CCK-8调节小鼠腹腔巨噬细胞B7.1、B7.2表达及其协同刺激功能的信号转导机制研究
|
摘要
胆囊收缩素(cholecystokinin, CCK)是广泛分布于体内多个系统的神经肽,具有多种生物学功能,如调节摄食、焦虑、记忆等。我们在前一项国家自然科学基金项目(CCK抗炎作用的受体及信号转导机制研究No.30270529)的研究中发现,八肽CCK(cholecystokinin octapeptide, CCK-8)具有一定的免疫调节及抗炎作用,CCK-8作用于表达CCK受体的巨噬细胞,通过激活cAMP-PKA来抑制PKC活性,从而抑制NF-κB活性,抑制脂多糖(lipopolysaccharide, LPS)诱生促炎细胞因子TNF-α、IL-1β、IL-6表达,表明CCK-8可调节天然免疫应答。但CCK-8对适应性免疫应答有何影响尚无报道。已有报道人Jurkat T淋巴细胞表达CCK-2受体及其mRNA转录,CCK mRNA在人淋巴细胞中有转录;CCK对植物血凝素PHA(phytohemagglutinin)诱导的人外周血淋巴细胞增生具有抑制性调节作用,对丝裂原-诱导的小鼠淋巴细胞增生及淋巴细胞的移动性也具有抑制作用,而对其自发性增生和粘附力则具有促进作用。CCK及CCK受体在淋巴细胞中表达,提示CCK可能通过自分泌或旁分泌的形式调节淋巴细胞的一些功能,参与适应性免疫应答。
CD4~+T细胞在抗原刺激下,可分化为Th1和Th2两种亚群,它们各自产生特征性细胞因子,介导不同的生理过程。Th1细胞主要分泌IL-2、IFN-γ、TNF-β、IL-12等,介导细胞免疫、细胞毒性T细胞和巨噬细胞活化及迟发型超敏反应,刺激IgG2a抗体产生。Th2细胞主要分泌IL-4、IL-5、IL-6、IL-10、IL-13等Th2型细胞因子,介导体液免疫、B细胞和嗜酸性粒细胞活化以及IgG1和IgE抗体产生,抑制巨噬细胞活化和抗原呈递。Th1/Th2细胞平衡对维持机体的免疫稳态十分重要。近年来研究发现,Th1/Th2细胞失衡与多种疾病有关,参与感染性疾病(内毒素血症、SIRS、结核、寄生虫感染等)、过敏性疾病(哮喘等)、自身免疫性疾病(类风湿性关节炎、葡萄膜炎等)及肿瘤等的发生、发展和预后。我们的系列研究发现,CCK-8具有一定的抗内毒素休克及缓解类风湿性关节炎的作用,但该作用是否与调节Th1/Th2细胞失衡有关尚属未知。研究CCK对Th1/Th2细胞平衡的调节作用及其机制,将有助于阐明神经免疫网络的生理及病理作用,为Th1/Th2失衡相关性疾病的临床治疗提供实验依据,具有深远的理论意义和广阔的应用前景。
初始CD4~+T细胞被激活、增殖和分化为Th0细胞。Th0细胞具有分化为Th1或Th2细胞的潜能,局部微环境中存在的细胞因子种类是调控Th0细胞分化的关键因素。IL-12可促进Th0细胞分化为Th1细胞;IL-4可促进Th0细胞分化为Th2细胞。T细胞活化及活化后产生有效的免疫应答不仅需要由TCR介导的抗原特异性信号刺激,还需要协同刺激分子介导的抗原非特异性信号刺激。在参与T细胞激活的诸多协同刺激分子中,最重要的是T细胞表面CD28与APC表面相应配体B7.1(CD80)和B7.2(CD86)的结合。B7.1和B7.2刺激T细胞增生的功能一致,但诱导T细胞分化的方向不同,B7.2优先作为Th2细胞极化的协同刺激分子,促进Th2细胞极化;B7.1则优先作为Th1细胞的协同刺激分子,因此,阻断B7.1抑制了Th1细胞极化,促进Th2细胞的极化,而阻断B7.2则引起相反的结果。因此,本课题拟观察CCK-8对静息及活化的巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响,以探讨CCK-8调节Th1/Th2细胞平衡的作用是否与巨噬细胞协同刺激分子B7的表达有关。
