摘要
T细胞的活化与分化需要抗原提呈细胞(antigen presenting cells, APCs)的辅助。T细胞必须接受APCs提供的三种信号才能有效活化与分化:第一信号是APCs表面的MHC-抗原肽与T细胞表面的TCR结合决定T细胞识别抗原的特异性;第二信号是APCs表面的CD80/CD86与T细胞表面的CD28结合提供协同刺激信号保证抗原特异性T细胞能够充分扩增;第三信号是APCs分泌的细胞因子作用于T细胞决定抗原特异性T细胞的分化方向。树突状细胞(dendritice cell, DC)是目前发现的功能最强大的APCs,也是唯一能够激活初始T细胞的专职APCs。研究认为初始T细胞分化成具有不同功能的抗原特异性T细胞的过程受DC所调控,DC的性质和功能状态直接影响T细胞的活化和分化方向。调节DC的功能将有可能对T细胞介导的疾病产生不同的影响。
胆囊收缩素(cholecystokinin, CCK)是一种典型的脑肠肽,在体内存在多种活性形式,包括4、8、33、39和58肽等,其中硫酸化的八肽胆囊收缩素(cholecystokinin octapeptide, CCK-8)是最主要的活性形式。CCK-8通过细胞表面的CCK受体(CCK-receptor, CCKR)在中枢和外周神经系统发挥多种调节功能。我们先前的系列研究表明CCK-8通过其表达于效应细胞表面的受体发挥抗炎和免疫调节功能。CCK-8能够抑制LPS诱导巨噬细胞分泌促炎细胞因子和趋化因子,促进抗炎细胞因子IL-10的分泌;并能抑制活化的巨噬细胞表面CD80和CD86的表达,降低其抗原提呈时的协同刺激功能。此外,CCK-8对LPS诱导的B细胞表面协同刺激分子表达和细胞因子分泌有调节作用。我们的最新研究表明DC表面也表达CCKR,CCK-8通过与DC表面的CCKR作用调节MAPK-NF-κB信号通路,上调DC分泌IL-12。因此我们推测CCK-8可能会对DC细胞表型和细胞因子分泌发挥调节作用,进而调节T细胞活化与分化,并对T细胞介导的疾病产生影响。
类风湿性关节炎(rheumatoid arthritis, RA)是一种以慢性侵蚀性周围关节炎为主要表现的自身免疫病。虽然其确切发病机制尚不完全清楚,但多数学者认为RA是在遗传和环境因素作用下由T淋巴细胞介导的自身免疫病,CD4+T细胞的分化异常和功能紊乱是其发病的重要原因。传统认为RA是Th1细胞占优势的疾病。然而,清除或中和Th1型细胞因子IFN-γ或IL-12,不能预防或减轻疾病进程。随后的研究发现IL-17在RA发病中有重要作用,IL-17可刺激单核细胞、巨噬细胞和滑膜细胞产生促炎细胞因子促进炎症反应,软骨破坏及血管翳形成。动物实验证实缺失分泌IL-17的T细胞可以使RA的发病受到抑制。因此,目前众多学者认为Th17细胞及其产生的IL-17是类风湿性关节炎发病的关键因素,抑制Th17细胞反应可能有利于RA病情缓解。
基于上述理论和研究结果,我们推测CCK-8有可能通过调节DC表型和细胞因子分泌来调控CD4+T细胞的活化与分化,并进一步对RA的发病和转归产生影响。据此,本课题在体外细胞实验和疾病实验动物模型整体水平系统分析了CCK-8对DC功能的影响及其对CD4+T细胞活化与分化的调节作用,并进一步观察了CCK-8对RA实验动物模型-胶原诱导型小鼠关节炎(collagen-induced arthritis,CIA)发病和关节病变的影响。1 CCK-8体外作用DC对抗原特异性T细胞分化的影响
目的:DC活化过程中表达的协同刺激分子水平和分泌的细胞因子类型影响T细胞的活化和分化方向。调节DC成熟过程中的表面分子表达和细胞因子分泌将会对T细胞介导的免疫反应产生不同的影响。本部分研究主要采用体外细胞实验观察CCK-8对LPS诱导DC成熟过程中细胞表型和功能的影响,及其对抗原特异性CD4+T细胞增殖和分化的调节作用。
方法:骨髓细胞自DBA/1J小鼠股骨和胫骨骨髓中分离,在含有GM-CSF的完全培养基中培养6~7天,收集悬浮和疏松贴壁细胞,作为骨髓来源的DC(bone marrow-derived dendritic cell,BM-DC)使用。(1)将BM-DC分为五组: Medium组(只加培养基) ; LPS组(1μg/ml) ;LPS(1μg/ml)+CCK-8(10-6、10-8、10-10 mol/L)。将细胞按上述条件培养24h,收集细胞,分别同时加入PE-抗CD11c抗体和FITC-抗CD80、FITC-抗CD86或FITC-抗MHCII抗体,孵育30min,采用流式细胞仪进行检测。分析CD11c+细胞表面CD80、CD86和MHCII表达量的变化。(2)用CD11c+免疫磁珠分选BM-DC获得纯化的DC(purified BM-DC,pBM-DC),将pBM-DC分为五组: Medium组(只加培养基) ; LPS组(1μg/ml) ;LPS(1μg/ml)+CCK-8(10-6、10-8、10-10 mol/L)。将细胞按上述条件培养24h。①收集上清,ELISA法检测IL-23含量;②收集细胞,用50μg/ml丝裂霉素C(mitomycin-C, MMC) 37°C孵育1h后,与免疫磁珠分选的CII特异性CD4+T细胞共培养,采用MTS法测定T细胞增殖,采用ELISA法检测培养上清中IFN-γ、IL-4、IL-17和TGF-β含量。
