1、对过敏性疾病发病机制的几点新认识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南京医科大学第一附属医院何韶衡何韶衡：过敏的发病率过敏的发病率1、占世界人口的、占世界人口的10%30%，而且正以每年大于，而且正以每年大于1%的速度的速度 增加，以儿童患者的发病率上升最为明显；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、发达国家比发展中国家多，城市比农村地区多，如 1994年到年到2000年间，在意大利一家医院接受治疗的非年间，在意
2、大利一家医院接受治疗的非 欧洲人移民中，有欧洲人移民中，有84%在到欧洲前从没有过过敏症状；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、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？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、我国儿童哮喘发病率比10年前明显增加，仅小儿哮喘患年前明显增加，仅小儿哮喘患 者达者达1000万之多，其中过敏为主要诱因。万之多，其中过敏为主要诱因。过敏反应发生过程示意图过敏反应发生过程示意图 第一部分第一部分:对肥大细胞在过敏性疾病对肥大细胞在过敏性疾病发病
3、机制中核心作用的新认识发病机制中核心作用的新认识MCTCMCTTryptaseChymaseHistamineHeparinCytokinesetc.CarboxypeptidaseTryptaseHistamineHeparinCytokinesetc.Tryptase 10 pg/cellHistamine 1-3 pg/cellTryptase 35 pg/cellChymase 5 pg/cellHistamine 1-3 pg/cellA:肥大细胞亚型的研究肥大细胞亚型的研究Investigation of mast cell subtypes.Flow cytometry anal
4、ysis of mast cell subtypes in human skinMCTMCCMCTCExpression of carboxypeptidase in MCT under con-focal microscopyCA1FITCAA5CY5 PERed+greenCA1FITCCC4PERed+greenExpression of carboxypeptidase in MCc under con-focal microscopyCA1FITCAA5CY5 PE+CC4PERed+greenExpression of carboxypeptidase in MCTC under
5、con-focal microscopySpecimenSpecimen UL-MC UL-MCC C UR-MC UR-MCTCTC LR-MC LR-MCT T CA1 CA1 SP SP CD117 CD117S13.421.283.625.021.651.97S21.250.31.32.351.572.07S30.210.041.091.370.349.97S411.252.975.869.8310.086.08S56.752.554.648.879.728.22S65.52.298.2114.8914.914.52S76.184.6512.5620.3812.171.09S85.08
6、3.4210.7115.946.80.45S99.371.767.89.765.410.21S104.050.995.086.191.320.17S1121.1712.41736.5621.40.55S1219.3710.7917.3733.7626.730.63S1317.775.6511.7223.7519.280.24S147.324.2810.6418.2710.50.56S1520.934.536.9222.9517.870.44Flow cytometry analysis of mast cell subtypes in human colonMCTMCCMCTCMCCMCTCM
7、CTSpecimen UL-MCC UR-MCTC LR-MCT CA1 SP CD117C12.761.371.760.571.680.16C26.495.692.635.737.262.79C34.080.780.490.440.490.15C44.160.970.50.691.950.47C50.490.312.31.980.350.12C60.50.542.12.52.550.88C711.296.83.3610.317.342.59C813.735.212.695.111.070.46C92.771.842.783.731.190.51C101.830.4322.070.350.22
8、C110.370.20.860.630.420.15C122.5922.093.142.430.76C139.42.461.862.121.161.32C2-fixed32.137.464.858.845.561.66C01.442.467.097.886.214.5Summary:There may be only one type of mas cells-MCTC;MCT and MCC represent different stages of MCTC.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
9、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。Immunological Reviews Self-amplification mechanisms of mast cell degranulation.Asthmatic BALF mast cells exhibit 100-fold increased sensitivity to IgE dependent stimilation compared with the mast cells from non-asthmatic controlsBALF(),lung
10、()or colon()DissectionChoppingDigestionSeparationChallengeLoadingMeasurementC:创立了创立了肥大细胞脱颗粒信号自我放大机制肥大细胞脱颗粒信号自我放大机制的假说的假说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 thro
11、ugh the autocrine mechanism.Tohoku J Exp Med,2009,217(3):185-191.2、干细胞因子干细胞因子Lukacs NW et 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、肿瘤坏死因子肿瘤坏死因子-alpha Wong CK,et al.Allergy.Molecular mechanisms for the r
12、elease 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物质物质Kulka M et al.Neuropeptides activate human mast cell degranulation and chemokine production Immunology.2008 Mar;123(3):398-410.Epub 20
13、07 Oct 65、骨桥蛋白骨桥蛋白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.Accumulation of mast cells in the peritoneum of mouseInfluence of compound 48/80 on mast cell accumulatio
