牙周致病菌Porphyromonas gingivalis、Tannerella forsythensis 對人類單核球衍生樹突狀細胞產生細胞激素的影響

Abstract

本實驗的目的是觀察牙周致病菌Porphyromonas gingivalis、Tannerella forsythensis〈舊名為Bacteroides forsythus〉、及其所萃取出的脂多醣內毒素，在體外對人類單核球衍生的樹突狀細胞，產生發炎前期細胞激素的影響。單核球衍生樹突狀細胞是從20個健康受試者之新鮮周邊血中分離並培養，分別以熱處理過的細菌、及不同濃度的脂多醣內毒素刺激24小時後，利用流式細胞儀觀察樹突狀細胞表型的改變，以ELISA方式測量分泌細胞激素產量、及反轉錄同步定量聚合酶連鎖反應偵測激素的基因表現，E. coli及其LPS在本實驗則為正向控制組。實驗結果顯示，體外經P. gingivalis、T. forsythensis及其LPS刺激樹突狀細胞，細胞的型態會改變、其中表面第1型及第2型MHC胜肽類複合物〈HLA-DR〉的表現及穩定度提昇，成熟化的記號〈CD83〉增加、共同刺激分子〈CD80、CD86〉的表現量也上昇。細胞激素測定方面，發現經細菌及其LPS刺激後，細胞激素的產量，與對照組相較，均顯著提昇，IL-10大於IL-12p40及IL-12p70，全菌的反應則較脂多醣內毒素明顯，皆具統計上的差異；樹突狀細胞受3種細菌的脂多醣內毒素刺激，分泌的細胞激素產量卻沒有明顯差別，IL-10具有劑量依存效應，但不具有統計上的意義；以E. coli刺激時，得到的IL-10、IL-12p70產量，顯著高於P. gingivalis和T. forsythensis，但在細胞激素分泌型式的比較上，沒有明顯的改變。本實驗所偵測到的IL-12，多為p40蛋白型式，IL-12p70的分泌量較低。細胞激素基因表現結果與ELISA結果類似，3種細菌刺激樹突狀細胞3小時後，IL-10 mRNA的表現最強，次為IL-12p40、IL-12p35最低，IL-12p35的mRNA，僅在以E. coli刺激時，有較顯著的提昇。由實驗結果得知，P. gingivalis及T. forsythensis感染寄主時，除了脂多醣內毒素外，其他細菌成份可能也在活化樹突狀細胞、及產生細胞激素的過程中參與反應。此外，牙周致病菌早期侵犯宿主時，可能因為較弱的免疫刺激能力、並能在初期免疫反應促使樹突狀細胞分泌高量抗發炎細胞激素，並誘導具有免疫抑制性質的調節性T淋巴球活化，使牙周感染區域形成慢性發炎的病灶，以避免遭到免疫細胞清除的命運。

The present study was to investigate the effect of periodontal pathogens Porphyromonas gingivalis, Tannerella forsythensis (Bacteroides forsythus) and their lipopolysaccharides (LPS) on the production of cytokines from human monocyte-derived dendritic cells (MDDC). MDDC were isolated from 20 healthy subjects and co-cultured with the heat treated whole bacteria or their LPS. The phenotypic changes were evaluated with flow cytometry, the cytokine profiles were determined by ELISA, and the mRNA expression of cytokine was analysis by real-time RT-PCR. E. coli and its LPS were used as positive control. The results showed that in vitro monocyte-derived DCs pulsed with P. gingivalis 33277, T. forsythensis 338 and their LPS would change the morphology and increase the surface expression and stability of MHC class I and class II-peptide complexes (HLA-DR), upregulated the maturation marker (CD83) and adhesion and co-stimulatory molecules(CD80, CD86). On cytokine detection, IL-10 production was higher than IL-12p40 and IL-12p70 in all groups. The response of whole bacteria was stronger than that of the LPS. Lipopolysaccharides of different bacteria induced similar IL-10 and IL-12 profiles from DCs with a dose dependent effect on IL-10. DCs pulsed with E. coli produced more IL-10 and IL-12 than P. gingivalis and T. forsythensis, without significant change of cytokine profile patterns. Most of IL-12 produced was in p40 form, rather than IL-12p70. IL-10 mRNA was highly expressed on DC after stimulation with the bacteria, but IL-12p35 mRNA level was elevated clearly only when DC pulsed with E. coli. The present study implicated that the components of P. gingivalis and T. forsythensis, other than LPS, maybe responsible for the interaction of the bacteria and the dendritic cells. The periodontal pathogens may induce the anti-inflammatory cytokine production to suppress the function of the dendritic cells in the innate immune response, and direct naïve T cells to regulatory T lymphocytes with immunosupressive properties. P. gingivalis and T. forsythensis with weaker immunostimulatory activity than E. coli may contribute to the induction of chronic inflammation at local lesion of periodontal infection.