Orai1蛋白体外干预对小鼠nuocyte细胞功能的影响
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摘要
目的探讨nuocyte细胞是否表达Orai1蛋白及该蛋白的功能。方法用卵清蛋白(ovalbumin,OVA)建立小鼠变应性鼻炎(allergic rhinitis,AR)模型,计数AR小鼠的打喷嚏、挠鼻次数,分离、提纯并体外培养AR小鼠鼻相关淋巴组织(nasal-associated lymphoid tissue,NALT)中的nuocyte细胞,用激光扫描共聚焦显微镜定性检测该细胞Orai1蛋白的表达,以ELISA法和实时荧光定量RT-PCR法分别定量检测该细胞Orai1蛋白及其mRNA的表达量,将小鼠重组(recombinant,rm)白介素(interleukin,IL)-33加入nuocyte细胞培养基,检测其中IL-5和IL-13蛋白及其mRNA的浓度,然后再将Orai1蛋白抗体加入nuocyte细胞培养基,再次检测其中IL-5和IL-13蛋白及其mRNA的含量。结果小鼠AR模型打喷嚏和挠鼻次数较正常小鼠显著增多,OVA诱导NALT来源的小鼠nuocyte细胞被鉴定为CD3CD4CD8CD19CD11bCD11cFcεR1(lineage)-ICOS+,该细胞表面表达Orai1蛋白,其蛋白和mRNA含量均较正常小鼠显著升高,rmIL-33加入nuocyte细胞培养基后,IL-5和IL-13蛋白及其mRNA的浓度均明显增多,而加入Orai1蛋白抗体后,其浓度出现下降。结论 nuocyte细胞表达Orai1蛋白,该蛋白的干预抑制nuocyte细胞的正常功能。
Objective To investigate the expression of Orai1 protein and its function in nuocytes.Methods Mice allergic rhinitis(AR) models were established using ovalbumin(OVA), and numbers of sneezing and nasal rubbing were counted. Then nuocytes were sorted and purified from the mouse nasal-associated lymphoid tissue(NALT), and the AR mice nuocytes were cultured in vitro. Qualitative examination of Orai1 protein was performed using confocal laser scanning microscopy, and quantitative examinations of this protein and its mRNA through enzyme-linked immunosorbent assay(ELISA) and real-time reverse transcription-polymerase chain reaction(RT-PCR). After that, mouse recombinant(rm) interleukin(IL)-33 was administered into nuocytes cultures, and the proteins and mRNAs of IL-5 and IL-13 were detected. Finally, Orai1 antibody was applied into the cultures, and the two cytokines proteins and their mRNAs were re-evaluated.Results Numbers of sneezing and nasal rubbing were increased in mice AR models. Nuocytes were identified as CD3 CD4 CD8 CD19 CD11 bCD11 cFcεR1(lineage)~-ICOS~+, and Orai1 protein was expressed in these cells. Its protein and mRNA were enhanced in AR mice compared to normal ones. After administration of rmIL-33, IL-5 and IL-13 proteins and mRNAs were elevated significantly in nuocytes cultures. However, their expressions were all reduced after application of Orai1 antibody.Conclusion The intervention of Orai1 protein in nuocytes inhibits their cellular function in vitro.
Objective To investigate the expression of Orai1 protein and its function in nuocytes.Methods Mice allergic rhinitis(AR) models were established using ovalbumin(OVA), and numbers of sneezing and nasal rubbing were counted. Then nuocytes were sorted and purified from the mouse nasal-associated lymphoid tissue(NALT), and the AR mice nuocytes were cultured in vitro. Qualitative examination of Orai1 protein was performed using confocal laser scanning microscopy, and quantitative examinations of this protein and its mRNA through enzyme-linked immunosorbent assay(ELISA) and real-time reverse transcription-polymerase chain reaction(RT-PCR). After that, mouse recombinant(rm) interleukin(IL)-33 was administered into nuocytes cultures, and the proteins and mRNAs of IL-5 and IL-13 were detected. Finally, Orai1 antibody was applied into the cultures, and the two cytokines proteins and their mRNAs were re-evaluated.Results Numbers of sneezing and nasal rubbing were increased in mice AR models. Nuocytes were identified as CD3 CD4 CD8 CD19 CD11 bCD11 cFcεR1(lineage)~-ICOS~+, and Orai1 protein was expressed in these cells. Its protein and mRNA were enhanced in AR mice compared to normal ones. After administration of rmIL-33, IL-5 and IL-13 proteins and mRNAs were elevated significantly in nuocytes cultures. However, their expressions were all reduced after application of Orai1 antibody.Conclusion The intervention of Orai1 protein in nuocytes inhibits their cellular function in vitro.
