幽門螺旋桿菌之熱休克蛋白60誘導前發炎性細胞激素的分泌但卻抑制單核球細胞之活性

Abstract

幽門螺旋桿菌之熱休克蛋白60在之前的文獻中被發現它可以誘導胃上皮細胞及人類單核球細胞產生介白素-8 (IL-8)，以及巨噬細胞產生介白素-6。在此研究中，我們將幽門螺旋桿菌之熱休克蛋白60 (rHpHSP60) 與人類單核細胞株THP-1共同培養，發現除了介白素-8以及介白素-6之外，腫瘤壞死因子-甲型以及介白素-1乙型都會被THP-1產生。腫瘤壞死因子-甲型與介白素1乙型與炎症反應的開始有很大的關連。其他生物的熱休克蛋白雖然也會誘導前發炎細胞激素(pro-inflammatory cytokines)的產生，但pro-inflammatory cytokines產生的時間卻不盡相同。在此篇研究中發現，腫瘤壞死因子-甲型在受到rHpHSP60刺激後兩小時就開始顯著的產生而在4小時產量最高；而介白素-1乙型，介白素-6，介白素-8會隨著時間延長而小幅增加，直到24小時才大量的表現。腫瘤壞死因子-甲型在這些細胞激素中最早產，因此對單核球細胞的活化最具影響力。我們藉由偵測單核球細胞的吞噬能力以及細胞表面抗原之表現來觀察細胞的活化。結果顯示，細胞的吞噬活性明顯下降而且主要組織相容性複合體II (MHCII) 的表現也顯著性的下降。而共同刺激分子CD40、CD80、CD86會顯著性的升高，而主要組織相容性複合體I(MHCI)表現則無改變。我們更進一步用商業化之人類腫瘤壞死因子-甲型觀察其對單核球細胞活化的影響，結果顯示隨著商業化之人類腫瘤壞死因子-甲型的增加，單核球細胞的吞噬能力竟然減弱了，而共同刺激分子CD40的表現則如我們預期的增加了。在胃部慢性發炎的病人中發現轉化生長因子–□1 (TGF-□1)會大量表現；為了模擬胃部慢性發炎的環境，我們將商業化之人類腫瘤壞死因子-甲型與轉化生長因子–□□同時刺激單核球細胞，結果發現腫瘤壞死因子-甲型可協助轉化生長因子–□□造成的抑制作用，轉化生長因子–□□則更進一步抑制由腫瘤壞死因子-甲型引起的單核球CD40之表現，阻礙其成熟與分化。根據以上的結果顯示，幽門螺旋桿菌之熱休克蛋白60會刺激單核球細胞產生介白素-1乙型，介白素-6，介白素-8以及腫瘤壞死因子-甲型；其中，腫瘤壞死因子-甲型最早產生，但單核球細胞之活性卻仍受到抑制。在我們的研究中，腫瘤壞死因子-甲型並非扮演活化性的角色反而是抑制單核球細胞的活性。在慢性發炎反應中， 腫瘤壞死因子-甲型結合轉化生長因子–□1對於單核球細胞的活化造成更嚴重的影響。

Heat shock protein 60 of Helicobacter pylori has been found that it can induce interleukin-8 (IL-8) secretion in human monocytic cells and gastric epithelium cells. In this study, we further found that the IL-6, IL-8, TNF-□□and IL-1□□were induced in THP-1 cells after H. pylori HSP60 stimulation. The kinetic of cytokine expression showed that TNF-□□was earliest secreted at 2 h, and reached a maximum at 4 h. This result consisted with the kinetic of TNF-□ mRNA expression analyzed by quantitative real-time PCR. TNF-□ may have a great effect on THP-1 cells activation. Dissimilarly, IL-1□, IL-6, and IL-8 were later produced by THP-1 cells. We further examined THP-1 cells activation by detecting its enodocytosis activity and surface marker expression. Surprisingly, the endocytosis ability of THP-1 cells was weakened after rHpHSP60 stimulation. However, the co-stimulatory molecules (CD40, CD80, and CD86) were up-regulated, whereas MHC class II which plays a central in presenting the foreign antigen to T helper cells was significantly down-regulated. MHC I expression was not influenced by rHpHSP60. Interestingly, the rhTNF-□ mimicry experiments indicated that the endocytosis activity of THP-1 cells was diminished by rhTNF-□ in□a□does-dependent manner. However, it can promote CD40 expression on THP-1 cells surface. To mimic the chronic inflammation area of H. pylori-infected patients, rhTNF-□ and TGF-□1 were used to treat THP-1 cells. The inhibitory effect on endocytotic activity of THP-1 cells was observed by rhTNF-□ and TGF-□1 synergy treatment. TNF-□ seemed to synergize with TGF-□1 to decrease the engulf ability of cells. However TGF-□1 further inhibited TNF-□-mediated CD40 expression. This study suggested rHpHSP60 induced TNF-□, IL-1□, IL-6, and IL-8 secretion in THP-1 cells. Among these cytokines, TNF-□ was earliest secreted. Even through the endocytosis ability of THP-1 cells was inhibited and the MHC class II was significantly decreased after rHpHSP60 stimulation. The role of TNF-□□in our study was not an “effector” on THP-1 cells activation but diminished its activity. In the chronic inflammation, the inhibition effect of TNF-□□combining with TGF-□1 on monocytes activation was more critical.