Study on the Effects of Pentoxifylline and Triptoliden Immune Effector Cells

現今有許多的疾病都被證實是由體內產生過度的免疫反應所造成，例如移植體對宿主反應(GVHD)、氣喘(asthma)，或是自體免疫方面的疾病，例如：類風濕性關節炎(RA)、多發性硬化症(MS)、及紅斑性狼瘡(SLE)等等。目前市面上很多免疫抑制藥物可供治療使用，卻也伴隨著副作用的產生。配妥西菲林(pentoxifylline)和雷公藤內酯醇(triptolide)這兩種藥物在臨床上已經被使用在治療許多不同的疾病上，也顯示出具有某種程度上能調節免疫的功能，但是當中詳細的機制還沒有完全了解清楚。因此在我們的實驗當中，我們利用T細胞接受器基因轉殖小鼠的CD4 T細胞以及BALB/c小鼠的樹突細胞來研究配妥西菲林(pentoxifylline)和雷公藤內酯醇(triptolide)的免疫調節功能。實驗中我們針對藥物影響細胞分泌細胞激素的量、細胞表面上標誌分子的表現、以及細胞增生的情形來觀察。實驗結果發現，配妥西菲林(pentoxifylline)和雷公藤內酯醇(triptolide)兩種藥物都能有效的抑制CD4 T細胞活化，不管是在介白素2的分泌上或者是細胞增生情況都有顯著被抑制的情形。同時實驗也發現這兩種藥物並不會去誘發T細胞表現類似調節性T細胞的特性，例如產生Foxp3分子表現或者是增加分泌介白素10的分泌量。而在藥物處理樹突細胞的實驗結果，發現配妥西菲林(pentoxifylline)會促進樹突細胞表現CD86分子，並且會促進樹突細胞分泌介白素12。而雷公藤內酯醇(triptolide)並不會對樹突細胞表面上分子的表現量有影響，但是可以有效的抑制樹突細胞分泌介白素12。在本次實驗當中我們可以了解到，配妥西菲林(pentoxifylline)以及雷公藤內酯醇(triptolide)可以藉由抑制CD4 T細胞活化來抑制免疫反應，但是卻不會透過誘導調節性T細胞產生。然而，配妥西菲林(pentoxifylline)和雷公藤內酯醇(triptolide)藉由不同機轉調節樹突細胞的功能，也能夠說明這兩種藥物可以各自用在各自不同的疾病治療上。Many diseases are well known to be caused by excessive and unexpected immune responses such as graft-versus-host diseases (GVHD), asthma, and other severe autoimmune diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE). Up to the present time, many immunosuppressive drugs have been used to treat these diseases effectively to treat but with many side-effects. Pentoxifylline (PTX) and triptolide (TPT) are used in different kinds of diseases for a long time and show some immune regulatory functions, but the mechanisms are still not completely understood. In this study, we isolated CD4 T cells from DO11.10 mice and DCs from BALB/c mice. We treated these immune cells with PTX or TPT and analyzed their phenotype and function including cytokine secretion, cell surface markers expression, and cell proliferation. We found that both PTX and TPT inhibited CD4 T cells activation including IL-2 secretion and cell proliferation; however, they did not induce Foxp3 expression and IL-10 secretion, which are the important characteristics of regulatory T cells. On the other hand, we found that PTX induced the expression of CD86 on DCs and promoted IL-12 secretion, while TPT did not affect the surface marker expression on DCs and inhibited the IL-12 secretion. In the present study, we suggest that both PTX and TPT inhibit CD4 T cell activation, and their immunosuppressive functions might not mediate through regulatory T cell induction. In addition, these two drugs have different effects on dendritic cell functions, and these results show that they have the potential to be applied in different diseases.摘要 ------------------------------------------------------------------------------------------------ Ibstract --------------------------------------------------------------------------------------------IIontents ------------------------------------------------------------------------------------------IVhapter I Introduction --------------------------------------------------------------------------1entoxifylline ------------------------------------------------------------------------------------2riptolide -----------------------------------------------------------------------------------------3endritic cells -----------------------------------------------------------------------------------6D4 T cells -------------------------------------------------------------------------------------13egulatory T cells -----------------------------------------------------------------------------16tudy Aims -------------------------------------------------------------------------------------20hapter II Materials & Methods ------------------------------------------------------------21aterials nimals---------------------------------------------------------------------------------------22ell line---------------------------------------------------------------------------------------22ell culture reagents------------------------------------------------------------------------22rugs------------------------------------------------------------------------------------------24LISA assay ---------------------------------------------------------------------------------24low cytometry------------------------------------------------------------------------------25agnetic cell sorting-----------------------------------------------------------------------26ethodssolation of naïve CD4 T cells-------------------------------------------------------------26ell toxicity test-----------------------------------------------------------------------------27ulture of bone narrow-derived dendritic cells-----------------------------------------27rug treatment on CD4 T cells------------------------------------------------------------28rug treatment on bone marrow-derived dendritic cells-------------------------------29roliferation assay--------------------------------------------------------------------------30easurement of cytokine production----------------------------------------------------30low cytometry analysis-------------------------------------------------------------------31hapter III Results -----------------------------------------------------------------------------33entoxifyllinehe cell toxicity test of PTX---------------------------------------------------------------34TX can inhibit CD4 T cell proliferation and IL-2 secretion upon stimulation----34TX do not have the regulatory T cell induction function-----------------------------35TX can not affect the marker expression on BMDCs but selectively increase cytokine secretion---------------------------------------------------------------------------36nhibition of NF-κB prevent the IL-12p40 secretion changes induced by PTX----37riptolidehe cell toxicity test of TPT---------------------------------------------------------------38PT can inhibit CD4 T cell proliferation and IL-2 secretion upon stimulation-----38PT do not induce regulatory T cell generation----------------------------------------39PT do not affect the surface marker expression on BMDCs but selectively increase cytokine secretion----------------------------------------------------------------39nhibition of ERK and p38 pathways prevent the IL-12p40 secretion affected by TPT--------------------------------------------------------------------------------------------40hapter IV Discussions & Conclusion------------------------------------------------------42entoxifylline--------------------------------------------------------------------------------43riptolide-------------------------------------------------------------------------------------46onclusion-----------------------------------------------------------------------------------48igures ---------------------------------------------------------------------------------------------49eferences ----------------------------------------------------------------------------------------6