另有研究证实:NF-κB可以正向调控协同刺激分子B7.1(CD80)和B7.2(CD86)的表达。结合我们前一项国家自然基金的研究结果:CCK受体作为一种G蛋白偶联受体,可介导CCK-8激活大鼠肺间质巨噬细胞内cAMP-PKA信号通路,进而抑制NF-κB活性,实现CCK-8抗炎作用。本课题将以小鼠腹腔巨噬细胞为研究对象,首先从体外和体内实验分别研究CCK-8对静息和LPS活化的巨噬细胞B7.1、B7.2表达和协同刺激活性的影响,并从受体、cAMP-PKA通路、转录因子NF-κB等多个层面,系统研究CCK-8对静息及LPS作用下小鼠腹腔巨噬细胞内CCKR―cAMP―PKA―NF-κB―B7.1和B7.2表达的这一信号转导通路的影响。深入探讨了CCK-8通过干预巨噬细胞协同刺激分子的表达进而间接调节Th1/Th2平衡的机制。
1 CCK-8对巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响
体外应用CCK-8(10~(-12) mol/L~10~(-6)mol/L)孵育小鼠腹腔巨噬细胞一定时间,采用流式细胞术分析细胞表面B7.1和B7.2含量的变化。用免疫磁珠从小鼠脾细胞分离CD4~+T细胞,按4:1数量比与腹腔巨噬细胞(预先用CCK-8和/或抗B7.1抗体、抗B7.2抗体、CCK1R拮抗剂CR1409、CCK2R拮抗剂CR2945孵育24h)共同体外培养,同时加入ConA 5mg/L,采用~3H掺入法测定CD4~+T细胞增殖程度以反映巨噬细胞的协同刺激活性。结果发现:CCK-8可上调静息巨噬细胞B7.1及B7.2的表达,并增强巨噬细胞的协同刺激活性。CCK-8的作用呈剂量依赖性,最大效应剂量是在10~(-7)mol/L～10~(-9)mol/L之间。抗B7.2抗体可减轻CCK-8增强巨噬细胞协同刺激活性的作用,CCK-8主要是通过上调巨噬细胞B7.2表达而增强其协同刺激活性。
体外应用CCK-8(10~(-12)～10~(-6))mol/L和(或)脂多糖(LPS)孵育小鼠腹腔巨噬细胞,采用流式细胞术分析细胞表面B7.1和B7.2含量的变化,用免疫磁珠从小鼠脾细胞分离CD4~+T细胞,按4:1数量比与腹腔巨噬细胞(预先用LPS、CCK-8和/或抗B7.1抗体、抗B7.2抗体孵育24h)共同体外培养,同时加入ConA 5mg/L,采用3H掺入法测定CD4~+T细胞增殖程度。发现:CCK-8可以下调LPS诱导的巨噬细胞的B7.1和B7.2表达,抑制LPS活化的巨噬细胞的协同刺激活性。CCK-8的作用呈剂量依赖性,最大效应剂量是在(10~(-7)～10~(-9) )mol/L之间。抗B7.1抗体和抗B7.2抗体可减轻LPS活化的腹腔巨噬细胞协同刺激活性。CCK-8通过下调LPS诱导的腹腔巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性。
2 CCK-8对巨噬细胞B7.1和B7.2表达的信号转导通路的研究
NF-κB可以诱导协同刺激分子B7.1(CD80)和B7.2(CD86)的表达。巨噬细胞是体内重要的炎性和免疫效应细胞,而脂多糖(lipopolysaccharide, LPS)是诱导巨噬细胞产生和释放炎症介质最强烈、最有效的激活物。研究表明,LPS可激活巨噬细胞cAMP-PKA信号通路并介导了一个抑制性信号,它可能抑制巨噬细胞表达一系列细胞因子,下调TNF-α和IL-1β的释放量。我们的研究证实CCK-8作用于表达CCK受体的巨噬细胞,通过激活cAMP-PKA、抑制PKC活性而抑制NF-κB活性,从而抑制脂多糖诱生促炎细胞因子TNF-α、IL-1β、IL-6。CCK-8是否通过CCKR-cAMP-PKA-NF-κB通路调节巨噬细胞B7.1和B7.2的表达,目前尚未见报道。据此,本部分实验将主要观察CCK-8调节静息巨噬细胞和LPS诱导的巨噬细胞B7.1和B7.2表达的细胞内信号转导通路。
本部分采用FACS检测巨噬细胞B7.1和B7.2的表达,[~3H]掺入法测定CD4~+T细胞增殖程度以反映巨噬细胞的协同刺激活性。ELISA法检测细胞内cAMP含量和PKA活性,EMSA检测小鼠腹腔巨噬细胞内NF-κB活性,Western blot检测小鼠腹腔巨噬细胞内IκB-α和p-IκB-α蛋白水平的变化。
