The Research Of CD8~+ Treg Cells In Asthmatic Mouse

Study BackgroundAsthma is a worldwide disease.It reported that the incidence of asthma had been increasing in recent years . In addition, the WHO reported that asthma related economic cost was higher than the total cost of tuberculosis and AIDS in the worldwide,and forecasted that there will be near 1 million new cases of asthmatic patients by 2025, an heavy burden will bring to governments, the families and the patients.There are variety of cells (eosinophils, mast cells, neutrophils, airway epithelial cells, etc.) and the productions of them involved in asthmatic airway.The important characteristics of this disease are a bronchospasm, mucosa edema, airway secretions increasing and airway hyperresponsieness. Because it has been harmfull at humans seriously, more and more researchers studied it from various fields of medicine,especially in immunology. The pathogenesis of asthma is very complex, it has not yet been clarified, now most researchers accepted the theory of chronic airway inflammation.Lots of scholars indicated that CD4+T lymphocytes played an important role in asthmatic occurrence and development about 10 years ago,they thought that the Th1/Th2 unbalanced played a key role in asthmatic pathogenesis,because of Th2 functions strengthened, Th1 function inhibited ,which could be associated with the pathogenesis of asthma. But this theory was hardly to explain all asthmatic futures by animals and relevant clinical experiments in recent years. Presently, the occurrence of asthma mainly due to the damaged immunological tolerance, which resulted in impairing the immune cells and damaging their organizational structure and function.Nearly,evidences that the asthma inflammation and maintaining the organism immune toleranced have a close relationship in regulatory T lymphocytes.T cells could have regulatory function by specific antigen stimulating and cytokines inducing,which were named regulatory T lymphocytes(Tregs). Tregs involving anti-infection immune,tumor immunity,transplantation immunity,allergy and many other pathologic immune process, they play roles in maintaining the stability of immune, preventing from harmfull immune.Tregs include CD4+Treg,Th3 cells,Tregl,Thl7 cells,NK (nature killer) cells,CD8+Treg and so on.CD4+Treg was recognized as regulatory T lymphocytes, it specific expression Foxp3, named CD4+ CD25highFoxp3+Treg cells. Large studies confirmed that CD4+CD25+T cell played a key role in maintaining the immune tolerance, and regulating the pathogenesis of asthma, it can inhibit Th2 reaction, eosinophils producing,and restrain the airway hyperresponsieness. Some studies found that Tregs declined and whose function reduced in asthma; however, asthmatic animals inputed Tregs from in vitro model could reduce airway responsieness, restrain eosinophils infiltrating and cytokines producing from Th2 cells.It showed that Tregs also could inhibit the allergic reaction when allergens attacked.With the development of research, researchers found that the role of CD8+Treg is particularly important, CD4+Treg induced the occurrence of inhibition,while CD8+Treg is an effect cells in the role of inhibition.It had demonstrated that CD8+CD28-T cell was one of regulatory CD8+T cells in recent years. Filaci found that more than 90 percent of CD8+Treg was CD28-,the lack of CD28 expressed group have suppressional function. CD8+CD28- T cell has immunosuppressive function,which is the same to CD4+CD25+T cell. Many studies indecated that CD8+CD28- T cell had involved the immune tolerance in the human cancers,it could inhibit T cell proliferation and its cytotoxic activity; CD8+CD28-T cell often exist in the emergence of immune tolerance, it was showed that CD8+ CD28-T cell related with them positively. Some research showed that CD8+CD28-T cell could be as an evaluational function for the emergence of tolerance. While, CD8+ Treg cells regulated the organic immune through different mechanism in physiological and various kinds of pathological conditions.According to reports, it had cytotoxic activity under centain conditions.when cytomegalovirus, EB virus, shingles virus infected, specificity dissolving mediated virus of CD8+CD28-T cell could be induced, this paper indicated that CD8+CD28-T cell had cytotoxic activity when virus infected. Agnes studies found that the percentage of CD8+CD28-T cell of sputum in such asthmatic patients as severe and light were significantly higher than normal persons. Research showed