【作者】 常颖；

【导师】 张桂珍； Anne Tsicopoulos；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 本研究通过体外实验以及应用人体化的重症联合免疫缺陷(SCID)小鼠进行的体内实验对CCL18在过敏性疾病发病机理中的作用进行了研究,取得了以下学术进展:1.研究发现在过敏原的刺激下,过敏性哮喘患者外周血单个核细胞(PBMC)在转录及翻译水平表达CCL18基因增高。免疫酶联及免疫组化检测进一步发现,未经治疗的过敏性哮喘患者血清及支气管肺泡盥洗液中CCL18水平显著增高。发现CCL18在体外能够趋化Th2细胞和嗜碱性粒细胞。本研究的结果进一步提出了CCL18参与过敏性疾病发病的科学依据。2.柴油废气颗粒(DEP)能够通过IL-13在转录及翻译水平显著提高CCL18在非过敏者PBMC中的表达,从而导致Th2细胞的募集。更进一步表明CCL18可能在过敏性疾病产生的最初始时期发挥致病作用。3.通过对CCL5利用CCR5在SCID小鼠模型中募集炎症细胞的实验,证明了该模型应用于趋化因子研究中的可行性。利用人体化SCID小鼠模型研究的结果表明CCL18能够趋化单核/巨噬细胞、Th2细胞、原态和记忆T细胞。进一步通过体内实验证明了CCL18能通过募集Th2细胞为主的炎症细胞来促进过敏性疾病的发生发展。4.构建了人CCL18及连有人或小鼠IgG1 Fc片段的CCL18的真核表达载体。经扩增表达及克隆选择后获得在无血清条件下高效稳定表达的细胞克隆。为在真核系统中克隆CCL18受体奠定了基础。总之,本研究首次发现CCL18参与了过敏性哮喘的发病,并首次揭示了CCL18在过敏性哮喘中的细胞来源及在过敏性疾病发病机制中的重要作用。表明CCL18可能是治疗过敏性疾病的潜在分子靶点。更多还原

【Abstract】 Chemokines are a group of small, mostly basic, structurally relatedmolecules that regulate cell trafficking of various types of leukocytes. Theinterreaction between chemokines and their related receptors controls the celltrafficking of immunocytes between circulatory system and tissues or organs,and play important roles in homeostasis or pathological conditions.Chemokines and cytokines together form an axis of mediating the trend ofimmune response induced by allergens.Numerous studies have shown that allergic diseases are Th2 lymphocyte-mediated inflammatory disorders. The involvement of some chemokines thatfavor Th2 cell recruitment leads to more Th2 cell trafficking to theinflammatory location and aggravates the imbalance of immune response.CCL18 is a chemokine recently cloned, preferentially expressed in lung tissueand induced by Th2-type cytokines like IL-4, IL-13 and IL-10, suggestingCCL18 may participate in the pathogenesis of allergic diseases especiallyallergic asthma. However, hereto few studies have been reported exceptseveral about allergic dermatitis. Therefore, we explored the roles of CCL18 inthe pathogenesis of allergic diseases.Firstly, the roles of CCL18 in the pathogenesis of allergic asthma wereinvestigated. Under the stimulation of allergen, the production of CCL18 inperipheral blood mononuclear cells (PBMC) from allergic asthmatics waselevated at both transcriptional and translational levels. There are at least twopathways involved in allergen-induced CCL18 production: one throughmonocyte stimulation by T cell-derived Th2 cytokines including IL-4 andIL-13, and one through direct pDC stimulation. These data suggest that duringthe Th2 polarization induced by allergen, CCL18 expression inantigen-presenting cells is increased. In vitro CCL18 attracted Th2 cells andbasophils but not Th1 cells or eosinophils. The fact that CCL18 inducedF-actin polymerization in Th2 cells and histamine and intracellular calciumrelease from basophils confirmed its chemotactic activity on these two types ofcells. These data suggest that CCL18 may be involved in secondary responsesinvolving Th2 cells and not only in primary immune responses as first thoughtfrom its ability to recruit naive T cells. The CCL18 levels in thebronchoalveolar lavage and serum from untreated and treated asthmaticpatients and control subjects were assayed. The results showed increasedCCL18 in the bronchoalveolar lavage from untreated asthmatic patientscompared to control subjects and asthmatics treated by inhaled corticoids.Altogether, this study shows a new function for CCL18, which is thepreferential recruitment of Th2 cells and basophils in the context of allergicasthma. Its high expression in bronchi from asthmatics suggests a predominantrole in the pathophysiology of allergic diseases.Consequently, the effect of environmental factors, particularly dieselexposure, was analysed. Diesel activation of PBMC from nonatopic subjectsinduced a late increase in CCL18 expression. These data suggest that even forthe nonatopic subjects, the expression of Th2 cytokines and CCL18 could stillbe elevated, furthermore the trend to allergy is exhibited, and CCL18 mayparticipate in the pathogenesis in the primary phase of allergic diseases.Studies to evaluate the in vivo role of CCL18 are limited due to theabsence of a murine homologue. Therefore, a model was established in SCID(Severe Combined ImmunoDeficient) mice grafted with human skin in orderto analyse the effect of chemokines and their receptors in vivo. We evaluatedthe role of CCR5 in the CCL5 (RANTES)-induced leukocyte recruitment inthis model. CCL5 induced a significant recruitment of memory T cells,monocytes/macrophages, eosinophils and IFN-γ+ cells by the use of CCR5.Thus, the feasibility of application of this model into in vivo studies ofchemokines was confirmed. We analysed the effect of CCL18 afterintradermal injection of SCID mice. Preliminary results showd a recruitmentof monocytes/macrophages, Th2 cells, na?ve and memory T cells, whichconfirmed the in vitro chemotactic activity of CCL18. These results suggestthat CCL18 could attract the inflammatory cells including Th2 cells toaggravate the development of allergic diseases.CCL18 receptor is still unknown. Our goal was to clone this receptorusing molecules produced in eukaryotic cells. CCL18 expressing vectors and asoluble fusion protein made of CCL18 fused to the Fc portion of human ormurine IgG1 were constructed. After amplification and clone selection, cloneswith stable and high productivity were obtained. These purified molecules willbe further used for the receptor cloning from a cDNA bank.Altogether, we firstly found that CCL18 participates in the pathogenesisof allergic asthma, and firstly disclosed the cell resource of CCL18 elevationin allergic asthma as well as its important roles in the pathogenesis of allergicdiseases. Thus, CCL18 could be a potential molecule target for the effectivetreatment of allergic diseases. These research results have been accepted forpublishing in the following SCI indexed journals: The Journal of Immunology(IF: 6.486), Journal of Allergy and Clinical Immunology (IF: 7.205) and TheJournal of Investigative Dermatology (IF: 4.238).更多还原