A Study About Inhibition Of TH2Reaction By Thymic Stromal Lymphopoietin Blockade In Vitro

Background:The drug treatment of allergic rhinitis is not ideal. In recent years, non-specificimmunotherapy about enhancing immune regulation and inducing immune tolerance wasinvolved more and more attraction. Thymic stromal lymphopoietin (TSLP) couldactivate and promote dendritic cells (DC) maturation and further TH2immune responseby combining TSLP receptor which was located in the surface of DC. So as Weinvestigated the process of blocking TSLP receptor for inhibition in TH2responses invitro.Objective:To establish an experimental model of dendritic cells culture and isolate the total Tlymphocytes in vitro. To explore the effect of dendritic cell (DC) with blocked thymicstromal lymphopoietin receptor (TSLPR) in the TH1/TH2immune balance. To establish amouse model of allergic rhinitis for further study. Methods:Take6-8week-old female BALB/c mice bone marrow as a source of dendritic cells,granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4(IL-4) invitro proliferation, choose the sixth day cultured cells to do the test of TEM, SEMmicroscopy and flow cytometry.The sixth day cultured cells were randomly divided into two groups, one group wasmixed with the antibody of TSLPR and was tested by flow cytometry in the eighth day.Take the6-8week-old female BALB/c mouse spleen as a source of T lymphocytes,use immunemagnetic beads to separate CD3+positive election, and flow cytometry to testthe purity.Mouse-derived immature dendritic cells T lymphocytes were co-cμltured in vitro,which were divided into4groups (TSLP stimμlationgroup, TSLP stimμlation and itsreceptor blocking group, ovalbumin stimμlation group and ovalbumin stimμlation andTSLP receptor blocking group). IL-4and IL-8in cell cμlture supernatant were detectedafter2days by ELISA.Twenty BALB/c mice were randomly divided into the allergic rhinitis group andcontrol group. Ovalbumin (OVA) was used to establish the mouse allergic rhinitis model.Hematoxylin-eosin (HE) staining, Giemsa staining and Alcianblue-periodicacid-Schiffstaining were carried out to observe the tissue remodeling, eosinophils and goblet cells,respectively. Enzyme linked immunosorbent assay (ELISA) was used to detect the seruminterleukin4(IL-4) level.Results:In the observation of TEM and electron microscope we found the morphologyirregular surface with more short processes, compliance with immature dendritic cellsmorphological features; the positive rate test of flow cytometry FITC-CD11c was76.83%±1.42%, so most cell culture was inferred myeloid dendritic cells; MHC-II, CD80andMHC-II, CD86double-labeled positive rates are2.61%±0.11%and10.7%±0.23%,respectively, and inferred cultured dendritic cells most are in immature state. The results of flow cytometry from two groups were significantly different. as a result,Blocking the TSLPR could effectively maintain DCs in immature stage.The use of immune magnetic beads to separate PE-CD3after the election was96.1%±2.8%, Inferred that by the way of immunomagnetic beads can obtain high-purity total Tlymphocytes.IL-4levels of TSLP receptor blocking group were significantly lower than those ofcorresponding TSLP stimulation group and ovalbumin stimulation group. However, therewas not significant differences of IL-8expression between corresponding two groups ofwhether or not TSLP receptor blocking. As a TH2factor, IL-4level reduced after TSLPRblockade in vitro，that means TH2reaction could inhibited by TSLPR blockade.The mouse allergic rhinitis model was successfully established, the score of biologicalbehavior was (6.5±1.3). Structural remodeling was occured in nasal mucosa of allergicrhinitis group. Compared with the control group, there were obviously cell desquamation,necrosis, squamous metaplasia and goblet cell hyperplasia in epithelial layer. In the laminapropria and submucosa, glandular hyperplasia, vasodilatation, edema and characteristiceosinophils infiltration were also found in the allergic rhinitis group. The eosinophils andgoblet cell counts were26.4±5.72and24.14±3.12in allergic rhinitis group respectively, andwhich were8.31±2.42and9.41±1.22in the control group respectively. The serum IL-4levelwas (18.9±3.1)pg/ml in allergic rhinitis group and (8.3±1.4)pg/ml in control group, whichwas significantly higher in allergic rhinitis group than that of the control group.Conclusion:TSLP receptor blockade in vitro can inhibit the process of T lymphocytetransformation to TH2, which may provide a new therapeutic strategy for clinical TH2dominant diseases such as allergic rhinitis and asthma.