The Research Of CD8~+T Cells In Asthmatic Mouse Model

Study BackgroundWith the advent of modern industrialisation in the middle of the 20th century the incidence of respiratory allergy increased substantially accounting for an ever greater healthcare burden. According to the world health organization, approximately 300 million people suffer from asthma, while estimates suggest that asthma prevalence increases globally by 50% every decade. While the prevalence of respiratory allergic disease may be levelling off in developed countries, it continues to rise in developing world.Bronchial asthma is characterized by airway hyperresponsiveness and chronic inflammation, It is generally believed that asthma is duo to chronic airway inflammation involving a variety of cells such as epithelial cells, fibroblasts, dendritic cells, eosinophils, mast cells and T lymphocytes. Among those cells, T lymphocytes are most important as a T helper 2 (Th2) biased T helper 1(Th1)/Th2 imbalance has been proved to play a cruicial role in the pathogenesis of asthma. While it is well established that CD4+ T lymphocytes play a crucial role in the initiation, progression and persistence of asthma, the role of CD8+ T cells is less understood. CD8+T cells form functionally similar subsets which exhibit similar cytokine profiles as Thl and Th2 cells, Evidence from animal studies suggest that CD8+ T cells are capable of regulating IgE production through the induction of IL-12 and IL-18 production in dendritic cells, and that CD8+ T cells may act to moderate Th2 polarisation within the localised lymph nodes during allergic sensitisation. Such findings have led to suggestion that Thl polarising, CD8+T cell-inducing vaccines would inhibit the development of airway hyperresponsiveness (AHR) and Th2 cell infiltration. Despite these positive findings, the role of CD8+ T cells within the lung remains poorly understood, the association between CD8+ T cells and asthma is almost universally defined as injurious, further suggesting a prejudicial role for these cells within the established disease. CD8+ T cells may be a valuable potential target for therapeutic intervention, either by potentiating their regulatory effects prior to the development of sensitisation, or through suppressing their proinflammatory properties within established atopy.In this study we designed to investigate the effect of CD8+ T cells in the murine asthma model and to explore their roles in the pathogenesis of asthma through exammning IL-4,IL-10,IFN-ysecreting in lungs and spleen.,at last,we analysed the proliferation situation of CD8+CD28- T cell.Part one Establishment of the asthmatic mouse modelObjectiveTo establish a BALB/c mouse asthma model by OVA sensitization and re-challenge.MethodsSPF female BALB/c mice (4 to 6 weeks),18～20g, were purchased from Laboratory Animal Services Center of Southern Medical University, and maintained on an ovalbumin-free diet.The mice were randomly divided into 2 groups:control group and asthmatic group. Mice were sensitized on days 0 and 14 by an intraperitoneal injection of a mixture containing 2 mg of ovalbumin and 5 mg of Al (OH) 3 in saline (a total volume of 0.2 ml). At 21 days after the first immunization the animals were firstly challenged with intranasal drop of 4% ovalbumin, then challenged by exposure to an aerosol of 4% ovalbumin generated by an ultrasonic nebulizer for 40 min. The 40-min rechallenge procedure was repeated once each day from day 22 to day 28. The control animals were treated with 5 mg of A1(OH)3 in saline and re-challenged with saline solution. Forty-eight hours after the last allergen challenge, mice were placed in a whole-body plethysmograph chamber.Enhanced pause (Penh) was measured after each nebulization with increasing doses of methacholine and this measure was used to evaluate AHR. Animals were then sacrificed. Six mice from each guoup were subjected to bronchoalveolar lavage. The bronchoalveolar lavages were stained with HE and eosinophil number counted with a hematocytometer. At least 400 lymphocytes were counted by microscopy before the percentages of various types of cells were calculted. Lung Tissues from other mice were sliced and HE stained for histological examination, including morphology, epithelial injury, perivascular and peritracheal infiltration of eosinophils.Results1. The behavior changes after re-challengeMice in asthmatic group displayed various types of allergic responses, such as scratching head and face, dyspnea, oral lip cyanosis, muscle spasm as well as gatism, while control group showed no such symptoms.2. AHR measurementRelative to animals in control group,OVA treatment resulted in a significant(P< 0.05, P< 0.01) increase in methacholine (≥6.25mg/ml)-induced airway hyperresponsiveness.3. The changes in total cells and different cell types in BALFa. The total BALF cell number in-the asthma group was 1.41±0.13×105/ml, that is significantly higher than that in the control group 0.62±0.43×105/ml (t=-14.152,P<0.01). b.The