结果:(一)、NF-κB参与CCK-8对巨噬细胞B7.1和B7.2表达的调节作用。
(1)NF-κB抑制剂Gliotoxin组剂量依赖性地抑制了LPS诱导的巨噬细胞B7.1和B7.2表达和LPS诱导的巨噬细胞协同刺激活性,表明NF-κB活性与LPS诱导的巨噬细胞B7.1、B7.2的表达有关,NF-κB参与调节巨噬细胞的协同刺激活性。
(2)在溶剂对照组,小鼠腹腔巨噬细胞核内几乎检测不到与特异性寡核苷酸探针结合的NF-κB。用LPS孵育小鼠腹腔巨噬细胞1h,细胞内NF-κB活性明显高于溶剂对照组(P<0.01),而用不同浓度的CCK-8(10~(-6) mol/L、10~(-8)mol/L、10~(-10)mol/L)处理后,则剂量依赖性地显著地抑制了LPS诱导的NF-κB活性增高,抑制率分别为56.58%、37.26%、15.59%(P<0.05),以LPS+CCK~(-8)(10~(-6)mol/L)组的抑制作用最为显著。不同浓度的CCK-8单独孵育小鼠腹腔巨噬细胞,10~(-6)mol/L和10~(-8)mol/L浓度的CCK-8使细胞NF-κB活性升高(P<0.05),而10~(-10)mol/L的CCK-8对细胞NF-κB活性无明显影响(P>0.05)。在反应体系中加入同源性寡核苷酸(含NF-κB结合位点)及异源性寡核苷酸(含AP-2结合位点),分别作为特异性和非特异性竞争物,以证实该实验中DNA-蛋白质结合的特异性。
(3)用LPS孵育小鼠腹腔巨噬细胞30min,与溶剂对照组比较,可使胞浆中IκB-α蛋白水平明显降低而p-IκB-α蛋白水平明显增加,p-IκB-α蛋白水平/IκB-α蛋白水平比值明显增加(P<0.05);在上述各组反应体系内加入CCK-8后,可使胞浆中IκB-α蛋白水平明显升高而p-IκB-α蛋白水平明显降低,p-IκB-α/IκB-α比值明显降低(P<0.05);不同浓度的CCK-8单独孵育小鼠腹腔巨噬细胞,10~(-6)mol/L和10~(-8)mol/L的CCK-8使胞浆中IκB-α蛋白水平降低,p-IκB-α蛋白水平增加,导致p-IκB-α蛋白水平/IκB-α蛋白水平比值明显增加(P<0.05);而10~(-10)mol/L的CCK-8对细胞内IκB-α和p-IκB-α蛋白水平均无明显影响(P>0.05)。上述结果表明:CCK-8诱导静息巨噬细胞IκB磷酸化及NF-κB活化,而抑制LPS诱导的巨噬细胞IκB磷酸化及NF-κB活化,参与巨噬细胞B7.1和B7.2的表达及其协同刺激活性。
(二)、CCK-8通过激活巨噬细胞内cAMP-PKA通路调节NF-κB活性,进而调节B7.1和B7.2表达
(1)用LPS孵育小鼠腹腔巨噬细胞15min或30min,细胞内cAMP含量及PKA活性升高,与对照组相比有显著性差异(P<0.01)。10~(-10)mol/L、10~(-8)mol/L和10~(-6)mol/LCCK-8预处理后加入LPS孵育15 min,CCK-8剂量依赖性地促进LPS引起的cAMP含量及PKA活性增加。不同浓度的CCK-8(10~(-10) mol/L、10~(-8) mol/L、10~(-6) mol/L)剂量依赖性地增加静息小鼠腹腔巨噬细胞内cAMP含量及PKA活性,表明CCK-8可以激活cAMP-PKA。
(2)在静息状态下,与对照组比较,Fsk(forskolin, cAMP激动剂毛喉素)组可以增加巨噬细胞内cAMP含量及PKA活性(P<0.05)。与CCK-8组比较,Fsk+CCK-8组cAMP含量及PKA活性升高(P<0.05)。在LPS活化的巨噬细胞内cAMP含量及PKA活性显著增高(P<0.01), Fsk,CCK-8分别与LPS合用均可使LPS诱导的巨噬细胞内cAMP含量及PKA活性进一步明显升高(P<0.05,P<0.01);但LPS+H89组,CCK-8+H89组,LPS+CCK-8+H89组cAMP含量分别与LPS组,CCK-8组,LPS+CCK-8组相比,cAMP含量均无明显改变,而PKA活性均下降(P<0.05),表明Fsk可激活cAMP,升高PKA活性,而H89对cAMP含量无明显影响,却抑制PKA活性。