that CD8+Treg inhibited delayed allergic reaction relating with CD94/NKG2A closely, and through what secreting IL-10, IFN-γ, IL-6.Yet there was little information about CD8+CD28-T cell whether had the role of immunosuppression or not and what is the mechanism.In this study we designed to detect and analysis CD8+CD28-T cell of blood and BALF,and the influence of CD8+CD28-T cell in asthmatic model mouse,so as to explore the immunosuppressional mechanism of NKG2A for CD8+Tregs.Part one Establishment and assessment of the asthmatic mouse modelObjectiveTo establish and assess a BALB/c asthmatic mouse model by OVA sensitization and rechallenge MethodsFemale 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 3 groups:asthmatic group(A group), the dexamethasone group (D group) and control group(C group), with 10 mice in each group. A,D group were sensitized on days 0 and 14 by an intraperitoneal injection of a mixture containing ovalbumin(OVA) and Al(OH)3 in saline (a total volume of 0.2ml). Then after three weeks the mice were firstly challenged with 1%OVA 100ul intranasal droped,then challenged by exposure to an aerosol with 2%OVA generated by an ultrasonic nebulizer for 30 min. The rechallenge procedure was repeated once each day,for 6 days.while evevry time, the D group were intraperitoneal injected dexamethasone(DXM) lmg/kg before 30 min inhaling 2%OVA.The C group mice were treated with 5mg Al(OH)3 in saline and with saline instead of 1%OVA,2%OVA and DXM,the operational approach like the D group.24 hours after the last allergen challenge ,three mice randomly chose in each group were mesured the airway responsiveness(AHR),enhanced pause(Penh) was measured after each nebulization with increasing doses of methacholine and this measure was used to evaluate AHR. The other animals sacrificed after the last allergen challenge 48 hours,the mice were subjected to bronchoalveolar lavage cytology performed and lung tissue sections were prepared for histopathologic examination to evaluate the airway inflammation,to examin the contents of IgE of blood and BALF with mouse IgE ELISA kit.Results1.The manifestation of mice after sensitization and re-challengeA,D group mice displayed various types of allergic responses after sensitization and re-challenge,such as the skin of mice lip and tail was caynosis, scratching head and face,dispnea,active decreased and so on, while C group showed no such symptoms.2. AHR measurementWhen inhaling methacholine>6.25mg/ml,The airway hyperrespomsiveness in A,D group was significantly higher than that in C group mice,every P＜0.05.3. The results in total cells and EOS in BALFThe total cells number and the EOS (4.89±3.49,1.75±0.75) 105/ml of BALF in A group mice were significantly higher than that in the control mice(0.91±0.64,0.30±0.16)105/ml, P=0.003 and P=0.000.The total cells number (2.11±1.22) 105/ml of BALF in D group were lower than that in A group, p=0.027; while relative to C group is insignificantly, P=0.314. The EOS(1.04±0.53)105/ml in D group is significantly lower than that in A group,P=0.024;and significantly higher than that in C group,P=0.020.4. Histological analysisMassive leukocyte infiltration was present in the lungs from the A,D group.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.and the A group mice's is more serious than D group.While lungs in the C group mice displayed a clear airway structure without any significant inflammatory cell infiltration or collagen deposition.5.The results of IgE in BALF and bloodRelative to IgE of blood in C group mice(18.16±4.45) ug·ml-1, A group and D group(37.47±6.18,P=0.000;25.93±2.71,P=0.006)were significantly higher.and which in D group were significantly lower than that in A group, P=0.000.Relative to IgE of BALF in C group (6.71±1.23)ug·ml-1,A group and D group (26.10± 7.18,P=0.001 and 13.57±2.11,P=0.000)were significantly higher. and whicht in D group was significantly lower than A group, P=0.008.ConclusionsThese variatios on the basis of the total cells,EOS,AHR, IgE and Histology of lung,it indicated that we successfully established an asthmatic mouse model by OVA sensitization and rechallenge.And intraperitoneal injected lmg/kg dexamethasone can alleviate the inflammation,AHR,symptoms of asthma.Part two The detection of CD8+CD28-T cell and the effect of dexamethasone in asthmatic mouseObjectiveTo explore CD8+CD28-T cell whether play a role in pathogenesis of asthma and the influence of CD8+CD28-T cell by dexamethasone (DXM).MethodsThe mice were randomly divided into 3 