结果:(1)未经LPS诱导的未成熟BM-DC表达较低水平的CD80、CD86和MHCII,经LPS诱导的成熟BM-DC CD80、CD86和MHCII表达显著增高,一定浓度的CCK-8与LPS同时作用则显著抑制了BM-DC CD80、CD86和MHCII表达,提示CCK-8可抑制LPS诱导BM-DC表型成熟。(2)未成熟pBM-DC分泌IL-23的能力非常低,低于试剂盒检测的下限。经LPS诱导的成熟pBM-DC分泌IL-23的水平明显升高,10-6和10-8 mol/L CCK-8处理则显著抑制LPS诱导的IL-23分泌(P<0.05),10-10 mol/L CCK-8处理作用效果不明显。(3) LPS诱导的成熟pBM-DC作为APC能显著刺激抗原特异性CD4+T细胞增殖,不同浓度CCK-8与LPS同时作用pBM-DC后则明显降低pBM-DC的刺激能力,CD4+T细胞的增殖活性下降(P<0.05)。(4)与未成熟pBM-DC相比,LPS诱导的成熟pBM-DC可刺激CD4+T细胞产生高水平IFN-γ和IL-17,一定浓度的CCK-8与LPS共同作用pBM-DC与LPS组相比显著提高IFN-γ的水平(P<0.05),降低IL-17的产生(P<0.05),不同浓度CCK-8与LPS同时作用pBM-DC对CD4+T细胞产生IL-4和TGF-β的水平无明显影响(P>0.05)。
我们先前的研究结果发现,LPS刺激DC成熟可显著促进DC分泌IL-6、IL-1β、IL-12和TNF-α;CCK-8处理则明显降低IL-6、IL-1β和TNF-α的产生,促进IL-12的产生。综合分析这些实验结果,提示CCK-8通过调节DC成熟过程中的细胞表型和细胞因子分泌对抗原特异性CD4+T细胞的增殖和分化发挥调节作用,通过上调IL-12的产生促进Th1细胞分化,下调IL-6和IL-23的产生抑制Th17细胞分化和扩增。2 CCK-8体内作用对DC功能及T细胞分化的影响
目的:第一部分体外细胞实验证实,CCK-8通过调节DC成熟过程中的细胞表型和细胞因子分泌对抗原特异性CD4+T细胞的增殖和分化发挥调节作用。本部分研究我们将在疾病实验动物模型整体水平观察CCK-8对DC和CD4+T细胞的调节作用。
方法:采用鸡CII加弗氏完全佐剂的乳化液免疫RA易感动物DBA/1J小鼠,建立CIA模型。小鼠随机分为四组:生理盐水对照组;CCK-8 1nmol组;CCK-8 5nmol组;CCK-8 10nmol组。各组小鼠于第二次增强免疫同时,腹腔注射以上药物,每天注射1次,连续注射7天。于第一次免疫后28天,取各组小鼠腹股沟引流淋巴结:(1)采用流式细胞双标术检测CD11c+细胞表面CD80、CD86和MHCII表达。(2)提取总RNA,采用荧光定量RT-PCR法检测IL-12p35、IL-12p40、IL-23p19、IL-6、T-bet(T-box expressed in T cells)、Gata-3(GATA binding protein 3)、RORγt(orphan nuclear receptor gammat)、Foxp3(foxhead/winged-helix box protein 3)、IFN-γ、IL-4、IL-17和TGF-βmRNA表达。(3)免疫磁珠分选CD4+T细胞,经CII刺激,MTS法检测细胞增殖,ELISA法检测培养上清中IFN-γ、IL-4、IL-17和TGF-β的含量。