14、n in the peritoneum of miceInfluence of tryptic enzymes on mast cell accumulation in the peritoneum of miceSkin ColonTonsil Tryptase+IL-29 Chymase+IL-29 LungFigure2.Double labeling immunohistochemical staining of IL-29 in human mast cells.Cells stained with G3 or B7 antibody only were in red(solid a
15、rrow 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).Table 1.Number of IL-29 containing cells in foreskin,colon,tonsil and lung tissues stained by anti-human tryptase antibody G3 and ant
16、i-human IL-29 antibody SC1 with a double labeling immunohistochemical techniqueTissuePositive stained cellsG3/mm2SC1/mm2G3 and SC1/mm2%G3 positive cells expressing IL-29Foreskin3.0(0.7 5.8)2.5(0.6 5.1)58.7(27 120)*95(94 97)Colon5.0(0.5 15.8)5.9(0.4 28.8)70.4(55.8 121)*95(78 99)Tonsil4.5(0.7 6.5)2.2(
17、0.2 8.2)34.7(8.6 129)*96.5(92 99)Lung10.2(6.0 14.7)2.2(0.8 8.0)18.4(11.1 37.3)*88(82 99)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 antib
18、ody alone.P 0.05 in comparison with the cells stained by SC1 antibody alone(paired Mann-Whitney U-test).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、组胺通过组胺通过H4受体受体诱导肥大细胞的聚集：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
19、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：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、金属蛋白酶金属蛋白酶9 能够诱导肥大细胞祖细胞在组织中聚集：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
20、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肥大细胞的细胞因子如RANTES、eotaxin、TNF等也具有募集肥大细等也具有募集肥大细 胞的作用：胞的作用：Brzeziska-Baszczyk E,Misiak-Toczek A.The regulation of mast cell migration.Part 2:mast cell chemoattractants.Postepy
21、 Hig Med Dosw,2007,61:493-499.；5、类糜蛋白酶诱导肥大细胞的聚集类糜蛋白酶诱导肥大细胞的聚集:尚未发表尚未发表 肥大细胞类胰蛋白酶和类糜蛋白酶相关论文肥大细胞类胰蛋白酶和类糜蛋白酶相关论文1)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-
22、indexed for MEDLINE2)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 MEDLINE3)He S,Peng Q,Walls AF.Potent induction of a neutrophil and eos
23、inophil-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 MEDLINE4)He S,Walls AF.Human mast cell tryptase:a stimulus of microvascular leakage and mast cell activation.Eur J P
24、harmacol,1997;328(1):89-97.PMID:9203574 PubMed-indexed for MEDLINE5)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):1491-500.6)McEuen AR,Gaca MD,Buckley MG,He S,Gore MG,Walls AF.Two distinct for
25、ms 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 MEDLINE7)He S,Walls AF.The induction of a prolonged increase in microvascular permeability by human mast cell chymase
26、.Eur J Pharmacol,1998;352(1):91-8.PMID:9718272 PubMed-indexed for MEDLINE8）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 ME
27、DLINE9)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 MEDLINE10)He S,Gaca MD,Walls AF.The activation of synovial mast cells:mo
28、dulation of histamine release by tryptase and chymase and their inhibitors.Eur J Pharmacol,2001;412(3):223-9.PMID:11166285 PubMed-indexed for MEDLINE11)Walls AF,He S,Buckley MG,McEuen AR.Roles of the mast cell and basophil in asthma.Clin.Exp.Allergy,2001,1:68-72.12)He S,Alan R.McEuen,Blewett SA,Li P
29、,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):1007-15.13)He S,Chen P,Chen HQ.Modulation of enzymatic activity of human mast cell tryptase and chy
30、mase by protease inhibitors.Acta Pharmacol Sin,2003;24(9):923-9.14)He S,Zheng J.Induction of inflammatory cell accumulation using human mast cell tryptases.Asian Pacific J Allergy Immunol,2003;21:145-51.15）He S,Aslam A,Gaca MD,He YS,Buckley MG,Hollenberg MD,Walls AF.Inhibitors of tryptase as mast ce
31、ll 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,Zha
