鼷鼠感染廣東住血線蟲後產生eotaxin的機轉的探討

Investigation on the mechanism of production of eotaxin in mice infected with Angiostrongylus cantonensis

張一旋(Eddy Essen)

指導教授 ： 顏全敏

高雄醫學大學/醫學院/醫學研究所/博士(2004年)

摘要

廣東住血線蟲 (Angiostrongylus cantonensis) 的非適當宿主體內的嗜伊紅性白血球於對抗廣東住血線蟲的感染佔有相當重要的地位。 Eotaxin 是嗜伊紅性白血球最強效的趨化素，可以專一的吸引嗜伊紅性白血球到標的部位。本研究發現BALB/c鼠感染廣東住血線蟲後，利用ELISA (enzyme-linked immunosorbent assay) 的方法檢測腦脊髓液中eotaxin的含量，發現其濃度顯著高於周邊血液，且隨著感染週數的增加，eotaxin的濃度亦隨著增加，一直到感染後第三週實驗結束為止，嗜伊紅性白血球在腦脊髓液的數量與eotaxin的濃度平行增加，相反的，蟲體回收的情形則逐漸減少。由此可知非適當宿主感染廣東住血線蟲後，誘發腦脊髓液中高濃度eotaxin的產生，對吸引嗜伊紅性白血球由周邊血液移行到腦脊髓液中，是一個重要的趨化素。在體外以微量細胞趨化盤分析感染廣東住血線蟲後BALB/c鼠的腦脊髓液對嗜伊紅性白血球的趨化能力，發現感染後第一週開始慢慢增加至感染後第三週為止。若在感染後二十一天的腦脊髓液中，分別加入抗-eotaxin，MIP (macrophage inflammatory protein) -1α，RANTES (regulated on activation, normal T-cells expressed and secreted) 或PAF (platelet-activating factor) 等不同單株抗體，再測定對嗜伊紅性球趨化的影響。在腦脊髓液中加入eotaxin或MIP-1α的抗體使嗜伊紅性白血球移行進入濾膜的數目顯著少於未加抗體 (Student t test， P <0.01及 P <0.05)；而在腦脊髓液加入抗 RANTES 或抗 PAF 抗體，無法抑制嗜伊紅性白血球的趨化。體外試驗證實，當感染廣東住血線蟲時，產生於鼷鼠腦脊髓液中的eotaxin，對嗜伊紅性白血球具有直接而且是最重要的趨化能力。
鼷鼠感染廣東住血線蟲後，免疫反應與Th (T-helper) 2的路徑有關，以廣東住血線蟲第三期幼蟲的抗原與取自脾臟和頸部淋巴結內的抗原呈現細胞(antigen presenting cell)和CD (cluster designation) 4+細胞在不同條件下共同培養，藉 ELISA 測量培養後收集的條件培養液中 IL (interleukin) -4， IL-1β， IL-13， IFN (interferon) -γ 和 TNF (tumor necrosis factor) -α 的濃度，結果發現，若先經 IL-4 共同培養後，再加入濃度為 85μg/ml 的廣東住血線蟲抗原二次刺激之後，所收集的條件培養液中含有高濃度的 IL-4，而此條件培養液可誘發 SVEC4-10 內皮細胞株產生 eotaxin。因此，鼷鼠感染廣東住血線蟲後產生eotaxin的機轉，應是感染後先產生IL-4，再由IL-4誘發細胞產生eotaxin進而吸引嗜伊紅性白血球移行到腦脊液。

關鍵字

廣東住血線蟲 ； eotaxin ； 嗜伊紅性白血球 ； 抗原呈現細 ； CD4+細胞

並列摘要

There is evidence showing that eosinophils play an important role in non-permissive host reactions against Angiostrongylus cantonensis after infection. Eotaxin is the strongest mediator involved in eosinophil chemotaxis and chemokinesis, which selectively recruits eosinophils. In our study, all eotaxin concentrations weekly in the cerebrospinal fluid (CSF) of the infected mice were significantly higher than those in the serum and got the peak level at third weeks. The increases in eosinophils in CSF paralleled the kinetics of the change in eotaxin levels in the CSF of infected mice. Furthermore, in parallel with this CSF eosinophilia, infected mice showed gradual reductions in intracranial worm counts. Therefore, the high concentration of eotaxin in the CSF is a very important reason for the migration and recruitment of eosinophils. We used microchambers to demonstrate direct eosinophil chemotactic activity. The chemotactic response of eosinophils from the CSF weekly increase from week 1 to week 3. Adding a variety of antibodies into CSF from 21 days post-infection with A. cantonensis, the antibodies directly neutralized eotaxin, RANTES (regulated on activation, normal T-cells expressed and secreted), MIP (macrophage inflammatory protein) -1α or PAF (platelet-activating factor), respectively. Eosinophil migrated into the polycarbonate mambrane covering CSF with anti-eotaxin or anti-MIP-1α antibodies was significantly lower than that for antibody-free CSF (Student’s t tests: p < 0.01, p < 0.05). Furthermore, anti-RANTES and anti-PAF antibodies failed to inhibit the migration of eosinophils to the CSF. In conclusion, eotaxin release in the CSF of A.cantonensis-infected mice have eosinophil chemotactic activity in this in vitro assay.
Th2 respones has been shown in mice infected with A. cantonensis. We wish to determine the kinetics of cytokine production and its possible role in expression of eotaxin in A. cantonensis infected mouse. Concentrations of IL (interleukin) -1β, IL-4, INF (interferon) -γ, IL-13 and TNF (tumor necrosis factor) -α in supernatants collected from different stimulated from APCs (antigen-presenting cells) and CD (cluster designation) 4+ T cells were assayed by ELISA (enzyme-linked immunosorbent assay). The conditioned medium collected form the medium preincubating APC and CD4+ T cells with IL-4 and then a second 85 μg/ml of A. cantonensis antigen stimulation expressing high level of IL-4. The conditioned medium were used to see the bioactivity of stimulating to the SVEC4-10 endothelial cells. Finally, only the conditioned medium with high level of IL-4 has the ability to stimulate SVEC4-10 endothelium cell expressing eotaxin. Although, the cytokine IL-1β, IL-13, IFN-γ and TNF-α also had the ability of stimulating cells to express eotaxin but in mouse infected with A. cantonensis seems only IL-4 may induce SVEC4-10 endothelial cells secreting eotaxin. We have demonstrated expressing of eotaxin for recruitment of eosinophils to CSF in mice infected with A. cantonensis and the necessary of IL-4 in expressing eotaxin in this study.

並列關鍵字

Angiostrongylus cantonensis ； eotaxin ； eosinophils ； antigen presenting cells ； CD4+ cells

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