结果:(1) 1nmol CCK-8处理对CIA小鼠腹股沟引流淋巴结DC表面CD80、CD86和MHCII表达无明显影响(P>0.05);5nmol和10nmol CCK-8处理可显著降低DC表面CD80、CD86和MHCII的表达量(P<0.05或P <0.01)。这一结果提示,一定浓度的CCK-8在实验动物整体水平对DC的表型成熟同样具有抑制作用。(2) 1nmol CCK-8处理对小鼠腹股沟引流淋巴结IL-12p35、IL-12p40、IL-6和IL-23p19 mRNA表达无明显影响(P >0.05); 5nmol CCK-8处理可显著提高IL-12p35和p40 mRNA表达(P <0.05)而抑制IL-6和IL-23p19 mRNA表达(P<0.01);10nmol CCK-8处理对IL-12p35和p40 mRNA表达无明显影响(P >0.05),但是可显著抑制IL-6和IL-23p19 mRNA表达(P<0.05或P<0.01)。IL-12、IL-6和IL-23主要由APC(主要包括DC和单核巨噬细胞)分泌,结合第一部分CCK-8体外作用对DC分泌细胞因子的影响,提示一定浓度CCK-8在实验动物整体水平对DC分泌的细胞因子同样具有调节作用。(3) 1nmol CCK-8处理对小鼠腹股沟引流淋巴结中T-bet、Gata-3、RORγt、Foxp3、IFN-γ、IL-4、IL-17和TGF-βmRNA表达均无明显影响(P>0.05)。5和10nmol CCK-8处理可显著提高T-bet、Foxp3、IFN-γ、IL-4和TGF-βmRNA表达(P<0.05或P<0.01),抑制RORγt、IL-17 mRNA表达(P<0.05或P<0.01),对Gata-3 mRNA表达无明显影响(P>0.05)。(4)对照组小鼠CD4+T细胞经CII刺激后体外明显增殖,并能产生较高水平的IL-17和较低水平的IFN-γ和TGF-β,而CCK-8处理组小鼠CD4+T细胞的增殖活性明显下降(P<0.05),并且显著抑制IL-17的分泌促进IFN-γ和TGF-β的产生(P<0.05),而对IL-4的产生无明显影响(P>0.05)。
以上结果表明:CCK-8腹腔注射CIA小鼠能够下调DC表面CD80、CD86和MHCII表达,并通过抑制IL-6和IL-23的产生,促进IL-12的产生,抑制CD4+T细胞向Th17细胞分化,促进其向Th1和Treg细胞分化。3 CCK-8对CIA发病和关节病变的影响
目的:目前研究表明Th17细胞通过分泌IL-17在RA发病中发挥关键作用。本课题前两部分体内外实验证实,CCK-8通过调节DC细胞表型和细胞因子分泌对抗原特异性CD4+T细胞的活化和分化发挥调节作用,尤其是抑制抗原特异性Th17细胞反应,提示CCK-8可能会延缓或防止RA发病。因此,本部分实验我们建立CIA模型,观察CCK-8对RA发病的影响。
方法:CIA模型的建立及分组处理同第二部分。于第二次增强免疫同时,隔日对小鼠发病率及关节肿胀程度进行观测。于第一次免疫后35天,(1)取小鼠后肢踝关节观察组织病理改变;(2)取小鼠后肢踝关节提取组织蛋白,检测各种炎性细胞因子和调节因子水平;(3)留取小鼠血清检测抗CII特异性抗体及其亚型水平。此外,分离CIA小鼠膝关节滑膜组织细胞,在CII刺激下,与不同浓度CCK-8共培养,检测培养上清中各种炎性细胞因子的含量。
结果:(1) 5和10nmol CCK-8于小鼠腹腔注射一周,能显著延迟CIA发病、降低发病率、减轻疾病严重程度,而1nmol CCK-8作用效果不明显。因5和10nmol CCK-8腹腔注射对CIA关节评分的影响无显著差异,因此后续实验均以5nmol剂量进行。(2)组织病理学观察结果显示:正常小鼠关节结构完整,无炎性细胞浸润,滑膜细胞排列整齐,软骨表面光滑,关节腔内未见渗出液;CIA对照组小鼠关节局部可见大量的炎细胞浸润,滑膜组织增生,关节软骨和骨组织破坏,关节腔内可见大量的炎性渗出液;CCK-8处理组小鼠关节局部炎症反应明显减轻仅显示轻度的滑膜组织增生和炎细胞浸润。组织病理评分结果显示CCK-8处理组组织病理评分显著低于对照组(P<0.05)。(3)关节局部组织蛋白检测发现,与对照组相比,CCK-8处理显著降低关节局部促炎细胞因子(IL-17, IL-23, IL-6和TNF-α)和趋化因子(MCP-1)水平(P<0.05);上调IFN-γ和TGF-β水平(P<0.05);对IL-4和IL-10的水平变化无明显影响(P>0.05)。(4)小鼠血清中抗CII特异性抗体检测结果显示,与对照组相比,CCK-8处理显著降低CIA小鼠血清中抗CII特异性抗体IgG的含量(P <0.05),特别是显著降低IgG2a的含量(P <0.05),而对IgG1的水平无明显影响(P >0.05)。(5) CCK-8体外作用CIA小鼠膝关节滑膜组织细胞后,与体内结果一致可显著抑制滑膜组织细胞分泌促炎细胞因子(IL-17, IL-23, IL-6和TNF-α)和趋化因子(MCP-1) (P <0.05),促进其分泌IFN-γ和TGF-β(P <0.05),对IL-4和IL-10的产生无明显影响(P >0.05)。
