage of epithelium. Bcl-2 expressing of EOS increased. Spleen gene expressing PAR-2, PAR-4 and TLR-4 reduced, but TLR-2 gene expressing unchanged. The above descriptions proved successful modeling.2. Compared with mice in group B, that in group C showed the similar but alleviated symptoms, such as gasp, deeper and faster breath, and dysphoria.3. Compared with that of group B, Penh of group C reduced (P<0.05).4. Compared with the total number of cells [(24.60±15.08)×105/ml], EOS cells [(22.20±3.76)×105/ml] in BALF of group B, total number of cells [(14.30±4.95)×105/ml], EOS cells [(5.20±1.56)×105/ml] in BALF of group C reduced (P<0.01).5. Compared with those in group B, rBla g 2-specific IgE reduced and IgG2a inceased in group C (P<0.01).6. The lung was characterized by a dense mixed cellular infiltration, tissue edema and damage of epithelium in group B, while lung inflammation of group C reduced obviously.7. Compared with that of group B, Bcl-2 expressing of EOS reduced in group C, and albumin masculine expressing weakened.8. Compared with those of group A, spleen gene expressing PAR-2, PAR-4 and TLR-4 of group B reduced, while which of group C increased, TLR-2 gene expressing unchanged.9. Asthmatic symptoms, airway Penh, total number of cells and cell classification in BALF, serum IgE and IgG2a, inflammation of lung, Bcl-2 expressing of EOS, and TLR-2, TLR-4, PAR-2 and PAR-4 gene expressing of spleen in group C appeared to be closed to those in group A.CONCLUSION:The rBla g 2 treatment by intraperitoneal injection can effectively alleviate the clinical symptoms in murine allergic asthma, and the apoptosis of EOS (evaluated by Bcl-2 expressing) plays an important role in this process.