32、ng XJ,Wang XJ.Inhibition of histamine release from human mast cells by natural chymase inhibitors.Acta Pharmacol Sin,2004;25(6):822-6.18)He S,Chen HQ,Zheng J.Inhibition of tryptase and chymase induced nucleated cell infiltration by proteinase inhibitors.Acta Pharmacol Sin,2004;25(12):1677-84.19)He S
33、,Xie H.Modulation of tryptase and histamine release from human lung mast cells by protease inhibitors.Asian Pacific J.Allergy Immunology,2004;22:205-12.20)He S,Xie H.Modulation of tryptase release from human tonsil mast cells by protease inhibitors.Pharmacological Reports,2005;57:523-30.21)Zhou YC,C
34、hen 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）（通讯作者）22）Wang L,Luo JM,He S.Induction of MMP-9 release from human dermal fibroblasts by thrombin through JAK/STAT
35、3 signaling pathway.BMC Cell Biology,2007;8:14:1-9.（通讯作者）（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
36、,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 Biotechnolog
37、y,2006,122:334-40.（通讯作者）（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）（通讯作者）27）Li MC,Chen YZ,Huang T,Liu YN,He S.Production,characterization and appli
38、cations of two novel monoclonal antibodies against human interleukin-28A.Tissue Antigens,2006;68(6):477-82.（通讯作者）（通讯作者）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。第二部分第二部分:蛋白酶活化受体在过敏性疾病蛋白酶活化受体在过敏性疾病 发病机制中的作用发病机制中的作用蛋白酶活化受体示意图PAR-1PAR-2PAR-3PAR-4具有激活作用的蛋白酶具有激活作用的蛋
39、白酶凝血酶胰蛋白酶凝血酶凝血酶因子a类胰蛋白酶胰蛋白酶颗粒蛋白酶A因子a组织蛋白酶GGingipains-R因子aGingipains-R胰蛋白酶蛋白酶-3顶体蛋白螨过敏原Der P3 D9Gingipains-R系锁配体序列系锁配体序列SFLLR(h)*SLIGKV(h)TFRGAP(h)GYPFQV(h)TFLLRN(h)FSLLRY(h)SFFLR(m,r)*SLIGRL(m,r)SFNGGP(m)GYPGKF(m)基因基因/染色体染色体F2R/5q13(h)F2RL1/5q13(h)F2RL2/5q13(h)F2RL3/19q12(h)F2R/13D2(m)F2R1/13D2(m)F2
40、R2/13D2(m)F2R3/8B3.3(m)表达部位表达部位血小板上皮血小板(m)血小板(h)内皮内皮内皮内皮上皮成纤维细胞肌细胞肌细胞神经元肌细胞星形细胞星形细胞星状细胞神经元巨核细胞白细胞星状细胞 肥大细胞成纤维细胞白细胞肥大细胞肥大细胞肌细胞角化细胞子宫内膜精子功能功能血小板激活促炎作用PAR-4共刺激因子血小板激活促炎作用疼痛感介体血小板激活胚胎发育气道保护调节血管紧张度调节血管紧张度精子活力蛋白酶激活受体蛋白酶激活受体 已经报道的蛋白酶活化受体与过敏性疾病文献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哮喘病人呼吸道上皮细胞PAR-2的表达增高的表达增高 Ro
41、che 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,111:367373.；2、PAR-2缺陷小鼠的嗜酸性粒细胞浸润被抑制、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气道对吸入的乙酰甲胆碱的高反应性下降、卵清、卵清 蛋蛋白刺激产生的白刺激产生的IgE水平降低水平降低 Takizawa T,
42、et al.2005.Abrogation of bronchial eosinophilic inflammation and attenuated eotaxin content in protease-activated receptor 2-deficient mice.J Pharmacol Sci,98(1):99102.；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蛋白联合应用可引起致敏小鼠气道的高反应性
43、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):11001108.；4、吸入过敏原的同时激活气道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，可诱导变应性致敏作用，但仅吸入过敏原
44、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缺陷小鼠在过敏原刺激后耳水肿和炎细胞浸润明显减轻耳水肿和炎细胞浸润明显减轻 Buddenkotte J,et al.2005.Agonists of proteinase-activated receptor-
45、2 stimulate upregulation of intercellular cell adhesion molecule-1 in primary human keratinocytes via activation of NF-kappa B.J Invest Dermatol,124(1):3845.；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 t
46、he proteinase-activated receptor-2 in human cutaneous mast cells.J Invest Dermatol.2006 Apr;126(4):746-55.1.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 hista
47、mine 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
48、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;38:157
49、1-83.（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
50、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)PAR相关的论文相关的论文8.Liu T,Wang BQ,Zheng PY,He S,Yang PC.Rhinosinusitis derived Staphylococcal enteroto