以上结果表明:CCK-8能有效抑制CIA发病,降低发病率、推迟发病时间、降低关节病理损伤程度。CCK-8发挥作用的机制主要与其抑制抗原特异性Th17细胞介导的自身免疫反应和炎症反应有关。
结论
本研究系统分析了CCK-8体内外作用对DC功能的影响及其对抗原特异性CD4+T细胞活化和分化的调节作用,在此基础上观察了CCK-8对CIA发病和关节病变的影响。得出以下结论:
1 CCK-8体外作用通过调节DC成熟过程中的细胞表型和细胞因子分泌对抗原特异性CD4+T细胞的增殖和分化发挥调节作用。通过上调IL-12的产生促进Th1细胞分化,下调IL-6和IL-23的产生抑制Th17细胞分化和扩增。
2 CCK-8体内作用CIA小鼠同样能够下调DC表面CD80、CD86和MHCII的表达,抑制IL-6和IL-23 p19 mRNA表达,促进IL-12 p35和p40 mRNA表达,抑制CD4+T细胞向Th17细胞分化,促进其向Th1和Treg细胞分化。
3 CCK-8能有效抑制CIA发病,降低发病率、推迟发病时间、降低关节病理损伤程度。
总之,CCK-8腹腔注射CIA小鼠可有效抑制发病,降低发病率、减轻关节病理损伤。CCK-8可通过多靶点发挥作用,其中最主要的途径是通过调节DC细胞表型和细胞因子分泌抑制抗原特异性Th17细胞介导的自身免疫反应和炎症反应。本研究为合理评价CCK-8对RA治疗的潜在价值提供了重要的动物实验和理论依据。
The activation and differentiation of antigen-specific T cells requires the help of antigen presenting cells (APCs). Only receiving three signals from APCs, T cells can be fully activated and differentiate into effector T cells. The first signal involves the presentation of antigen on the surface of an MHC class II molecule, which facilitates T cell recognition of the cognate antigen through the TCR. In order for antigen-specific T cells to become activated and this population to expand in number, a second siganl must be generated through the interaction of adhesion and costimulatory molecules present on the APCs, such as CD80 and CD86, with CD28 on the surface of T cells. The third signal is the secretion of cytokines by APCs, which directs the differentiation of activated antigen-specific lymphocyte into an effector T cell subtype. Dendritic cells (DCs) are the most potent professional APCs,only DCs can activate naive T cells and initiate adaptive immune responses. Rissoan considered that DC controled T cell proliferation and effector function by providing costimulation and establishing the cytokines environment at the time of T cell priming. Thus, regulation of DC costimulatory molecules expression and cytokines production may have different consequences on the T cell mediated autoimmune disorders.
