Study Of Molecular Mechanism Of Dendritic Cells In The Pathogenesis Of Asthmatic Mice

Asthma is a commonly globe disease which seriously threatens people health; it has increased dramatically in prevalence, but we have not completely understood the pathogenesis of asthma. The research in recent years shows that asthma is defined as a chronic airway inflammatory condition with characteristic inflammatory cells such as T lymphocytes, eosinophilic and mast cells infiltrated in airway; The greatest immune abnormal of asthma is the CD4~+ helper T lymphocytes activated excessively and the imbalance of both the ration and function of Th1/Th2. It also shows that dendritic cells (DCs) are the functionally strongest antigen presenting cells (APC) in the antigen presenting process in vivo, and it has proved to be they are the unique APCs those can activate naive T cells in vitro. DCs play a key role in the balance or deviation (imbalance) process in inducing Thl/Th2 cells differentiation. To investigate the role of DC in the pathogenesis of asthma from molecular level, we sensitized BALB/C mice with ovalbumin (OVA) and established asthmatic models. The bone marrow cells were isolated from both asthmatic mice and normal controls, then these cells were induced and amplified with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin 4 (rmIL-4) to DCs (bone marrow-derived DCs) in vitro. The morphological changes were observed and the expression of the surface molecules CD80, CD40, CD86 and 33D1 on bone marrow-derived DCs were examined by fluorescent activated cell sorter (FACS). Their potentials of DCs to stimulate the proliferation reaction of allogeneic T cells were observed with no-radioactive mixed lymphocytes reactivation (MLR). The transcription of cytokine mRNA was examinedby ribonuclease protection assay (RPA) combined with multi-probe templates; last whether nuclear factor kB (NF-kB) was activated or not was studied by electrophoretic mobility shift assay (EMSA). It is hoped that these studies can offer some molecular foundations for understanding the role of DCs in asthma and new ideas that DCs may be the foreground as a novel cellular target in future ' anti-asthmatic therapies. The main results and findings are like the following:1. Bone marrow cells isolated from asthmatic and control mice were induced to be DCs with CM including rmGM-CSF and rmTL-4, on 2nd day, there were many dot colony and cells were seen in round shape. Continued culture, the colony became increasing and cells got larger; after 4 day's culture, they were seen dendritic character and some cells began to suspend; on 7 day, most of cells suspended and distributed in medium, displayed characteristic dendritic or stellate morphological characteristiand, they could be collected by whiffed. Both groups of DCs had big or small dendrites, but the number of dendrites was not different in two groups.2. The DCs in both asthmatic group and control group induced by bone marrow expressed the related differentiated antigen of DCs: CD8CK CD40> CD86 and mouse special marker-33Dl. Moreover the expression of CD86 on asthmatic group of DCs was higher than that control group, but the expression of CD80> CD40 and 33D1 were no difference between two groups.3. The DCs in both asthmatic group and control group could intensely stimulate the proliferation of allogeneic T cells, and the power of stimulation of DCs in asthmatic group was markedly stronger than that of control group, which indicated the ability of antigen presenting in DCs in asthmatic group was obviously enhanced. Under the same T cells, the power of proliferation was enhanced with the increase of numbers of DCs; in asthmatic group, there was discrepancy in different the ratio of DC and T cells, but not in control group.4. The DCs in both asthmatic group and control group expressed cytokine IL-13 ^ IL-9 and IL-3 mRNA; Furthermore the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic group was significantly higher than control group, but the level of IL-3 mRNA was no discrepancy between two groups.5. NF-kB was activated in DCs between two groups and the extent of activation in asthmatic group increased, which were related with their stronger functions.It can be proved that DCs are truly induced and amplified from asthmatic mice bone marrow from above results and findings. In asthmatic mice, DCs express CD80> CD40 and CD86 costimulatory molecules and strengthen their capacity of antigen presenting through different expression of CD86 costimulatory molecules on cell surface, farther enhance the ability of stimulating naive T cells. Moreover, the transcription of IL-13 and IL-9 mRNA in DCs in asthmatic mice is higher than that control mice at the same time, which will contribute to prime the deviation of allergic special T cells and the production of Th2 cytokine excessively, finally leading to deviation of Thl/Th2. NF-kB is activated increasedly in DCs of asthmatic mice, which probably relates with higher expression of costimulatory molecules and transcription of cytokine in asthmatic group. The study provides new avenue for the research of pathogenesis of asthma and clues to therapy for asthma based on DCs will be a new goal.