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对过敏性疾病发病机制的几点新认识 New understanding of the pathogenesis of allergic disease

对过敏性疾病发病机制的几点新认识 New understanding of the pathogenesis of allergic disease. 南京医科大学第一附属医院 何韶衡. 过敏性疾病是由 IgE 介导的一组疾病，其本质是过 敏性炎症反应 (Allergic inflammation ), 它包括 ：. Asthma 哮喘 Allergic rhinitis 过敏性鼻炎 Atopic dermatitis 过敏性皮炎

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对过敏性疾病发病机制的几点新认识 New understanding of the pathogenesis of allergic disease

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  1. 对过敏性疾病发病机制的几点新认识 New understanding of the pathogenesis of allergic disease 南京医科大学第一附属医院 何韶衡

  2. 过敏性疾病是由IgE介导的一组疾病，其本质是过 敏性炎症反应(Allergic inflammation ), 它包括： Asthma 哮喘 Allergic rhinitis 过敏性鼻炎 Atopic dermatitis 过敏性皮炎 Food and drug allergy 食物和药物过敏 Anaphylaxis 过敏症 Hay fever 枯草热 Etc 等等

  3. 过敏的发病率 1、占世界人口的10%~30%，而且正以每年大于1%的速度 增加，以儿童患者的发病率上升最为明显； 2、发达国家比发展中国家多，城市比农村地区多，如 1994年到2000年间，在意大利一家医院接受治疗的非 欧洲人移民中，有84%在到欧洲前从没有过过敏症状； 3、随着生活水平的提高和环境的改善，我国近年来过敏性 疾病的发生率也有明显上升趋势；与城市化有关？ 4、我国儿童哮喘发病率比10年前明显增加，仅小儿哮喘患 者达1000万之多，其中过敏为主要诱因。

  4. 过敏反应发生过程示意图

  5. 第一部分: 对肥大细胞在过敏性疾病 发病机制中核心作用的新认识

  6. Mast cell and its mediators MCT MCTC Tryptase Chymase Histamine Heparin Cytokines etc. Carboxypeptidase Tryptase Histamine Heparin Cytokines etc. Tryptase 10 pg/cell Histamine 1-3 pg/cell Tryptase 35 pg/cell Chymase 5 pg/cell Histamine 1-3 pg/cell

  7. A: 肥大细胞亚型的研究Investigation of mast cell subtypes.

  8. MCTC MCC MCT Flow cytometry analysis of mast cell subtypes in human skin

  9. Expression of carboxypeptidase in MCT under con-focal microscopy AA5－CY5 PE CA1－FITC Red + green

  10. Expression of carboxypeptidase in MCc under con-focal microscopy Red + green CA1－FITC CC4－PE

  11. Expression of carboxypeptidase in MCTC under con-focal microscopy AA5－CY5 PE + CC4－PE CA1－FITC Red + green

  12. MCTC MCTC MCC MCC MCT MCT Flow cytometry analysis of mast cell subtypes in human colon

  13. Summary:There may be only one type of mas cells-MCTC; MCT and MCC represent different stages of MCTC.

  14. 鉴定亚型的意义： • 人体为什么会出现只有蛋白酶差别的3种亚型？ • 各亚型的结构和功能有何差别？ • 各亚型在组织中的分布如何？ • 各亚型与疾病的关系如何？ • 在肥大细胞稳定剂的研发上，是否需要同时说明是 稳定哪一种亚型肥大细胞的？ 我们仅发现一种肥大细胞，因此问题就简单多了。

  15. Immunological Reviews

  16. www.nature.com/reviews/immunol

  17. B: 《肥大细胞脱颗粒信号自身放大机制》假说 Self-amplification mechanisms of mast cell degranulation.

  18. Asthmatic BALF mast cells exhibit 100-fold increased sensitivity to IgE dependent stimilation compared with the mast cells from non-asthmatic controls BALF (●), lung (■) or colon ( ▲)