Cholecystokinin (CCK) is a typical neuropeptide, and it is identified as several different size of the peptide including 4, 8, 33, 39, and 58 amino acid forms. The sulfated cholecystokinin octapeptide (CCK-8) is the biologically predominant active form of endogenous CCK, which exerts a variety of physiological actions in both the peripheral and central nervous system through activating cell surface CCK receptors (CCKR). A series of studies from our laboratory indicate that CCK-8 can act as an immunomodulator with predominant anti-inflammatory effects through the specific receptors. CCK-8 decreases pro-inflammatory cytokines like TNF-α, IL-6 and IL-1β, increases anti-inflammatory cytokines IL-10, down-regulate B7.1 and B7.2 expression and inhibits the costimulatory activity in lipopolysaccharide (LPS)-activated macrophages. In addition, CCK-8 regulates LPS-activated B cells costimulatory molecule expression and cytokines production. DC also expresses CCKR, and CCK-8 increases the production of IL-12 by modulating MAPK-NF-κB signaling pathways through its specific receptor on DC. So we presumed that CCK-8 might have different effect on T-cell mediated diseases via regulating dendritic cells.
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease primarily affecting the peripheral joints. Although the precise pathogenesis of RA remains unknown clearly, most of scientists believe that RA is a T cell-mediated autoimmune disease. Traditionally, RA has been identified as a Th1-mediated autoimmune disorder. However, neutralization or clearance of IFN-γor IL-12 can not prevent the disease. Soon after a subset of interleukin (IL)-17-producing T (Th17) cells distinct from Th1 and Th2 cells was identified by Laurie E Harrington research group and shown to play a crucial role in the induction of autoimmune tissue injury by up-regulating the release of IL-1β、TNF、IL-6、IL-8、G-CSF、PGE2 and VEGF by synoviocytes, macropahges and monocytes. Most of investigators consider that Th17 and IL-17 may play critical role in pathogenesis of RA.