percentages of BALF eosinophils and neutrophils were 25.42%±4.05% and 29.63%±4.91% in the asthma group, that were also significantly higher than those in the control group 0.83%±1.18% and 5.67%±1.42%(t=-14.259, t=-11.479, P<0.01 for both comparisons).3. Histological analysisMassive leukocyte infiltration was present in the lungs from the asthmatic mice. Neutrophils, eosinophils and lymphocytes were mainly found in the alveolar, interstitial and peribronchial regions. Goblet cell hypertrophy, epithelial damage, mucus hypersecretion, mucus plugs, as well as collagen deposition were also easily identified. While lungs in the control mice displayed a clear airway structure without any significant inflammatory cell infiltration or collagen deposition.ConclusionsBALB/c mice first sensitized by an intraperitoneal injection of a mixture containing ovalbumin and Al(OH)3, and then re-challenged with intranasal ovalbumin inhalation in combination with exposure to an aerosol of ovalbumin successfully developed asthmatic clinical characteristics and pathological changes, which is consistent with airway pathophysiological changes during asthma.Part two A study on the mechanism of CD8+ T cells in the pathogenesis of asthmaObjectiveTo study the level of IL-4,IL-10,IFN-γin the lung,spleen in both asthmatic mice and normal mice and explore the mechanism of action of CD8+ T cell in asthma.MethodsIn 16 normal BALB/c female mice and 16 asthmatic mice, the changes in the airway pathology and the cell proportion in the bronchoalveolar lavage fluid (BALF) were observed. Mice were killed, lungs and spleen were harvested and cut into small pieces and smashed on the mesh to make single cell suspension(MNC). CD8+ T cells were positively selected from lung and spleen MNC of by magnetic cell sorting. The selected fraction were confirmed by FACS contained 95% CD8~+T cells, The purified CD8~+T cells were restimulated with OVA (50 mg/ml) at 37℃with 5%CO2. Supernatants were harvested after 96 h for the detection of cytokines by sandwich enzyme-linked immunosorbent assay (ELISA).Resultsthe level of IL-4 produced by CD8+ T cell of lungs derived from asthmatic mice(pg/ml)(203.2±16.59) was significantly higher than those in the control mice (t=33.918, P< 0.01), while the levels of IL-4 produced by CD8+ T cell of spleen in two groups have no significant difference(t=1.084, P> 0.05); the level of IFN-y produced by CD8+ T cell of spleens derived from asthmatic mice(pg/ml)(4997±189) were significantly higher than those In the control mice (t=3.261, P< 0.01), while the level of IFN-y produced by CD8+ T cell of lungs from asthmatic mice(pg/ml)(237.3±36.96) were significantly lower than those in the control mice (t=-12.951, P< 0.01); the level of IL-10 produced by CD8+ T cell of lungs and spleens derived from asthmatic mice(pg/ml)(265±36.98 vs 22.49±6.69)were both significantly higher than those in the control mice (t=19.047, t=6.340, P< 0.01).The levels of IL-4,IL-10 and IFN-γproduced by lung and spleen were significantly different (P<0.01)ConclusionsHigh amount of IL-4,IL-10 are secreted in the lungs of asthmatic mice. While high amount of IL-10,IFN-γare secreted in the spleen of asthmatic mice.Therefore, CD8+ T cell may play an important role in development of bronchial asthma, The CD8+ T cells are kinds of functional diversity cell subsets which are associated with asthma ongoing closely. And the cytokings produced by CD8+ T cell are different among organs because of the behavior of migration and local cell differentiation,which proved CD8+ T cell is a population consist of cells with different form and function.Part three The analysis of proliferation of spleen CD8+CD28- cell in miceObjectiveTo study the the proliferation of spleen CD8+CD28- T cell in mice.Method6 normal BALB/c female mice were sacrificed,spleen were harvested and cut into small pieces and smashed on the mesh to make MNC. Mononuclear cells were prepared by standard centrifugation over Ficoll-Paque..Carboxy fluorescein diacetate succinimidyl ester (CFSE) was used to assess the number of T cells'population divisions. Cells were labeled without stimulation or before PHA stimulation. To the cells resuspended at a density of 107 cell/ml in PBS with 0.1% BSA, CFSE was added to the final concentration of 10 mM and the cells were mixed and incubated.The labeled cells were washed twice with the culture medium before they were cultured.The cells were stimulated by PHA added at the time of seeding (day 0). From day 3, cells were washed with PBS and recombinant IL-2 together with the fresh medium was added, at 37℃with 5%CO2 for 2 days. Cells were harvested at 5th day and stained by monovlonsl antibody for flow cytometry analysis of proliferation.Resultssome of CD8+CD28- cells present proliferate well after mitogen and IL-2 treatment.ConclusionsA lack of CD28 expression is the hallmark of CD8+ replicative senescence should be re-evaluated..the lack of CD28 expression is neither a cause nor a predictor for a lack of proliferative capacity.