  19. Dissection Chopping Digestion Separation Challenge Loading Measurement

  20. C: 创立了《肥大细胞脱颗粒信号自我放大机制》的假说

  21. 其它能够诱导肥大细胞脱颗粒的肥大细胞分泌产物其它能够诱导肥大细胞脱颗粒的肥大细胞分泌产物 1、 半胱氨酸白三烯Kaneko I, Suzuki K, Matsuo K, et al. Cysteinyl leukotrienes enhance the degranulation of bone marrow- derived mast cells through the autocrine mechanism. Tohoku J Exp Med, 2009, 217(3):185-191. 2、 干细胞因子Lukacs NWet al. Blood. The role of stem cell factor (c-kit ligand) and inflammatory cytokines in pulmonary mast cell activation 1996 Mar 15;87(6):2262-8. 3、 肿瘤坏死因子-alpha Wong CK, et al. Allergy. Molecular mechanisms for the release of chemokines from human leukemic mast cell line (HMC)-1 cells activated by SCF and TNF-alpha: roles of ERK, p38 MAPK, and NF-kappaB 2006 Mar;61(3):289-97. 4、P物质Kulka M et al. Neuropeptides activate human mast cell degranulation and chemokine production Immunology. 2008 Mar;123(3):398-410. Epub 2007 Oct 6 5、 骨桥蛋白Nagasaka A, Matsue H, Matsushima H, et al. Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells. Eur J Immunol, 2008, 38(2):489-499.

  22. Accumulation of mast cells in the peritoneum of mouse

  23. Influence of compound 48/80 on mast cell accumulation in the peritoneum of mice

  24. Influence of tryptic enzymes on mast cell accumulation in the peritoneum of mice

  25. Tryptase + IL-29 Chymase + IL-29 Skin Figure2. Double labeling immunohistochemical staining of IL-29 in human mast cells. Cells stained with G3 or B7 antibody only were in red (solid arrow heads), with SC1 antibody only were in brown (open arrow heads) and double labeled with G3 and SC1 antibodies or B7 and SC1 antibodies were in brown and red (arrows). Colon Tonsil Lung

  26. Table 1. Number of IL-29 containing cells in foreskin, colon, tonsil and lung tissues stained by anti-human tryptase antibody G3 and anti-human IL-29 antibody SC1 with a double labeling immunohistochemical technique Data shown are the median (range) value from 6 different donors. Positively stained cells in at least 30 fields ( 400 magnification) of each section were counted. * P < 0.05 in comparison with the cells stained by G3 antibody alone. † P < 0.05 in comparison with the cells stained by SC1 antibody alone (paired Mann-Whitney U-test).

  27. 其它能够诱导肥大细胞募集的肥大细胞分泌产物其它能够诱导肥大细胞募集的肥大细胞分泌产物 1、组胺通过H4受体受体诱导肥大细胞的聚集：Hofstra CL, et al. Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther, 2003, 305(3):1212-1221.； 2、 五羟色胺通过其受体诱导肥大细胞的聚集：Kushnir-Sukhov NM, et al. 5-hydroxytryptamine induces mast cell adhesion and migration. J Immunol, 2006, 177(9):6422-6432.； 3、 金属蛋白酶9 能够诱导肥大细胞祖细胞在组织中聚集：Di Girolamo N, et al. Human mast cell-derived gelatinase B (matrix metalloproteinase-9) is regulated by inflammatory cytokines: role in cell migration. J Immunol, 2006, 177(4):2638-2650. ； 4、 肥大细胞的细胞因子如RANTES、eotaxin、TNF等也具有募集肥大细 胞的作用：Brzezińska-Błaszczyk E, Misiak-Tłoczek A. The regulation of mast cell migration. Part 2: mast cell chemoattractants. Postepy Hig Med Dosw, 2007, 61:493-499.； 5、 类糜蛋白酶诱导肥大细胞的聚集:尚未发表

  28. 肥大细胞类胰蛋白酶和类糜蛋白酶相关论文 • Walls AF, He S, Teran LM, Buckley MG, Jung KS, Holgate ST, Shute JK, Cairns JA. Granulocyte recruitment by human mast cell tryptase. Int Arch Allergy Immunol, 1995;107(1-3):372-3. PMID: 7613178 [PubMed - indexed for MEDLINE] • McEuen AR, He S, Brander ML, Walls AF. Guinea pig lung tryptase. Localisation to mast cells and characterisation of the partially purified enzyme. Biochem Pharmacol, 1996; 52(2): 331-40. PMID: 8694858 [PubMed - indexed for MEDLINE] • He S, Peng Q, Walls AF. Potent induction of a neutrophil and eosinophil-rich infiltrate in vivo by human mast cell tryptase: selective enhancement of eosinophil recruitment by histamine. J Immunol, 1997; 159(12): 6216-25. PMID: 9550425 [PubMed - indexed for MEDLINE] • He S, Walls AF. Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation. Eur J Pharmacol, 1997; 328(1): 89-97. PMID: 9203574 [PubMed - indexed for MEDLINE] • He S, Walls AF. Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo. Br J Pharmacol, 1998; 125(7): 1491-500. • McEuen AR, Gaca MD, Buckley MG, He S, Gore MG, Walls AF. Two distinct forms of human mast cell chymase--differences in affinity for heparin and in distribution in skin, heart, and other tissues. Eur J Biochem, 1998; 256(2): 461-70. PMID: 9760188 [PubMed - indexed for MEDLINE] • He S, Walls AF. The induction of a prolonged increase in microvascular permeability by human mast cell chymase. Eur J Pharmacol, 1998; 352(1): 91-8. PMID: 9718272 [PubMed - indexed for MEDLINE]

