| Allergic asthma is a highly complex disease that is still poorly understood and whose cause remains unknown. It is characterized by reversible airway obstruction, elevated serum levels of immunoglobulin E (IgE), chronic eosinophilic airway and airway hyperesponsiveness (AHR) to bronchospasmogenic stimuli. Type 2 T-helper (Th2) lymphocytes play a crucial role in the initiation, progression and persistence of the disease. Initially, it was hypothesized that a disturbance between Th1-and Th2- mediated immune response underlies aberrant Th2 reactions to harmless inhaled allergens. Recent research founded that T regulatory cell also play an important role in the inflammation of asthma.Dendrtic cells (DC) are the most potent APCs that play a central role in initiating the primary immune response. There're two distinct subsets of DC, myeloid DC (DC1) and plasmacytoid DC (DC2), have been identified. DC1, derived from myeloid precursors, expressmyeloid cell markers CD 13 and CD33, and require exogenous granulocyte-macrophage colony-stimulating factor for their survival. Mature DC1 (mDC) produce high levels of IL-12 and drive a potent Thl-polarized immune response. On the other hand, DC2, which originate from lymohoid precursors, show a plasma cell-like morphology and a lack of myeloid cell markers, and express high amounts of IL-3 receptor a chain. DC2 can elicit an IL-4-independent Th2 polarization of naive T cells. On the basis of these distinct properties of the DC subtypes, DC1 and DC2 were considered to be specialized APCs inducing a Thl and Th2 response, respectively. Moreover, it has been suggested that mature DC(mDC) could polarize Thl response and induce immunity, whereas immature DC(imDC) have the potential to induce tolerance. So, DC, being the most powerful professional APCs, play a pivotal role in the decision between T-cell activation and anergy. Interleukin (IL)-18 is one of important Thl cytokines which can induce production of interferon-Y(IFN-y) and company with IL-12 to induce ThO cells differentiation into Thl cells. Therefore, we hypothesize that IL-18 gene modified mDC vaccine are important in correcting the disturbance of Thl/Th2 and may favor of alleviating the chronic eosinophilic airway and airway hypersecrection in vivo in patients with asthma. In this study, we used ovalbumin(OVA)-specific mDC vaccine(mDCovA) and IL-18 gene transferred mDCovA(IL-18-mDCovA) to immunize the mouse model of asthma, observed the therapeutic potential of IL-18-mDCovA and study the related mechanism.PURPOSEObserve the impact of IL-18 gene transfered mDCovA on murine model with allergic asthma and study the associated mechanism, and try to establish the basis for exploring the new therapeutic method for the asthma.METHODS1. Gengenation and genetic modification of bone marrow-derived DC: BALB/c mice (6-8W) were used in the experiment. Bone marrow-derived DC were generated by culturing mouse bone marrow cells from femus with DC culture for 6 days in 6 well plate. After 6 days culturing, the loosely adherent cells were stimulated by LPS and OVA for 10 hours, DC were incubated with AdLacZ or AdIL-18 at multiplicity of infection(MOI) of 100:1 for 2 hours and then washed twice with complete RPMI 1640 twenty-four hours after gene modification, LacZ gene-modified DC(LacZ-DC) were collected for 5-bromo-4-chloro-3-indolyl- P -D-galactopyranoside(X-Gal) staining to evaluate gene transfer efficiency, IL-18 gene-modified DC were subjected to RT-PCR analysis for IL-18 expression, and phenotypic analysis of DC after transfection with Ad vectors was performed by FACS using the following FITC-McAbs: Iad, CD86, CD40.2. Establish the murine model of allergic asthma: Mice were sensitized by subcutaneous injection (s.c.) with antigen 10% ovalbumin and aluminium hydroxide adjuvant mixture in the two back-footpads, each side for 50ul. Then sensitized them intraperitoneally by the same antigen on lid. On 15d, exposure the mice to an aerosol of 10% ovalbumin solution for 30 min, and one time everyday for 7 days.3. The immunzation of mDCovA? LacZ-mDCovA or IL-18-mDCovA:120 BALB/c have been divided into two teams including once immunization team and twice immunization team. Then divided each team into 6 groups(n=10), Control group, PBS group, DXM group, mDCovA group, LacZ-mDCovA group, IL-18-mDCovA group. mDCovA, LacZ-mDCovA or IL-18-mDCOvA (1 X 106cells/mouse) was s.c. once in the two groins of mouse 7days (named once immunization team), or s.c. twice lday and 21 days before the first time of OVA sensitization, respectivly (named twice immunization team). 