Based on these theory and results, we presumed that CCK-8 might regualte antigen-specific CD4+T cell proliferation and differentiation by modulating the co-stimulatory molecular expression and cytokines production of DC, furtherly might have effect on the pathogenesis of RA. In the present study, we investigated the effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for CD4+T cell in vitro and in vivo. Furthermore, we used the CIA model to evaluate the potential effect of CCK-8 on the pathogenesis of RA.
1 Effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for antigen-specific CD4+T cell in vitro
Objective: Following maturation, DCs influence T cell proliferation and effector function by providing costimulation signals and establishing the cytokine environment at the time of T cell priming. The regulation of DC costimulatory molecules expression and cytokines production may have different effect on T cell mediated immune response. In this part, we examined the effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for antigen-specific CD4+T cell in vitro.
Methods: Bone marrow cells were flushed from the tibiae and femurs of DBA/1J mice. The cells were cultured in RPMI-1640 supplemented with heat-inactivated FCS, penicillin, streptomycin and GM-CSF. On day 6~7, the non-adherent cells and loosely adherent cells were harvested and used as BM-DC. (1) The cells were incubated in medium or LPS in the absence or presence of various concentration of CCK-8 for 24 hour, and then simultaneously stained with FITC-conjugated anti-mouse CD80, CD86 or MHC II, and PE-conjugated anti-mouse CD11c. Expression of the costimulatory molecules and MHC II on CD11c+ Cells were analysed by flow cytometry. (2) Purified BM-DC (pBM-DC) was isolated from BM-DC using anti-CD11c mAb immunomagnetic beads. Then the cells were cultured in medium or LPS in the absence or presence of various concentration of CCK-8 for 24 hour.①Cell culture supernatants were collected and the content of IL-23 was determined by ELISA kit.②The cultured pBM-DC were collected and treated with 50μg/ml MMC for 1 hour at 37°C, then added to purified CD4+T cells from CII-immunized mice in 96-well plates for 3 days. T-cell proliferation was measured by MTS and the cytokines profile was assayed by ELISA.
Results: (1) Bone marrow cells cultured for 6~7 days in the presence of GM-CSF as immature BM-DC expressed low levels of CD80, CD86 and MHC II. In contrast, BM-DC, matured following LPS treatment, express high levels of CD80, CD86 and MHC II, while CCK-8 treatment, concomitant with LPS, inhibited CD80, CD86 and MHC II expression.These results indicated that CCK-8 could inhibit the LPS-induced phenotypic maturation of DCs. (2) The immature CD11c+ DC purified from BM (pBM-DC) produced too little IL-23 to be detected. In contrast, pBM-DC, matured following LPS treatment, produced much higher level of IL-23, while CCK-8 treatment at 10-6 and 10-8 mol/L, concomitant with LPS, decreased the production of IL-23, but CCK-8 treatment at 10-10 mol/L had no this effect.(3) Purified BM-DC matured by LPS treatment as APC induced T cell proliferation, however, treatment with LPS and different concentration of CCK-8 reduced the stimulatory capacity.(4) T cells exposed to LPS-treated pBM-DC produced high levels of IFN-γand IL-17. If the DC were treated with LPS plus CCK-8, IFN-γproduction was significantly increased, while IL-17 production was significantly decreased without significant changes in IL-4 and TGF-β.
Our previous study showed that DC, matured following LPS treatment, produced high levels of IL-6,IL-1β,IL-12 and TNF-α, while CCK-8 treatment, concomitant with LPS, significantly decreased the production of IL-6,IL-1βand TNF-α, but increased the production of IL-12. Considering these results, CCK-8 can affect antigen-specific CD4+T cell proliferation and differentiation by regulating DC costimulatory molecules expression and cytokines production. CCK-8 might promote Th1 profile by increasing IL-12 and inhibit Th17 profile by decreasing IL-6 and IL-23.