groups: A group, D group and C group, with 7 mice in each group. The methods and process of asthmatic mouse models were the same to the part one.After the modle succeded 48 hours, mice were bloodletted by cut eyeball, then performed bronchoalveolar lavage cytology and lung tissue sections were prepared for histopathologic examination to evaluate the airway inflammation. the blood placed into anticoagulational EP test, prepared for measuring the percentage of CD8+CD28-T cell by flow cytometry within 8 hours. And the BALF measured by flow cytometry as well as quickly. To examine the IgE IFN-y of BALF with mouse IgE,IFN-y ELISA kit . To analysis the correlation between total cells,EOS,IgE,IFN-γof BALF among CD8+CD28-T cells of blood in A group. Results1.The manifestation of mice after sensitization and re-challenge:The manifestation of every group were similar to part one.2. The results of the total cells and EOS in BALFThe total cells number (5.56±4.06)105/ml and the EOS(3.29±2.23) 105/ml of BALF in A group mice were significantly higher than that in the C mice(0.91±0.65,P=0.003;0.43±0.37,P=0.001).And the total cells number,the EOS(2.59±1.6,1.11±0.73)105/ml of BALF in D group were lower than that in A group,p＝0.044,P=0.008. But D guoup relative to C group animals were insignificantly, p=0.234,P=0.363.3. Histological analysisMassive leukocyte infiltration was present in the lungs from the A,D group 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,and the A group mice's is more serious than D group's.While lungs in the C group mice displayed a clear airway structure without any significant inflammatory cell infiltration or collagen deposition.4. The results of IgE and IFN-γin BALF:Relative to IgE of BALF in C group(6.54±1.03)ug·ml-1,A group (23.85±5.97,P=0.001)and D group(13.15±2.22,P=0.000)were significantly higher. and whicht in D group was significantly lower than that in A group,P=0.007.Relative to IFN-y of BALF in C group(130.88±32.23)ng·L-1, A group and D group (37.44±21.01,77.35±37.71)ng·L-1 were significantly lower,P=0.000,P=0.005. which in D group was also significantly higher than A group,P=0.027.5. The percentage of CD8+CD28-T in blood and BALF The percentage of CD8+CD28-T of blood in A group and D group (18.68±4.12,13.43±2.90)% were significantly higher than that in C group(8.43±4.60)%,P=0.000,P=0.029. The percentage of CD8+CD28-T cell of BALF in A group and D group((1.25±0.40,0.66±0.49)%were also significantly higher than C group(0.21±0.19)%,P=0.000,P=0.041.The percentages of CD8+CD28-T cell of blood and BALF in the D group were significantly lower than A group.6.The results of Person's analysis in A groupThe correlation between the total cells (r=0.806, P=0.029),EOS (r=0.804, P=0.029),IgE(r=0.864,P=0.012),IFN-γ(r=0.124,P=0.792) among CD8+CD28-T cells of blood.ConclusionsThe percentage of CD8+CD28-T cell is associated with asthmatic ongoing closely. CD8+CD28-T cell may be one of the factors for leading asthma.AHR and some syndrome in asthmatic mice could be relieved by dexamethasone,which could through inhibiting the express of CD8+CD28-T cell. Key words:asthma CD8+CD28-Tcell dexamethasonePart three The research of regulatory mechanism of CD8+Treg cells in asthmatic mouse modelObjectiveTo assess the CD8+Treg cells whether or play regulatory function by detecting NKG2A of lung tissue in asthmatic mouseMethodsFemale BALB/c mice were randomly divided into 3 groups:A group, D group and C group, with 7 mice in each group. The establishment and assessment of the asthmatic mouse model from the part two. The lung tissue fixed in 4% formaldehyde solution, then the conventional dehydration,paraffin embedding and paraffin wax, taken off, add the corresponding antigen repairⅠ,Ⅱantibody, colored, dehydration, sealing piece, microscopy. NKG2A expressed is tan-yellow positive dyed as immunohistochemical results. NKG2A expression in each slice observation six vision, all slice for 400 magnification times by using microscopy and Imaga - ProPlus 6.0 software,to analysis perspective with IOD insteaded of the contents of NKG2A expressed directly.ResultsRelative to IOD of NKG2A expressed in lung of C group(10.85±3.71),A group and D group(24.29±6.34,17.28±4.13)were significantly higher ,P=0.000,P=0.024. andwhich in D group was significantly lower than that in A group,P=0.015ConclusionsNKG2A of lung tissue expressed more in Asthmatic mice,which may suppress the inhibiting function of CD8+Treg cells; dexamethasone can inhibit the expression of NKG2A in the lung tissue.