引文
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[2] Matsushita K, Kato Y, Akasaki S, et al. Proallergic cytokines and group 2 innate lymphoid cells in allergic nasal diseases[J]. Allergol Int, 2015, 64(3): 235-240.
[3] Neill DR, Wong SH, Bellosi A, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity[J]. Nature, 2010, 464(7293): 1367-1370.
[4] Barlow JL, Bellosi A, Hardman CS, et al. Innate IL-13-producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity[J]. J Allergy Clin Immunol, 2012, 129(1): 191-198.e1-4.
[5] Lin L, Dai F, Wei JJ, et al. Allergic inflammation is exacerbated by allergen-induced type 2 innate lymphoid cells in a murine model of allergic rhinitis[J]. Rhinology, 2017, 55(4): 339-347.
[6] Hiromura Y, Kishida T, Nakano H, et al. IL-21 administration into the nostril alleviates murine allergic rhinitis[J].J Immunol, 2007, 179(10): 7157-7165.
[7] Giavina-Bianchi P, Aun MV, Takejima P, et al. United airway disease: current perspectives[J]. J Asthma Allergy, 2016, 9: 93-100.
[8] Berridge MJ, Bootman MD, Roderick HL. Calcium signaling: dynamics, homeostasis and remodeling[J]. Nat Rev Mol Cell Biol, 2003, 4(7): 517-529.
[9] Parekh AB, Putney JW Jr. Store-operated calcium channels[J]. Physiol Rev, 2005, 85(2): 757-810.
[10] Peinelt C, Vig M, Koomoa DL, et al. Amplification of CRAC current by STIM1 and CRACM1 (Orai1)[J]. Nat Cell Biol, 2006, 8(7): 771-773.
[11] Mercer JC, DeHaven WI, Smyth JT, et al. Large store-operated calcium-selected currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, stim1[J]. J Biol Chem, 2006, 281(34): 24979-24990.
[12] Feske S, Gwack Y, Prakriya M, et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function[J]. Nature, 2006, 441(7090): 179-185.
[13] Lin L, Zheng C, Zhang L, et al. Up-regulation of Orai1 in murine allergic rhinitis[J]. Histochem Cell Biol, 2010, 134(1): 93-102.
[14] Lin L, Zheng C, Zhang L, et al. 2-Aminoethoxydiphenyl borate administration into the nostril alleviates murine allergic rhinitis[J]. Am J Otolaryngol, 2011, 32(4): 318-328.
[15] Wang Y, Lin L, Zheng C. Downregulation of the expression of Orai1 in the airway alleviates murine allergic rhinitis[J]. Exp Mol Med, 2011, 44(3): 177-190.
[16] Lin L, Zhao X, Yan W, et al, Influence of Orai1 intervention on mouse airway epithelium reactions in vivo and in vitro[J]. Ann Allergy Asthma Immunol, 2012,108(2): 103-112.
[17] Lin L, Zhao X, Yan W, et al. Amelioration of Muc5b mucin hypersecretion is enhanced by IL-33 after 2-APB administration in a murine model of allergic rhinitis[J]. Biotech Histochem, 2014, 89(4): 273-286.
[18] Lin L, Dai F, Chen Z, et al. In vitro treatment with 2-APB inhibits the inflammation in nasal polyps[J]. Otolaryngol Head Neck Surg, 2015, 153(3): 461-467.
[19] Lin L, Dai F, Chen Z, et al. The intervention of CRAC channels alleviates inflammatory responses in nasal polyps[J]. Int Arch Allergy Immunol, 2015, 167(4): 270-279.
[20] 蔺林, 戴飞, 孙庭钰. Orai1抗体对变应性鼻炎小鼠模型的治疗作用[J].中华耳鼻咽喉头颈外科杂志, 2016, 51(1): 43–49. Lin L, Dai F, Sun TY. The Orai1 antibody treatment for a mouse model of allergic rhinitis[J]. Chinese Journal of Otorhinolaryngology Head and Neck Surgery,2016, 51(1): 43-49.
[21] Fahy JV, Locksley RM. The airway epithelium as a regulator of Th2 responses in asthma[J]. Am J Respir Crit Care Med, 2011, 184(4): 390-392.
[22] Mj?sberg JM, Trifari S, Crellin NK, et al. Human IL-25-and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161[J]. Nat Immunol, 2011, 12(11):1055-1062.
[23] Fort MM, Cheung J, Yen D, et al. IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo[J]. Immunity, 2001, 15(6): 985-995.
[24] Schmitz J, Owyang A, Oldham E, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines[J]. Immunity, 2005, 23(5): 479-490.