2 Effect of CCK-8 on DC and T cells in vivo
Objective: We have demonstrated that CCK-8 can affect antigen-specific CD4+T cell proliferation and differentiation by regulating DC costimulatory molecules expression and cytokines production in vitro. In this part, we used collagen-induced arthritis (CIA) to examine the effect of CCK-8 on DC and T cells in vivo.
Methods: DBA/1J mice were used to induce CIA. Experimental mice were immunized subcutaneously at the base of the tail with emulsified chicken CII and Freund’s complete adjuvant on day 0 and boosted with the same agent on day 21. Treatment with different doses of CCK-8 (1nmol; 5nmol; 10nmol) commenced with the secondary immunization, and it was administered intraperitoneally on 7 consecutive days. The control groups of mice were injected saline alone. On day 28 after the first immunization, draining lymph node (DLN) were isolated from mice. (1) Using flow cytometry to analyze the expression of CD80, CD86 and MHC II on CD11c+ cells. (2) Total RNA was prepared from DLN, and IL-12p35, IL-12p40, IL-23p19, IL-6, T-bet, Gata-3, RORγt, Foxp3, IFN-γ, IL-4, IL-17 and TGF-βmRNA expression were measured by quantitative RT-PCR. (3) CD4+T cells were isolated from DLN cells by magnetic-antibody cell sorting. Then T-cell proliferations specific to CII were measured by MTS assay and cytokine levels in the culture supernatants were determined by ELISA.
Results: (1) The expression of CD80, CD86 and MHC II on CD11c+ cells were significantly decreased in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice (P<0.05 or P<0.01). These results indicated that certain concentration of CCK-8 could also inhibit the phenotype maturation of DC in vivo. (2) The expression of IL-12p35 and IL-12p40 mRNA were significantly increased in CCK-8 treated mice at dose of 5 nmol but not 1- and 10nmol, compared to control mice. In contrast, the expression of IL-6 and IL-23p19 mRNA were significantly decreased in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice. IL-12、IL-6 and IL-23 are mainly produced by APCs, and DCs are the most potent professional APCs. Considering the fact that we demonstrated the effect of CCK-8 on DC in vitro at part 1, we presumed that certain concentration of CCK-8 can also medulate the cytokine production of DC in vivo.(4) The expression of T-bet, Foxp3, IFN-γ, IL-4 and TGF-βmRNA were significantly increased, but the expression of RORγt and IL-17 mRNA were obviously decreased in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice. There is no difference of GATA-3 mRNA expression among 4 groups. (4) T cells from control mice showed marked CII-specific proliferation, and they produced high level of IL-17 and low levels of IFN-γand TGF-β. Differently, T cells from CCK-8 treated mice proliferated much less, produced low level of IL-17 and high levels of IFN-γand TGF-β; the amount of IL-4 was not significantly affected.
These results indicated that CCK-8 administration to CIA can down-regulate the expression of CD80, CD86 and MHCII on DC. By decreasing the production of IL-6, IL-23, and increasing the production of IL-12, CCK-8 inhibited Th17 profile and promoted Th1 and Treg profile. 3 Effect of CCK-8 on the pathogenesis of CIA
Objective: Many investigations report that Th17 cell plays a critical role in the pathogenesis of RA by producting IL-17. We have demonstrated that CCK-8 could regualte antigen-specific CD4+T cell proliferation and differentiation by inhibiting the stimulatory or co-stimulatory molecular expression on DC or altering the cytokines production, especially could suppress Th17 cell reaponse. These suggest that CCK-8 may be a potential therapeutic agent for RA. Therefore, we used the CIA model to evaluate the potential effect of CCK-8 on the pathogenesis of RA.
