| 摘要: | 類風濕性關節炎(Rheumatoid arthritis, RA)是一種複雜的慢性自體免疫疾病，目前影響全球1-2%人口，該病主要侵犯關節部位軟、硬骨組織，其致病機制至今仍不完全明瞭。文獻指出，增加免疫細胞內cAMP濃度可有效降低多種免疫發炎反應，進而改善慢性發炎疾病如氣喘、COPD與過敏性皮膚炎等。第四型環狀核?磷酸雙酯? (phosphodiesterase 4, PDE4)是免疫細胞中主要水解cAMP的酵素，抑制PDE4可增加細胞內cAMP，因此臨床上PDE4抑制劑被廣泛研發為抗發炎藥劑。研究指出，PDE4抑制劑處理膠原蛋白誘發關節炎小鼠能改善關節炎症狀，然而其作用機制仍不甚明瞭。已知Th1與Th17細胞激素IFN-與IL-17A在RA致病過程中扮演重要的角色，因此本研究欲探討PDE4或cAMP信號傳導是否在RA致病過程中參與Th1與Th17細胞激素的調控。我們首先利用RA動物模型，即膠原蛋白誘發關節炎(collagen-induced arthritis, CIA)模型誘導C57BL/6與DBA/1小鼠發病，結果顯示，兩種小鼠的誘發率分別為45% (C57BL/6)與91% (DBA/1)，發病高峰期分別約在21-42天與27-33天，而早期認為對CIA有阻抗性之C57BL/6小鼠於本實驗中也能誘發出嚴重的關節炎。接著我們利用不同的致敏方法，即id./id.模型(將雞隻膠原蛋白(CII)先後注射於小鼠尾巴根部皮下)與id./ip.模型(將CII先後注射於小鼠尾巴根部皮下與腹腔內)，使DBA/1小鼠對CII產生免疫反應，之後以小鼠脾臟或腹股溝淋巴結T細胞進行實驗，結果顯示兩種致敏方法均可刺激CII專一性Th1與Th17細胞之活化。同時，我們發現CII或-CD3抗體可刺激兩種淋巴組織T細胞釋放IFN-與IL-17A，而PDE4抑制劑rolipram可有效抑制此等反應（除了腹股溝淋巴結細胞對IL-17A的釋放外）。此外，非選擇性PDE抑制劑IBMX對IFN-與IL-17A的釋放也有相當程度的抑制作用，表示在11個PDE家族中PDE4應是主要調控此等反應的酵素。再者，使用cAMP增升劑如dbcAMP與forskolin處理細胞，其抑制兩種細胞激素的程度與rolipram相似，因此我們推測rolipram的抑制作用是由於增加細胞內cAMP所致。而藉由PKA、Epac活化劑與抑制劑的使用，我們進一步發現rolipram的抑制作用是經由活化PKA訊號傳導所致。此外，PDE3抑制劑cilostazol與PDE7抑制劑BRL 50481對CIA小鼠之Th1與Th17免疫反應多不具抑制作用，然而cilostazol卻能協同rolipram使IFN-與IL-17A的抑制作用更為顯著。為此，我們推測PDE4選擇性抑制劑能改善RA的發炎反應，而PDE3與PDE4雙重抑制劑應可更有效地減緩RA病症。
Rheumatoid arthritis (RA) is a complicated, chronic autoimmune disorder that affects 1-2% population worldwide. It primarily attacks cartilage and bone of joints. The etiology of the disease still remains to be defined. Accumulating evidence indicates that elevation of intracellular cAMP concentration can suppress a vast spectrum of inflammatory responses in most immune cells and, thereby ameliorate chronic inflammatory diseases, such as asthma, COPD, and atopic dermatitis. Type 4 phosphodiesterases (PDE4s) are the major cAMP-hydrolyzing enzymes in various inflammatory cells. Inhibition of PDE4, thus increasing intracellular cAMP, has been considered an attractive strategy for developing anti-inflammatory drugs. Using collagen-induced arthritis (CIA) models, previous studies show potential benefits of PDE4 inhibitors in RA, while the mechanism of the effect remains unclear. Moreover, the Th1 and Th17 cytokines IFN- and IL-17A, respectively, are known important in the pathogenesis of RA. Therefore, in this study we employed CIA models to access whether PDE4 or cAMP signaling is involved in regulation of Th1 and Th17 responses in RA. By immunization of C57BL/6 and DBA/1 mice with chick collagen II (CII), we found that the disease incidence for the two mouse strains were 45% and 91%, respectively, and the arthritic symptoms developed during 21-42 and 27-33 days, respectively, after collagen immunization. In addition, the C57BL/6 mice, considered genetically resistant to CIA in early studies, also developed severe arthritic conditions. Immunization of DBA/1 mice with CII by different protocols (i.e. the id./id. and id./ip. models) indicated that both models induced the activation of CII-specific Th1 and Th17 cells in the spleen and draining (inguinal) lymph nodes of these mice. In vitro stimulation with CII and/or -CD3 antibody revealed a significant increase in IFN-and IL-17A release in T cells of the two lymphoid tissues from the two models, and these responses were significantly inhibited by the PDE4 inhibitor rolipram except for the release of IL-17A in the inguinal lymph node cells. The non-selective PDE inhibitor IBMX also attenuated IFN- and IL-17A release in these T cells to an extent similar to that of rolipram, indicating PDE4 is the major PDE isozymes in regulation of the Th1 and Th17 responses. Studies with the cAMP-elevating agents dbcAMP and forskolin and with PKA and Epac activators and inhibitors further suggested that the inhibitory effect of rolipram was mediated by activation of the cAMP/PKA axis. The PDE3 inhibitor cilostazol and PDE7 inhibitor BRL 50481 mostly had no effect on the Th1 and Th17 responses, whereas cilostazol showed synergistic effect to the rolipram inhibition. In conclusion, these findings indicate that PDE4 selective inhibitors can efficaciously alleviate inflammation in RA, yet PDE3 and PDE4 dual inhibitors may be more beneficial to RA patients. |