(3)用不同刺激因素孵育小鼠腹腔巨噬细胞60min,与control组相比,LPS可以可以升高细胞内p-IκB-α蛋白表达及增强NF-κB活性(P<0.05)。单独应用CCK-8孵育小鼠腹腔巨噬细胞,在10~(-6) mol/L和10~(-8)mol/L时,胞浆中p-IκB-α蛋白表达及NF-κB活性升高(P<0.05)。Fsk单独孵育小鼠腹腔巨噬细胞,可以升高细胞内p-IκB-α蛋白表达及NF-κB活性(P<0.05)。与control组相比,LPS+Fsk组,CCK-8+Fsk组细胞内p-IκB-α蛋白表达及NF-κB活性均升高(P<0.05)。用LPS+CCK-8共同孵育巨噬细胞,细胞内p-IκB-α蛋白表达及NF-κB活性低于LPS组(P<0.05)。H89与LPS和CCK-8共同孵育,p-IκB-α蛋白表达及NF-κB活性高于LPS+CCK-8组,且有显著性差异(P<0.01),低于LPS组(P<0.05)。表明CCK-8通过进一步激活cAMP-PKA抑制LPS诱导的巨噬细胞NF-κB活化。
(4)cAMP激动剂Fsk与CCK-8的作用相似,对静息巨噬细胞B7.1的表达无明显影响,而升高B7.2的表达;剂量依赖性地抑制LPS诱导的小鼠腹腔巨噬细胞B7.1和B7.2的表达,以Fsk 10~(-6)mol/L的作用最强。PKA抑制剂H89可以剂量依赖性地抑制CCK-8诱导的巨噬细胞表面B7.2表达,对B7.1表达无影响(P>0.05)。H89可以逆转CCK-8对LPS活化的巨噬细胞的作用,即剂量依赖性地升高巨噬细胞B7.1和B7.2表达,以LPS+CCK-8+H89 10-8mol/L的作用最强,有显著性差异(P<0.05)。表明CCK-8通过激活腹腔巨噬细胞cAMP-PKA信号通路调节B7.1和B7.2表达。
以上结果表明:CCK-8通过激活cAMP-PKA-NF-κB信号通路上调B7.1和B7.2表达,增强巨噬细胞的协同刺激活性;并通过进一步激活LPS诱导的巨噬细胞cAMP-PKA-NF-κB信号通路,抑制IκB蛋白降解及NF-κB活性,下调B7.1和B7.2表达,抑制巨噬细胞的协同刺激活性。
(三)、CCK-8通过CCK受体调节cAMP―PKA―NF-κB―B7信号通路
(1)体外实验中,CCK1R拮抗剂CR1409及CCK2R拮抗剂CR2945均可逆转CCK-8作用下增强的巨噬细胞协同刺激活性和逆转CCK-8对LPS活化的巨噬细胞协同刺激活性的抑制作用,而且相同浓度的CR1409和CR2945相比较,CR1409的作用强于CR2945,表明CCK1R、CCK2R共同介导了CCK-8调节静息及LPS活化的巨噬细胞协同刺激活性的作用,并且CCK1R起主导作用。
(2)CCK-8显著抑制了LPS诱导的巨噬细胞中IκB磷酸化及NF-κB活性增高,对NF-κB活性的抑制率达37.26%(P<0.05)。CCK1R拮抗剂CR1409及CCK2R拮抗剂CR2945均可逆转CCK-8抑制的LPS诱导的巨噬细胞内IκB磷酸化及NF-κB活性增高,即剂量依赖性地升高巨噬细胞核内p-IκB-α蛋白表达及NF-κB活性(P<0.05),CR1409的作用强于CR2945。表明CCK1R、CCK2R共同介导了CCK-8调节LPS活化的巨噬细胞内IκB磷酸化及NF-κB活性,并且CCK1R起主导作用。