Methods: The induction of CIA and CCK-8 treatment were the same as part 2. After the second immunization, Mice were analyzed every other day and monitored for signs of arthritis onset using two clinical parameters, incidence and severity. On day 35 after the first immunization, (1) hind paws from mice were collected to examine histopathological changes; (2) protein extracts were isolated by homogenization of joints to determination of cytokines in joints; (3) serum samples were collected to determine the levels of anti-CII IgG, IgG1 and IgG2a Abs. Moreover, on day 35 after primary immunization, synoviums in knee joints were isolated from CIA mice, synovial cells were stimulated with inactivated CII in the absence or presence of different concentrations of CCK-8, and cytokine levels in supernatants were determined.
Results: (1) Delayed onset, lower incidence and decreased severity of CIA were observed in CCK-8 treated mice at doses of 5- and 10nmol but not 1nmol, compared to control mice, as assessed by clinical score. Because we observed few differences between the 5- and 10-nmol doses CCK-8 administrations, all further experiments were carried out using 5nmol dose. (2) The histologic examination of the joints showed even and clearly joint space and smooth articular cartilage in normal DBA/1J mice, while severe articular cartilage and bone erosion as well as massive inflammatory cell infiltration and obvious intra-articular exudates in the control mice. The CCK-8 treated mice exhibited only mild synoviocyte hyperplasia and inflammatory cell infiltration and the degree of arthritis was significantly reduced compared with the control ones. (3) CCK-8 treatment significantly reduced the production of pro-inflammatory cytokines (IL-17, IL-23, IL-6 and TNF-α) and chemokines MCP-1 in the joints of arthritic mice, while upregulated the production of IFN-γand TGF-β. No difference was observed in the levels of IL-4 and IL-10 in the joints between the two groups. (4) CII-specific IgG level, particularly that of IgG2a, in sera from CCK-8 treated mice was significantly lower than those from the control mice, whereas the levels of IgG1 were not different between the two groups. (5) CCK-8 inhibited the production of pro-inflammatory mediators (IL-17, IL-23, IL-6 and TNF-α) and chemokines MCP-1, while increased the production of IFN-γand TGF-βin culture supernatant of synovial cells isolated from mice with CIA on restimulation with CII in vitro.
These results suggested that CCK-8 could effectively inhibit the progression of CIA by reducing the incidence of arthritis, delaying the onset, improving symptoms and preventing occurrence of joint damage. These therapeutic effects are brouhgt about through inhibition of Th17 mediated autoimmune and inflammatory responses.
CONCLUSIONS
In present study, we firstly investigated the effect of CCK-8 on DC expression of costimulatory molecules and the stimulatory activity for CD4+T cell in vitro and in vivo. Furthermore, we used the CIA model to evaluate the potential effect of CCK-8 on the pathogenesis of RA. The conclusions were as follows:
1 CCK-8 could modulate antigen-specific CD4+T cell proliferation and differentiation by regulating DC costimulatory molecules expression and cytokines production in vitro. CCK-8 might promote Th1 polarization by increasing the production of IL-12 and inhibit Th17 polarization by decreasing the production of IL-6 and IL-23.
2 CCK-8 administered intraperitoneally to CIA could down-regulate the expression of CD80, CD86 and MHCII on DC. CCK-8 also could inhibit Th17 polarization and promoted Th1 and Treg polarization, by decreasing the production of IL-6, IL-23, and increasing the production of IL-12.
3 CCK-8 could effectively inhibit the progression of CIA by reducing the incidence of arthritis, delaying the onset and preventing occurrence of joint damage.
In summary, the present study indicates that treatment of CIA mice with CCK-8 has great benefit at the clinical and pathological levels. The effect of CCK-8 was exerted at multiple targets, mainly being associated with down-regulation of Th17-mediated autoimmune and inflammatory responses by modulating DC costimulatory molecules expression and cytokines production. These findings provide a preliminary rationale for studies assessing the efficacy of CCK-8 as a novel therapeutic approach to the treatment of RA.
引文
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