THE MOLECULAR BIOLOGY OF CCR-4 MEDIATED ALLERGIC REACTION IN NASAL MUCOSA AND ITS REGULATION

Eotaxin （CCL11）是一种强效的嗜酸性细胞趋化剂，属于C-C趋化因子。为了评估eotaxin在鼻粘膜嗜酸性粒细胞炎症中的作用，我们研究了抗原激发后鼻灌洗液中嗜酸性粒细胞化学引诱剂的水平，并与嗜酸性粒细胞计数和嗜酸性粒细胞蛋白X （EPX）水平进行了比较。在变应性鼻炎患者中，过敏原挑战导致鼻灌洗液中嗜酸性粒细胞计数、EPX水平和eotaxin浓度的平行增加。灌洗液中eotaxin水平与灌洗液中嗜酸性粒细胞计数和EPX水平有很强的相关性。正常受试者在抗原激发前后鼻腔灌洗液中嗜酸性粒细胞和EPX以及测量参数很少（如果有的话）。在我们用鼻微血管内皮细胞进行的嗜酸性粒细胞内皮迁移（TEM）实验中，eotaxin在各种嗜酸性粒细胞化学引诱剂中表现出最有效的作用。此外，抗CCR3单抗治疗嗜酸性粒细胞可显著阻断鼻黏膜匀浆诱导的嗜酸性粒细胞TEM。这些结果表明eotaxin在鼻粘膜嗜酸性粒细胞依赖性炎症中具有重要作用，并提示阻断eotaxin或CCR-3可能有助于变应性鼻炎的新治疗工具。研究表明，CCR4与MDC/CCL22和TARC/CCL17之间以及CCR8与I-309/CCL1之间的相互作用对于变应性疾病气道黏膜对变应原刺激的Th2细胞募集至关重要。为了更好地了解TARC在变应性鼻炎发病机制中的作用，我们研究了该趋化因子在鼻黏膜中的细胞来源。此外，我们还研究了细胞因子对TARC生成的影响。变应性鼻炎患者鼻黏膜上皮细胞表达的信号水平高于非变态反应患者。IL-4、TNF-α、IL-13、TNF-α联合刺激可协同诱导上皮细胞TARC表达。此外，鼻过敏患者上皮细胞中由这些细胞因子诱导的TARC含量高于非过敏患者。这些结果表明鼻上皮细胞在TARC的表达中起着至关重要的作用，并且Th2细胞因子IL-4和IL-13可能通过诱导上皮细胞产生TARC来促进Th2反应。接下来，我们检测了健康人与变应性鼻炎患者外周血T细胞CCR4表达的差异。CCR4在健康人及变应性鼻炎患者外周血CD4+CD45RO+ T细胞中的表达百分比轻度组和中度组与健康组之间无显著差异。而重症组的CCR4表达明显高于健康组。用抗CCR-4单克隆抗体处理单核细胞，并用流式细胞仪将其分为CCR-4阳性细胞和阴性细胞。在PMA和IOM刺激下，CCR4+ T细胞主要产生IL-4而不产生IFN-γ，而CCR4-T细胞优先产生IFN-γ而不产生IL-4。这些结果表明，在严重变应性鼻炎中，CCR4优先在Th2细胞上表达，并且细胞因子谱明显倾向于Th2优势。少

Eotaxin (CCL11) is a potent eosinophil chemoattractant belonging to the C-C chemokine. To evaluate the role of eotaxin in eosinophilic inflammation in nasal mucosa, we investigated the levels of eosinophil chemoattractants in nasal lavage fluids obtained after antigen challenge, and compared with eosinophil counts and eosinophil protein X (EPX) levels. In subjects with allergic rhinitis, allergen challenge led to parallel increases in eosinophil counts, levels of EPX and eotaxin concentrations in nasal lavage fluid. The levels of eotaxin in lavage samples showed strong correlation with lavage levels of eosinophil counts and EPX. Normal subjects had few, if any, eosinophils and EPX as well as the measured parameters in their nasal lavage fluids before and after antigen challenge. In our experiments of eosinophil endothelial transmigration (TEM) assay using the nasal microvascular endothelial cells, eotaxin showed most potent effect among various eosinophil chemoattractants. In addition, … More treatment of eosinophils with anti CCR3 mAb significantly blocked eosinophil TEM induced by homogenate of nasal mucosa. These results indicate that eotaxin has an important role in eosinophil-dependent inflammation in nasal mucosa and suggest that blocking eotaxin or CCR-3 might be useful for new therapeutic tools of allergic rhinitis.It has been shown that interactions between CCR4 and MDC/CCL22 and TARC/CCL17, as well as between CCR8 and I-309/CCL1, are critical for the recruitment of Th2 cells in the airway mucosa of allergic diseases in response to allergen stimulation. To gain a better understanding of the role of TARC in the pathogenesis of allergic rhinitis, we have investigated the cellular sources of this chemokine in nasal mucosa. In addition, the effect of cytokines on the TARC production has been investigated. Epithelial cells in nasal mucosa in subjects with allergic rhinitis expressed higher signal level than those in non-allergy patients. The combined stimulation with IL-4 and TNF-α, as well as IL-13 and TNF-α, synergistically induced TARC expression in epithelial cells. Furthermore, the amount of TARC induced by these cytokines was higher in epithelial cells obtained from nasal allergy patients than in those from non-allergy patients. These results demonstrate a crucial role of nasal epithelial cells in the expression of TARC, and that Th2 cytokine IL-4 and IL-13 may promote Th2 responses by inducing TARC production from epithelial cells.Next we examined the difference of CCR4 expression on peripheral blood T cell among healthy subjects and allergic rhinitis subjects. The percent expression of CCR4 on peripheral blood CD4+CD45RO+ T cells in healthy subjects and allergic rhinitis subjects. There is no significant difference between the mild and moderate groups and healthy subjects. However, the CCR4 expression in the severe group was significantly higher than that seen in the healthy subjects. Treatment of mononuclear cells with anti CCR-4 monoclonal antibody and soating using flowcytometer divided them to CCR-4 positive and negative cells. CCR4+ T cells predominantly produce IL-4 but not IFN-γ when they were stimulated with PMA and IOM, whereas CCR4-T cells preferentially produce IFN-γ but not IL-4. These results suggest that CCR4 was expressed preferentially on Th2 cells and that cytokine profile is markedly inclined to Th2 predominance, in severe allergic rhinitis. Less