(3)两种拮抗剂均能不同程度地抑制CCK-8诱导的巨噬细胞内cAMP含量增高及PKA活性升高。CR1409和CR2945在10~(-9) mol·L~(-1)时对CCK-8的抑制作用不明显(P>0.05),当二者浓度达到10~(-7)mol·L~(-1)以上时,细胞内cAMP含量及PKA活性较LPS+CCK-8组明显降低(P<0.01),且有显著性差异(P<0.05)。两种拮抗剂对CCK-8的抑制效应均有剂量依赖性,CR1409的作用较CR2945强,说明两种CCK受体拮抗剂均能影响cAMP含量及PKA活性,并且CCK1R起主导作用。以上结果表明: CCK-8主要是通过上调静息巨噬细胞B7.2表达而增强其协同刺激活性,下调LPS诱导的巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性,该作用由CCK1R及CCK2R介导,其中CCK1R起主要介导作用。
综上,CCK-8通过激活静息巨噬细胞cAMP-PKA-NF-κB信号通路增强NF-κB活性,上调B7.1和B7.2表达,增强巨噬细胞的协同刺激活性;并通过进一步激活LPS诱导的巨噬细胞cAMP-PKA信号通路,抑制IκB蛋白降解及NF-κB活性,下调B7.1和B7.2表达,抑制巨噬细胞的协同刺激活性。CCK1R及CCK2R共同参与介导作用,其中CCK1R起主要介导作用。
3体内实验CCK-8对静息和LPS活化的巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响
第一部分的体外实验证实了CCK-8对静息及LPS诱导的巨噬细胞B7.1和B7.2表达及其协同刺激活性的影响,本部分将从整体实验进一步验证上述结果。实验将小鼠(n=4)分为:Control组、CCK-8组、CCK-8+CR1409/CR2945组、LPS组、LPS+CCK-8组、LPS+CCK-8+CR1409/ CR2945组,分别腹腔注射(i.p.)一定剂量的N.S.,LPS和/或CCK-8及CCK1R、CCK2R,12h后按收集并纯化腹腔巨噬细胞。采用流式细胞术分析细胞表面B7.1和B7.2含量的变化;用免疫磁珠从小鼠脾细胞分离CD4~+T细胞,按4:1数量比与上述各组腹腔巨噬细胞共同体外培养,同时加入ConA 5mg/L,采用~3H掺入法测定CD4~+T细胞增殖反映巨噬细胞的协同刺激活性。整体应用CCK-8作用小鼠腹腔巨噬细胞12h,与对照组相比,CCK-8可使小鼠腹腔巨噬细胞B7.2的表达增加,而对B7.1的表达则无影响。并且CCK-8还可使CD4~+T细胞[~3H]-TdR的掺入率升高,即促进其增殖,与对照组相比,有显著性差异(P<0.05)。小鼠腹腔注射CCK-8和/或CCKR拮抗剂,然后观察他们对于LPS i.p.(100μg/mouse)小鼠的作用,12h后收集小鼠腹腔巨噬细胞,流式细胞术检测B7的表达。与LPS组相比,CCK-8可以降低LPS诱导的小鼠腹腔巨噬细胞B7.1和B7.2的表达(P<0.05);并降低CD4~+T细胞的[~3H]-TdR掺入率,即抑制其增殖,说明CCK-8可降低小鼠腹腔巨噬细胞的协同刺激活性。整体应用CCKR拮抗剂可部分逆转CCK-8对巨噬细胞和LPS刺激的巨噬细胞的B7.1和B7.2表达及其协同刺激活性,以CCK1R的作用为著。综上,体内实验表明CCK-8通过上调小鼠腹腔巨噬细胞B7.2的表达而增强巨噬细胞的协同刺激活性;通过下调LPS诱导的腹腔巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性,该作用由CCK1R及CCK2R介导,其中CCK1R起主要介导作用。
结论
本研究分别从体内实验和体外实验入手,首次系统研究了CCK-8对静息及LPS活化的小鼠腹腔巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响,并从受体、cAMP-PKA、转录因子NF-κB等多个层次系统研究了CCK-8调节小鼠腹腔巨噬细胞B7.1和B7.2表达的信号转导机制,深入揭示了CCK-8发挥免疫调节作用的机制。