  29. 8） He S, Gaca MD, Walls AF. A role for tryptase in the activation of human mast cells: modulation of histamine release by tryptase and inhibitors of tryptase. J Pharmacol Exp Ther, 1998; 286(1): 289-97. PMID: 9655871 [PubMed - indexed for MEDLINE] • He S, Gaca MD, McEuen AR, Walls AF. Inhibitors of chymase as mast cell-stabilizing agents: contribution of chymase in the activation of human mast cells. J Pharmacol Exp Ther, 1999; 291(2): 517-23. PMID: 10525066 [PubMed - indexed for MEDLINE] • He S, Gaca MD, Walls AF. The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors. Eur J Pharmacol, 2001; 412(3): 223-9. PMID: 11166285 [PubMed - indexed for MEDLINE] • Walls AF, He S, Buckley MG, McEuen AR. Roles of the mast cell and basophil in asthma. Clin. Exp. Allergy, 2001, 1: 68-72. • He S, Alan R. McEuen, Blewett SA, Li P, Buckley MG, Leufkens P, Walls AF. The inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase, chymase and cathepsin G. Biochem Pharmacol, 2003; 65(6): 1007-15. • He S, Chen P, Chen HQ. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors. Acta Pharmacol Sin, 2003; 24(9): 923-9. • He S, Zheng J. Induction of inflammatory cell accumulation using human mast cell tryptases. Asian Pacific J Allergy Immunol, 2003; 21: 145-51.

  30. 15） He S, Aslam A, Gaca MD, He YS, Buckley MG, Hollenberg MD, Walls AF. Inhibitors of tryptase as • mast cell stabilizing agents in the human airways: effects of tryptase and other agonists of PAR2 on • histamine release. J Pharmacol Exp Ther, 2004; 309(1): 119-26. • 16） He S, Zheng J. Stimulation of mucin release from airway epithelial cells by mast cell chymase. Acta • Pharmacol Sin, 2004; 25(6): 827-32. • 17） He S，Xie H, Zhang XJ, Wang XJ. Inhibition of histamine release from human mast cells by • natural chymase inhibitors. Acta Pharmacol Sin, 2004; 25(6): 822-6. • He S, Chen HQ, Zheng J. Inhibition of tryptase and chymase induced nucleated cell infiltration by proteinase inhibitors. Acta Pharmacol Sin, 2004; 25(12): 1677-84. • He S, Xie H. Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors. Asian Pacific J. Allergy Immunology, 2004; 22: 205-12. • He S, Xie H. Modulation of tryptase release from human tonsil mast cells by protease inhibitors. Pharmacological Reports, 2005; 57: 523-30. • Zhou YC, Chen ZQ, He S. Preparation, characterization and epitope mapping of monoclonal antibodies specific to human mast cell carboxypeptidase. Scandinavian Journal of Immunology, 2006; 64: 564-70. （通讯作者）

  31. 22） Wang L, Luo JM, He S. Induction of MMP-9 release from human dermal fibroblasts by thrombin through JAK/STAT3 signaling pathway. BMC Cell Biology, 2007; 8:14: 1-9. （通讯作者） 23） Liu T, He S, Zheng PY, Zhang TY, Wang BQ, Yang PC. Stapylococcal enterotoxin B increases TIM4 expression in human dendritic cells that drives maive CD4 T cells to differentiate into Th2 cells. Molecular Immunology, 2007; 44:3580-3587. 24）Feng B, He S, Zheng P, Wu L ,Yang P.. Mast cells play a crucial role in staphylococcus aureus petidoglycan- induced diarrhea. The American Journal of Pathology, 2007,171:537-547. 25） Li MC, He S. Purification and characterization of recombinant human interleukin-29 expressed in Escherichia coli. Journal of Biotechnology, 2006, 122: 334-40. （通讯作者） 26）Li MC, Wang HY, Wang HY, Li T, He S. Liposome-mediated IL-28 and IL-29 expression in A549 cells and antiviral effect of IL-28 and IL-29 on WISH cells. Acta Pharmacol Sin, 2006; 27(4): 453-9. （通讯作者） 27） Li MC, Chen YZ, Huang T, Liu YN, He S. Production, characterization and applications of two novel monoclonal antibodies against human interleukin-28A. Tissue Antigens, 2006;68(6): 477-82. （通讯作者）

