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

嗜酸性粒细胞趋化因子(CCL11)是一种强效嗜酸性粒细胞趋化因子，属于C-C趋化因子。为探讨嗜酸性粒细胞趋化因子在鼻黏膜嗜酸性炎症中的作用，我们检测了抗原攻击后鼻腔灌洗液中嗜酸性粒细胞趋化因子的水平，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。在过敏性鼻炎受试者中，过敏原刺激导致鼻灌洗液中嗜酸性粒细胞计数、EPX水平和嗜酸性粒细胞浓度平行增加。灌洗液中嗜酸粒细胞计数和EPX与嗜酸细胞趋化因子水平呈显著正相关。正常受试者在抗原攻击前后鼻灌洗液中的嗜酸性粒细胞和EPX以及测量的参数很少(如果有的话)。在我们用鼻腔微血管内皮细胞进行的嗜酸性粒细胞内皮迁移试验中，嗜酸性粒细胞趋化因子的作用是最强的。此外，…进一步用抗CCR3单抗处理嗜酸性粒细胞，可明显阻断鼻粘膜匀浆诱导的嗜酸性粒细胞的透射电子显微镜。这些结果表明，嗜酸性粒细胞趋化因子在嗜酸性粒细胞依赖的鼻黏膜炎症中起重要作用，阻断嗜酸性粒细胞趋化因子或CCR-3可能成为治疗变应性鼻炎的新工具。已有研究表明，CCR4与MDC/CCL22和TARC/CCL17之间以及CCR8与I-309/CCL1之间的相互作用在变态反应性疾病的呼吸道黏膜中Th2细胞的募集中起关键作用。为了更好地了解TARC在变应性鼻炎发病机制中的作用，我们研究了鼻黏膜中这种趋化因子的细胞来源。此外，还研究了细胞因子对TARC产生的影响。变应性鼻炎患者鼻粘膜上皮细胞表达的信号水平高于非变应性鼻炎患者。IL-4和肿瘤坏死因子-α以及IL-13和肿瘤坏死因子-α联合刺激可协同诱导上皮细胞TARC的表达。此外，在鼻变态反应患者的上皮细胞中，这些细胞因子诱导的TARC的数量高于非变态反应患者。这些结果表明鼻腔上皮细胞在TARC的表达中起着至关重要的作用，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应答。正常人和变应性鼻炎患者外周血CD4+CD45RO+T细胞CCR4的表达轻、中度组与健康组比较，差异无统计学意义。而CCR4在重症组的表达明显高于健康组。用抗CCR-4单抗处理单个核细胞，并用流式细胞仪分离，将其分为CCR-4阳性细胞和阴性细胞。CCR4+T细胞主要产生IL-4，但不产生干扰素-γ；CCR4-T细胞优先产生干扰素-γ，但不产生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，细胞因子谱明显倾向于Th2优势。较少

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