Effect Of Mouse IL-12 Plasmid On CD44, CD49d Expression And Associated Cytokine Production In The Asthmatic Model Of Mouse

ObjectiveTo investigate the changes of cellular adhesion molecules CD44 and CD49d expression, recruitment of airway inflammatory cells, production of associated cytokine and hyperplasia of airway goblet cells in the sensitized mice after antigen challenge, and to study the probable mechanism of the effect of mouse IL-12 plasmid (mIL-12 plasmid) in the asthmatic model of mouse and provide experimental proofs for immunological genetic therapy of asthma.MethodsSixty-four male BALB/c mice were divided into eight groups including asthma model group (n=8, sensitized with OVA plus challenging with OVA by aerosol), normal control group (n=8, no any treatment), model control group (n=8, sensitized with OVA plus aerosolizing with normal saline), positive control group treatment with DXM (n=8, intraperitoneal injection of DXM with the dose of 0.5mg.kg^-1 oncea day from day 14 to the end of the experiment), empty plasmid prevention control group (n=8, empty plasmid lOOug per mouse respectively administrated intramuscularly on day 1, 3 and 5), empty plasmid treatment control group (n=8, empty plasmid lOOug per mouse respectively administrated intramuscularly on day 14, 16 and 18), mIL-12 plasmid prevention group (n=8, mIL-12 plasmid lOOug per mouse respectively administrated intramuscularly on day 1, 3 and 5), mIL-12 plasmid treatment group (mIL-12 plasmid lOOug per mouse respectively administrated intramuscularly on day 14, 16 and 18). Mice were sacrificed by cervical dislocation and the peripheral blood were obtained at 24h after the last aerosol exposure, the right lungs were deligated and the left lungs were lavaged, the number of inflammatory cell in the mouse bronchoaleolar lavage fluids (BALF) were counted with microscope, and the expression of CD44 and CD49d on surface of leukocyte in the peripheral blood and BALF were detected by flow cytometry. The concentrations of IL-12, IL-13 in blood serum and IL-4, IL-5 in BALF supernatant were also detected by using a sandwich ELISA according to the manufacturer's instruction. Made the pathological section with the right lungs fixed by 10% formalin, and then investigated the eosinophil infiltration in the lung tissue after stained with hematoxylin-eosin (HE) and the hyperplasia of goblet cells in the bronchial mucous membrane after stained with alcian blue- periodic acid Schiff (AB/ PAS).Results(1) The effect of mIL-12 plasmid on recruitment of airway inflammatory cells:After exposure to OVA, the number of total cells was significantly increased in BALF compared with normal and asthma control mice (.P<0.01). Prevention and treatment with mIL-12 plasmid suppressed the number of inflammatory cells compared with the asthma model group, the empty plasmid prevention and treatment control groups (P<0.0l), same as the inhibiting effect of the positive control drugDXM on recruitment of airway inflammatory cells. Evidence of inflammatory cells infiltration and the effect of mIL-12 plasmid were further investigated by histologic examination. OVA-sensitized and challenged mice increased the number of eosinophils in the lung tissue, while prevention and treatment with mIL-12 plasmid. but not empty plasmid, inhibited this eosinophils infiltration.(2) The effect of mIL-12 plasmid on CD44 and CD49d on the surface of leukocyte in the peripheral blood: Flow cytometry for CD44 and CD49d expression revealed that the percentages of CD44 or CD49d positive leucocytes derived from peripheral blood and associated Mean fluorescence Intensity (MFI) were evaluated significantly in OVA-sensitized and challenged BALB/c mice. Prevention and treatment with mIL-12 plasmid can markedly inhibit the expression of CD44 and CD49d on leucocytes compared with the asthma model group, the empty plasmid prevention and treatment control group (PO.01), similar to the effect of the positive control drug DXM on expression of CD44 and CD49d on surface of leucocytes.(3) The effect of mIL-12 plasmid on CD44 and CD49d on the surface of cells in BALF: The percentages of CD44 or CD49d positive inflammatory cells in BALF and associated MFI from asthma model group were evaluated significantly compared with normal control and asthma control group mice (P<0.01) and prevention and treatment with mIL-12 plasmid significantly inhibited the increases (.PO.01), just as the effect of the positive control drug DXM, but the empty plasmid had no these effects.(4) The effect of mIL-12 plasmid on cytokines production: After OVA-sensitized and challenged, the increases of Th2 type cytokines IL-4 and IL-5 in BALF supernatants were significantly suppressed by mIL-12 plasmid administration but not by empty plasmid (PO.01), just as the effect of the positive control drug DXM. The production of IL-12 P70 in serum from asthma model mice was decreasedcompared with normal and asthma control mice (P<0.0l), and prevention and treatment with mIL-12 plasmid significantly increased the concentration of IL-12 P70 in serum compared with asthma model mice (PO.01). The levels of IL-12 P70 in serum from mIL-12 plasmid prevention and treatment group mice were also higher than that from the positive control group treated with DXM mice, even higher that from normal mice (no statistical significance). The serum IL-13 concentration was too low to be detected.(5) The effect of mIL-12 plasmid on the hyperplasia of airway goblet cells: The hyperplasia of airway goblet cells was obvious in mouse model of asthma, and mIL-12 plasmid administration significantly inhibited the hyperplasia of airway goblet cells, just as the effect of the positive control drug DXM, but the empty plasmid had no these effects. The results of semi-quantitative analysis also confirmed it.(6) Results of correlations analysis: There were significant correlations between the percentages of CD44 or CD49d positive cells and the number of inflammatory cells in BALF, the levels of IL-4 and IL-5 in BALF supernatants, the number of goblet cells and the percentages of AB/PAS-positive stained area of epithelium.ConclusionMIL-12 plasmid intramuscularly administrated can efficiently inhibit the airway inflammatory infiltration, especially decrease the EOS, and down-regulated the expression level of cell adhesion molecules such as CD44 and CD49d on the surface of leukocyte in the peripheral blood and BALF, and decrease the hyperplasia of airway goblet cells. IL-12 protein expressed endogenously by mIL-12 plasmid regulates the secretion of Th2 type cytokines IL-4 and IL-5 and then regulates the Thl/Th2 cells imbalance, which may be result in those changes. Our experimental results suggest that mIL-12 plasmid can influence the expression of cell adhesion molecules in a mouse model of asthma and cell adhesion molecules are stickytargets for pathogenesis, prevention and therapy of asthma, and then it may be a potential and powerful preventive and therapeutic medicament of asthma.