1、体内及体外实验证实,CCK-8主要通过上调小鼠腹腔巨噬细胞B7.2表达而增强其协同刺激活性;通过下调LPS诱导的巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性。CCK-8调节巨噬细胞的协同刺激活性与B7.1和B7.2表达密切相关。
2、CCK-8通过激活静息巨噬细胞cAMP-PKA-NF-κB信号通路增强NF-κB活性,上调B7.1和B7.2表达,增强巨噬细胞的协同刺激活性;并通过进一步激活LPS诱导的巨噬细胞cAMP-PKA-NF-κB信号通路,抑制IκB蛋白磷酸化及NF-κB活性,下调B7.1和B7.2表达,而抑制巨噬细胞的协同刺激活性。CCK1R及CCK2R共同参与介导作用,其中CCK1R起主要介导作用。
因此,CCK-8通过干预巨噬细胞协同刺激分子的表达影响Th细胞的功能,这可能是CCK-8发挥免疫调节作用的机制之一。
Cholecystokinin (CCK) being a kind of neuropeptide presents extensively in many systems of the body, with a broad spectrum of biological functions, such as regulating ingestion, anxiety, memory and so on. Our research have found that CCK-8 has some immunomodulatory and antiinflammatory functions in one of our National Nature Scinece Foundation (CCK receptor and anti-inflammatory intracellular signaling mechanisms activated by CCK-8, No. 30270529), CCK-8 inhibits activity of NF-κB by activiting cAMP-PKA and inhibiting PKC activity, and then inhibits the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, which shows that CCK-8 can modulate natural (or innate) immunity. But there are still no reports about how CCK-8 modulating acquired immunity. It has been reported that CCK2 receptor and mRNA express on human Jurkat T lymphocytes and CCK mRNA expresses on human T lymphocytes. CCK inhibits the proliferation of human peripheral blood lymphocytes induced by PHA (phytohemagglutinin) and so do the murine lymphocytes. Furthermore, it also inhibits the mobility of murine lymphocytes, but enhances its spontaneous hyperplasia and adhension. The existence and expression of specific CCK receptor on lymphocytes indicates CCK functioning as modulators of T lymphocytes by autocrine or paracrine secretion and affecting acquired immunity.