  32. 《肥大细胞脱颗粒信号自身放大机制》的意义：《肥大细胞脱颗粒信号自身放大机制》的意义： • 对微量过敏原进入体内即能在短时间内诱发过敏给与了解释； • 该假说可能也适用于嗜碱性粒细胞和其它具有募集功能的炎症性细胞； • 也是对部分过敏患者有器官特异性倾向的一种解释。

  33. 第二部分: 蛋白酶活化受体在过敏性疾病 发病机制中的作用

  34. 蛋白酶活化受体示意图

  35. 蛋白酶激活受体

  36. 已经报道的蛋白酶活化受体与过敏性疾病文献 1、哮喘病人呼吸道上皮细胞PAR-2的表达增高 Roche N, et al. 2003. Effect of acute and chronic inflammatory stimuli on expression of protease-activated receptors 1 and 2 in alveolar macrophages. J Allergy Clin Immunol, 111: 367~373.； 2、 PAR-2缺陷小鼠的嗜酸性粒细胞浸润被抑制、气道对吸入的乙酰甲胆碱的高反应性下降、卵清 蛋白刺激产生的IgE水平降低 Takizawa T, et al. 2005. Abrogation of bronchial eosinophilic inflammation and attenuated eotaxin content in protease-activated receptor 2-deficient mice. J Pharmacol Sci, 98(1): 99~102.； 3、 PAR-2激动肽通过环氧酶依赖途径抑制过敏小鼠气道嗜酸性粒细胞增加和气道高反应性，与卵 清蛋白联合应用可引起致敏小鼠气道的高反应性和气道炎症 De Campo BA, Henry PJ. 2005. Stimulation of protease-activated receptor-2 inhibits airway eosinophilia, hyperresponsiveness and bronchoconstriction in a murine model of allergic inflammation. Br J Pharmacol, 144(8): 1100~1108. ； 4、 吸入过敏原的同时激活气道PAR2，可诱导变应性致敏作用，但仅吸入过敏原则导致免疫耐受 Ebeling C, et al. Proteinase-activated receptor-2 promotes allergic sensitization to an inhaled antigen through a TNF-mediated pathway. J Immunol. 2007 Sep 1;179(5):2910- 7.； 5、 PAR-2缺陷小鼠在过敏原刺激后耳水肿和炎细胞浸润明显减轻 Buddenkotte J, et al. 2005. Agonists of proteinase-activated receptor-2 stimulate upregulation of intercellular cell adhesion molecule-1 in primary human keratinocytes via activation of NF-kappa B. J Invest Dermatol, 124(1): 38~45.； 6、 PARs激活肽可引起人类皮肤及结肠组织中的肥大细胞呈浓度依赖性释放组胺 Moormann C, et al. Functional characterization and expression analysis of the proteinase-activated receptor-2 in human cutaneous mast cells. J Invest Dermatol. 2006 Apr;126(4):746-55.

  37. 我们的工作

  38. PAR相关的论文 1. He S, Aslam A, Gaca MD, He YS, Buckley MG, Hollenberg MD, Walls AF. Inhibitors of tryptase as mast cell stabilizing agents in the human airways: effects of tryptase and other agonists of PAR2 on histamine release. J Pharmacol Exp Ther, 2004; 309(1): 119-26. (IF:4.309) 2. He S, Xie H, Fu YL. Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2. Acta Pharmacol Sin, 2005; 26(5): 568-74. (IF:1.68) 3. Hou YF, Zhou YC, Zheng XX, Wang HY, Fu YL, Fang ZM, He S. Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine. Mol Immunol, 2006; 43: 1982-92. （通讯作者） (IF:4.768) 4. Wang L, Luo JM, Fu YL, He S. Induction of interleukin-8 secretion and activation of ERK1/2, p38 MAPK signaling pathways by thrombin in dermal fibroblasts. Int J Biochem Cell B, 2006; 38: 1571-83. （通讯作者）(IF:4.804) 5. Wang HY, Zheng YS, He S. Induction of release and up-regulated gene expression of interleukin (IL)-8 in A549 cells by serine proteinases. BMC Cell Biology, 2006; 7:22: 1-13. （通讯作者）(IF:3.157) 6. Li T, Wang HY, He S. Induction of interleukin-6 release from monocytes by serine proteinases and its potential mechanisms. Scand J Immunol, 2006; 64: 10-6. （通讯作者）(IF:2.186) 7. Li T, He S. Induction of IL-6 release from human T cells by PAR-1 and PAR-2 agonists. Immunol Cell Biol, 2006; 84: 461-6. （通讯作者）(IF:3.859)