[2] Matsushita K, Kato Y, Akasaki S, et al. Proallergic cytokines and group 2 innate lymphoid cells in allergic nasal diseases[J]. Allergol Int, 2015, 64(3): 235-240.
[3] Neill DR, Wong SH, Bellosi A, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity[J]. Nature, 2010, 464(7293): 1367-1370.
[4] Barlow JL, Bellosi A, Hardman CS, et al. Innate IL-13-producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity[J]. J Allergy Clin Immunol, 2012, 129(1): 191-198.e1-4.
[5] Lin L, Dai F, Wei JJ, et al. Allergic inflammation is exacerbated by allergen-induced type 2 innate lymphoid cells in a murine model of allergic rhinitis[J]. Rhinology, 2017, 55(4): 339-347.
[6] Hiromura Y, Kishida T, Nakano H, et al. IL-21 administration into the nostril alleviates murine allergic rhinitis[J].J Immunol, 2007, 179(10): 7157-7165.
[7] Giavina-Bianchi P, Aun MV, Takejima P, et al. United airway disease: current perspectives[J]. J Asthma Allergy, 2016, 9: 93-100.
[8] Berridge MJ, Bootman MD, Roderick HL. Calcium signaling: dynamics, homeostasis and remodeling[J]. Nat Rev Mol Cell Biol, 2003, 4(7): 517-529.
[9] Parekh AB, Putney JW Jr. Store-operated calcium channels[J]. Physiol Rev, 2005, 85(2): 757-810.
[10] Peinelt C, Vig M, Koomoa DL, et al. Amplification of CRAC current by STIM1 and CRACM1 (Orai1)[J]. Nat Cell Biol, 2006, 8(7): 771-773.
[11] Mercer JC, DeHaven WI, Smyth JT, et al. Large store-operated calcium-selected currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, stim1[J]. J Biol Chem, 2006, 281(34): 24979-24990.
[12] Feske S, Gwack Y, Prakriya M, et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function[J]. Nature, 2006, 441(7090): 179-185.
[13] Lin L, Zheng C, Zhang L, et al. Up-regulation of Orai1 in murine allergic rhinitis[J]. Histochem Cell Biol, 2010, 134(1): 93-102.
[14] Lin L, Zheng C, Zhang L, et al. 2-Aminoethoxydiphenyl borate administration into the nostril alleviates murine allergic rhinitis[J]. Am J Otolaryngol, 2011, 32(4): 318-328.
[15] Wang Y, Lin L, Zheng C. Downregulation of the expression of Orai1 in the airway alleviates murine allergic rhinitis[J]. Exp Mol Med, 2011, 44(3): 177-190.
[16] Lin L, Zhao X, Yan W, et al, Influence of Orai1 intervention on mouse airway epithelium reactions in vivo and in vitro[J]. Ann Allergy Asthma Immunol, 2012,108(2): 103-112.
[17] Lin L, Zhao X, Yan W, et al. Amelioration of Muc5b mucin hypersecretion is enhanced by IL-33 after 2-APB administration in a murine model of allergic rhinitis[J]. Biotech Histochem, 2014, 89(4): 273-286.
[18] Lin L, Dai F, Chen Z, et al. In vitro treatment with 2-APB inhibits the inflammation in nasal polyps[J]. Otolaryngol Head Neck Surg, 2015, 153(3): 461-467.
[19] Lin L, Dai F, Chen Z, et al. The intervention of CRAC channels alleviates inflammatory responses in nasal polyps[J]. Int Arch Allergy Immunol, 2015, 167(4): 270-279.
[20] 蔺林, 戴飞, 孙庭钰. Orai1抗体对变应性鼻炎小鼠模型的治疗作用[J].中华耳鼻咽喉头颈外科杂志, 2016, 51(1): 43–49. Lin L, Dai F, Sun TY. The Orai1 antibody treatment for a mouse model of allergic rhinitis[J]. Chinese Journal of Otorhinolaryngology Head and Neck Surgery,2016, 51(1): 43-49.
[21] Fahy JV, Locksley RM. The airway epithelium as a regulator of Th2 responses in asthma[J]. Am J Respir Crit Care Med, 2011, 184(4): 390-392.
[22] Mj?sberg JM, Trifari S, Crellin NK, et al. Human IL-25-and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161[J]. Nat Immunol, 2011, 12(11):1055-1062.
[23] Fort MM, Cheung J, Yen D, et al. IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo[J]. Immunity, 2001, 15(6): 985-995.
[24] Schmitz J, Owyang A, Oldham E, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines[J]. Immunity, 2005, 23(5): 479-490.