Upon antigenic stimulation, CD4~+Th cells can differentiate into two distinct types of effector cells, Th1 and Th2 subsets, each producing its own set of cytokines and mediating separate functions. Th1 cells secrete cytokines (IL-2, IFN-γ, and TNF-β, IL-12) critical for the generation of a cellular immune response, thereby activating CTL (cytotoxic T lymphocytes) and macrophages, inducing delayed-type hypersensitivity (DTH) responses, and stimulating IgG2a Ab production in mice. Th2 cells produce IL-4, IL-5, IL-13 and IL-10, which are critical for IgG1 and IgE Ab production and immunity against helminthic parasites, and inhibit macrophage, B cell and eosinophilic granulocyte activation and Ag presentation, thereby down-regulating the cellular immune responses. It is very important that Th1/Th2 balance maintain the immunostasis of the body. In the current research it has been found that Th1/Th2 balance relates with many diseases and participates in the initiation, development and prognosis of infective diseases (endotoxemia, SIRS, tuberculosis, parasitic infection et al.), hypersensitivity diseases (asthma et al.), the autoimmune diseases (RA, uveitis et al.). A series of studies in our laboratory has been focused on the effect of CCK-8 against endotoxin shock (ES) and releasing rheumatoid arthritis, but we still do not know whether this effect is related to the regulation of CCK-8 on Th1/Th2 balance? Focus on the modulating fuctions and its mechanism of CCK-8 on Th1/Th2 balance will conduce to interpreting the physiological and pathology functions of neuroimmune network, and presenting theory for clinical therapies of Th1/Th2 unbalance diseases.
Na?ve CD4~+T lymphocytes become activated and undergo clonal expasion, then differentiate into Th0 lymphocytes. Th0 cells have the potential ability of differentiating into Th1 or Th2 phenotype cells. Determining factors include the nature of the APC, the nature and amount of Ag, and the genetic background of the host, with the cytokine microenvironment as the dominant factor. Cytokine microenvironment is the dominant factor on regulating the differentiation of precursor Th cells (Th0). Increasing evidence demonstrates that IL-12 and, to a lesser extent IFN-γ, direct CD4~+T cells to differentiate into Th1. In contrast, IL-4 is necessary to induce the development of Th2. When both IL-4 and IL-12 are added to in vitro cultures, IL-4 dominates over IL-12, driving naive CD4~+T cells toward the Th2 phenotype. The activation of naive CD4~+T lymphocytes requires two signals delivered by APCs, leading to enhanced cytokine production and proliferation and produce effective immune response. The first signal, which confers specificity, is provided by the interaction of the antigenic peptide/MHC complex with the T cell receptor. The second signal is provided by costimulatory molecules expressed on APCs. Among the accessory molecules, the B7 family appears to be the most potent. The B7 costimulatory pathway involves at least two molecules, B7.1 (CD80) and B7.2 (CD86), both of which can interact with their counterreceptors, CD28 and CTLA-4, on T cells. B7.1 and B7.2 appear to be equivalent in the costimulation of T cell proliferation. However, B7.1 and B7.2 may differ in the signals provided for T cell differentiation, with B7.2 favoring Th2 development. As a general rule, B7.2 is induced earlier during the activation process and at higher levels than B7.1. Therefore, we will investigate the role of CCK-8 on macrophage B7.1 and B7.2 expression and costimulatory function for Ag-primed CD4~+T cells, and on the macrophage-induced regulation of Th1/Th2 differation in vitro and in vivo.