  39. Liu T, Wang BQ, Zheng PY, He S, Yang PC. Rhinosinusitis derived Staphylococcal enterotoxin B plays a possible role in pathogenesis of food allergy. BMC Gastroenterol, 2006; 6:24: 1-11. (IF:2.136) • Wang HY, He S. Induction of lactoferrin and IL-8 release from human neutrophils by tryptic enzymes via proteinase activated receptor-2. Cell Biology International, 2006; 30: 688-97. （通讯作者）(IF:1.619) • 10. Wang HY, Yi TT, Zheng YS, He S. Induction of monocyte chemoattractant protein-1 release from A549 cells by agonists of proteinase activated receptor-1 and –2. European Journal of Cell Biology, 2007; 86: 233-42. （通讯作者）(IF:3.955) • Wang L, Luo JM, He S. Induction of MMP-9 release from human dermal fibroblasts by thrombin through JAK/STAT3 signaling pathway. BMC Cell Biology, 2007; 8:14: 1-9. （通讯作者）(IF:3.157) • 12. Zhang HY, Yang XY, Yang HW, Lin Qing, Zhang ZF, Zheng YS, Chen SY, Yang PC, He S. • Modulation of mast cell proteinase-activated receptor expression and IL-4 release by IL-12. • Immunol Cell Biol, 2007; 85 (7): 558-66. （通讯作者）(IF:3.859) • 13. Niu QX, Chen HQ, Chen ZY, Fu YL, Lin JL, He S. Induction of proinflammatory cytokine release from human umbilical vein endothelial cells by agonists of proteinase activated receptor-2. Clin Exp Pharmacol P, 2008 Jan; 35(1): 89-96. （通讯作者） (IF:2.196) • 14. Feng B, He S, Zheng P, Wu L ,Yang P. Mast cells play a crucial role in staphylococcus aureus • petidoglycan-induced diarrhea. Am J Pathol, 2007; 171: 537-547. (IF:5.917)

  40. Zhang ZF, Zhang HY, Yang HW, Zhang LX, Chen C, Zheng XX, HE S. Induction of Th2 • cytokine release and upregulated expression of protease activated receptors on mast cells by • recombinant American cockroach allergen Per a 7. Clin Exp Allergy, 2008; 38: 1160-1167. • （通讯作者）(IF:3.73) • Huiyun Zhang, Haiwei Yang, Lianxia Zhang, Xiaoyu Yang, Zhongfang Zhang, Qing Lin. • Induction of IL-4 Release and Upregulated Expression of Protease Activated Receptors by • GM-CSF in P815 Cells. Cytokine. 2009. 48；196–202 （通讯作者） • 17. Liya Qiao, Huiyun Zhang, Shandong Wu and Shaoheng He. Downregulation of Protease • Activated Receptor Expression and Cytokine Production in P815 Cells by RNA Interference. • BioMed Immunol. BMC Cell Biology. 2009, 10:62-69 （通讯作者） • 18. He S, Zhongfang Zhang, Huiyun Zhang, et al. Induction of Th2 cytokine release and • upregulated expression of protease activated receptors on mast cells by recombinant • American cockroach allergen Per a 1. Submitted.

  41. 小结： • 发现T细胞、单核细胞、中性粒细胞、嗜酸性粒 　细胞上PAR的表达变化； • 发现肥大细胞对PAR介导的刺激的应答特点及调 　节机制； • 发现美洲大蠊主要过敏原对肥大细胞PAR表达变 　化的影响。

  42. 评论： • 上面所述PAR与过敏性疾病的相关性均是间接 证据。 • 直接评价PAR与过敏性疾病的相关性是检测 血清中PAR的N末端片段在疾病中的水平变化。 • 我们已经进行了7年，再有1-2年内就可以完成。

  43. 研究PAR与过敏性疾病关系的意义 • 揭示了过敏性疾病中蛋白酶在炎症细胞上的作用靶点； • 揭示了一种非IgE介导的肥大细胞激活机制； • 提示某些具有与系锁配体序列相同的肽段理论上可以激活肥大细胞，从而引起过敏样的临床表现。

  44. 第三部分: Toll样受体(TLR)在过敏性疾病 发病机制中的作用

  45. 哺乳动物所表达TLRs的配体

  46. 已经报道的TLRs与过敏性疾病相关的工作

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