Role Of BRAP In The Antigen Presentation Of Human Bronchial Epithelial Cells

There are three bombesin-like peptide(BLPs)receptors in mammals: bombesin receptor subtype 3(BRS-3), neuromedin B receptor (NMBR) and gastrin-releasing peptide receptor (GRPR). NMBR and GRPR could combined with NMB and GRP respectively with high affinity and induced the corresponding biological function. The native ligand of BRS-3 has not been found, it is still an orphan receptor, little is known about its gene expression regulation, biological function, and its biological significance.We have found previously that BRS-3 may play a role during the regulation of cell growth, wound repair and stress response in human bronchial epithelial cells (HBECs) and airway epithelia. We found two new proteins interacted with BRS-3, whose biological function and biological significance are not known yet with the bacterial two-hybrid technique and Co-Immunoprecipitation. One of them was named as bombesin receptor activated protein (BRAP), and its gene bank locus is NM-152734.The results of bioIFNormatics analysis of BRAP showed that BRAP may be a signal molecular. Northern blot showed that the expression of BRAP increased in O3 stressed lung tissues. We found that BRAP protein mostly located in the membrane and cytoplasm observed by the laser confocal microscope, suggesting that this protein may be a cytoplasmic protein, involved in cellular signal transduction and may play an important role during the stress response.The stress responses of the HBECs included cell growth, wound repair, expressing adhesion moleculars, adhering to extracellular matrix or leucocyte, transfering signals of IFNlammation or stress response, uptaking antigen and then serving as antigen presenting cells to stimulate lymphocyte immune response. According to our preliminary studies, we have found that BRAP overexpression could promote the cell proliferation and increase the wound repair ability of HBECs. In this subject, we will discuss whether BRAP could affect the antigen presenting ability of HBECs, and whether it play an important role during the stress response to airway IFNlammation and airway hyperresponsiveness.Objective:To study the functions of BRAP during the stress response and the stress signal conduction, we will study the temporal and spatial distribution of BRAP in airway hyperresponsiveness animal modle induced by ozone stress. To explore the IFNluence and significance of BRAP on the antigen presenting ability of HBECs and whether BRAP play a important role during the stress respose and airway hyperresponsiveness, we constructed a pcDNA3.1(+)/BRAP overexpression plasmid and BRAP silenced plasmid and transferred them into HBECs respectively, then we study the IFNluence of BRAP on the antigen uptaking of HBECs, the IFNluence on the expression of major histocompatibility complexⅡand costimulatory molecules B7 family and the capacity of HBECs to serve as antigen presenting cells for presenting antigen to stimulate lymphocyte proliferation and differentiation.Method:(1) Detect the change of BRAP at the levels of mRNA and protein in airway hyperresponsiveness animal modle induced by ozone exposure stress through Real-time PCR and immunocytochemistry.(2) The recombinant plasmid pcDNA3.1(+)/BRAP and the vector plasmid pcDNA3.1(+) were constructed and stably transfected into HBECs, and the positive clone was selected by G418. BRAP silenced plasmid and the negative control plasmid were transfected into HBECs.Then the levels of BRAP mRNA and protein were detected by Real-time PCR and Western-blot to identify whether the recombinant plasmid pcDNA3.1 (+)/BRAP was stably transferred into HBECs and the efficiency of BRAP silenced plasmid.(3) pcDNA3.1(+)/BRAP overexpression plasmid and pcDNA3.1(+) vector plasmid stably transfected HBECs and the normal HBECs were cultured in 24 or 6-well plates, adding FITC-OVA(100μg/ml) to the wells, and then incubatated at 37℃for 30 min,60 min and 90 min. Results were observed with fluorescence microscope or analyzed by flow cytometer at different time points, the mean fluorescence intensity or percentage of positive cells indicate the antigen uptaking ability of HBECs.(4) Real-time PCR and flow cytometer were used to measure the expression of HLA-DR, costimulatory molecules mRNA and protein on HBECs respectively.(5) pcDNA3.1(+)/BRAP overexpression, BRAP silenced and the control HBECs were plated in 6-well plates(1×105/well), after treated with 1mg/ml OVA for 2 h, HBECs were washed twice with PBS, then cocultured with freshly isolated lymphocyte(1×106/well)for 48 h. The lymphocyte and supernatant liquid were harvested respectively, then the cell cycle and proliferation of lymphocyte was detected by flow cytometer or MTT, and the concentration of IFN-γ, IL-4, IL-17 was detected by ELISA.Result:(1) The expression of BRAP mRNA and protein were obviously increased in the lung tissue of airway hyperresponsiveness animal modle induced by ozone stress for 2 days and 4 days.(2) Compared with pcDNA3.1(+) vector plasmid transfected HBECs, the expression of BRAP mRNA and protein were significally increased in pcDNA3.1(+)/BRAP recombinant plasmid transfected HBECs; Compared with the negative control plasmid transfected HBECs, the expression of BRAP mRNA and protein were significally decreased in BRAP silenced recombinant plasmid transfected HBECs.(3) HBECs can uptake FITC-OVA, but BRAP overexpression could inhibit HBECs uptaking FITC-OVA.(4)Compared with pcDNA3.1(+) vector plasmid transfected HBECs, pcDNA3.1(+)/BRAP overexpression HBECs could increase the expression of HLA-DR and B7 family mRNA, increase the expression of HLA-DR and B7DC, but decrease the expression of CD86 and B7H1; Compared with the negative control HBECs, BRAP silenced HBECs could increase the expression of CD86.(5) Compared with pcDNA3.1(+) vector plasmid transfected HBECs, pcDNA3.1(+)/BRAP overexpression HBECs could decrease the percentage of lymphocyte cell cycle on the S phase, but increase on the G1 phase, inhibit the proliferation of lymphocyte; Compared with the negative control HBECs, BRAP silenced HBECs could increase the percentage of lymphocyte cell cycle on the S phase, but decrease on the G1 phase, promote the proliferation of lymphocyte.(6)Compared with pcDNA3.1(+) vector plasmid transfected HBECs, pcDNA3.1 (+)/BRAP overexpression HBECs could inhibit lymphocyte to secret IL-4 and IL-17, and have no IFNluence on the secretion of IFN-γ. But BRAP silenced HBECs have no IFNluence on the secretion of IFN-γand IL-17. Conclusion:(1) Ozone stress could increase the expression of BRAP mRNA and protein, which shows that BRAP may play an important role during the stress response and the stress signal conduction.(2) pcDNA3.1(+)/BRAP overexpression could inhibit HBECs' ability of uptaking antigen, and inhibit HBECs serving as antigen presenting cells to present antigen and stimulate lymphocyte proliferation and differentiation.