28 days later of atomization, sacrificed themice and collected the samples. Take count of total number of inflammatory cells and the percent of EOS in the bronchoalveolar lavage fluid (BALF);Use ELISA to determine the concentration of IFN-y, IL-4 and IL-13 secreted by the splenic cells;RT-PCR to determine the expression of IFN-y mRNA;FACS to analyze the ratio of CD4+CD25+Treg in the splenic cells;the lung was histoligically examinated after fixing in 10% formalin followed by PAS and HE stain.RESULTS1. Decreasing the number of inflammatory cells in the BALF from the mouse of asthma immunized with mDCovA* LacZ-mDCovAor IL-18-mDCovA- In the once injection team the number of inflammatory cells of mDCovA, LacZ-mDCovA, IL-18-mDCovA groups are 6±0.412, 6.18±0.37, 5.640±0.15 (106cells/ml), significantly lower than PBS group (P<0.01);In the twice immunization team the number of inflammatory cells of mDCovA, LacZ-mDCovA> IL-18-mDCovA groups are 5.46±0.335, 5.54±0.35, 4.3±0.308 (106cells/ml), significantly lower than PBS group (PO.01). Percent of the EOS of the three groups is also lower than PBS group(P<0.01).2. Polarizing Th2 response in the mouse of asthma immunized with mDCovA, LacZ-mDCovA or IL-18-mDCovA' concentration of IFN-y secreted by splenic cells of mDCovA, LacZ-mDCovA, IL-18-mDCovA groups is higher than that of PBS group (PO.01);concentration of IL^ IL-13 secreted by splenic cells of mDCovA, LacZ-mDCovA, IL-18-mDCovA groups is lower than that of PBS group..3. Enhancing the expression of IFN-y mRNA in the lung from the mouse of asthma immunized with mDCovA, LacZ-mDCovA or IL-18-mDCovA: from the electrophoretic result we find that the expression of IFN-y mRNA in mDCovA, LacZ-mDCovA, IL-18-mDCovA groups is remarkably higher than that of the otherthree groups.4. Increasing the ratio of CD4+CD25+T cells in the splenic cells from the mouse of asthma immunized with mDCovA, LacZ-mDCovAor IL-18-mDCovA^ the ratio of CD4+CD25+T cells in the splenic cells of mDCovA, LacZ-mDCovA, IL-18-mDCovA groups is higher than that of three other groups.5. Inhibiting the infiltration of pulmonary inflammatory cells and hyperplasia of goblet cell in the lung tissue after immunization with mDCovA, LacZ-mDCovAor IL-18-mDCovA: the inflammatory cell infiltration, quantity of EOS in lung tissues from mice treated by mDCovA, LacZ-mDCovA or IL-18-mDCovA are significantly lower than that in PBS group. Moreover, the hyperplasia of airway goblet cells was obvious in mouse model of asthma, and mDCovA or LacZ-mDCovA administration simificantly inhibited the hyperplasia of airway goblet cells. The most significant decreaseing of number of airway goblet cells were observed in the IL-18-mDCovA group compared with those from all of other groups.CONCLUSIONS1. IL-18-mDCovA vaccine can inhibit the infiltration of pulmonary inflammatory cells and the hyperplasia of goblet cell. Thl cytokine gene modified mDCovA immunization may alleviate the chronic eosinophilic airway and airway hypersecrection in vivo in mouse with asthma.2. IL-18-mDCovA immunization can augment Th-1 type CKs secretion and reduce the production of Th-2 type CKs expression, also increase the expression of the IFN-y mRNA extracted from lung tissue.The induction of Thl immune response may suggest that immunization with IL-18-mDCovA has therapeutic potential in the patient of asthma.3. IL-18-mDCovA immunization can increase the ratio of CD4+CD25+T cells inthe splenic cells, which is one of the possible mechanisms involved in the effection of IL-18-mDCovA vaccine.
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