Some researchers have proved that the activation of Toll/NF-κB results in induction of cytokines and costimulatory molecules― B7.1(CD80) and B7.2(CD86). It is well known that CCK exerts a variety of physiological actions through its cell surface receptors, which have been pharmacologically classified into two subtypes CCK1R and CCK2R according to their affinity to the peptide agonist sulfated carboxyl-terminal octapeptide (sCCK-8) and gastrin. CCK receptors belong to G protein-coupled receptor (GPCR) superfamily and distribute extensively in mammalian body. Our current research suggested that CCK-8 inhibited activity of NF-κB by activiting cAMP-PKA and inhibiting PKC activity through its receptors in rat PIMs. To elucidate the intracellular signaling mechanism of CCK-8 regulating macrophage B7.1 and B7.2 expression, we performed a series of studies on mouse peritoneal macrophages, from the effect of CCK-8 on cAMP-PKA pathway, and transcription factors NF-κB which involved in regulating B7.1 and B7.2 expression, and the costimulatory activity on macrophages induced by LPS, and on the macrophage-induced regulation of Th1/Th2 differation in vitro and in vivo.
1 Effects of CCK-8 on B7.1 and B7.2 expression and costimulatory activity of macrophage
Aim: To investigate in vitro effects of CCK-8 on the expressions of B7.1 and B7.2 and on the costimulatory activity for T lymphocytes in unstimulated and LPS-activated macrophages. Methods: Mouse peritoneal macrophages were isolated and incubated with CCK-8(10~(-12) mol/L ~10~(-6) mol/L), or with LPS and/or CCK-8(10~(-12)～10~(-6))mol/L for indicated times. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry. CD4~+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, or with LPS, CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, for 24h. ConA was added into the culture medium to stimulate CD4~+T cell proliferation. The proliferation was determined by measuring [~3H]-TdR incorporation in aβ-scintillation counter. Results: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations of 10~(-7) mol/L to 10~(-9) mol/L. Anti-B7.2 antibody, but not anti-B7.1 antibody, reduced the modulatory role of CCK-8 on costimulatory activity. LPS-induced B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were inhibited by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations of 10~(-7) mol/L to 10~(-9) mol/L. Anti-B7.1 antibody and anti-B7.2 antibody inhibited the modulatory role of LPS on costimulatory activity. Conclusions: CCK-8 enhanced macrophage costimulatory activity by upregulating B7.2 expression, and inhibited LPS-induced macrophage costimulatory activity by down-regulating B7.1 and B7.2 expressions.
2 CCK-8 regulated macrophage B7.1 and B7.2 expression by cAMP-PKA-NF-κB signaling pathway
Documents reported that NF-κB induces the expression of costimulatory molecule, B7.1 (CD80) and B7.2 (CD86). Macrophages are the most important inflammatory and immune effector cells, while LPS is the most effective and intensive activator inducing macrophages to produce and release mediators of inflammation. Some studies proved that cAMP-PKA pathway and an inhibitory signaling are involved in activation of macrophages by LPS. This signaling can down-regulate cytokine production of macrophages, such as TNF-αand IL-1β. Our current studies demonstrated that CCK-8 inhibited LPS-induced NF-κB binding activity via activation of cAMP-PKA signaling pathway and inhibition of PKC activity, and then inhibited the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6. However, there are still no reports about whether CCK-8 regulates B7.1 and B7.2 expression through CCKR-cAMP-PKA-NF-κB signaling pathway? Thus, we will focus on the signaling pathway relating to the B7.1 and B7.2 expression in resting and LPS-inducecd macrophage.
Methods:①Mouse peritoneal macrophages were isolated and incubated with LPS and NF-κB inhibitor Gliotoxin, or with CCK-8 at different concentration (10~(-12)mol·L~(-1)~10-6mol·L~(-1)), Fsk and/or H89, or with CCK receptor antagonists (10~(-9) mol·L~(-1)~10~(-5)mol·L~(-1)) for 10 min prior to addition of LPS (5mg·L~(-1)) and CCK-8 (10-8mol·L~(-1)) for indicated time. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry (FACS). CD4~+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages, which were pretreated with the superior reagents. ConA was added into the culture medium to stimulate CD4~+T cell proliferation. The proliferation was determined by measuring [~3H]-TdR incorporation in aβ-scintillation counter representing the assay of macrophage costimulatory activity.②Mouse peritoneal macrophages were isolated and stimulated with LPS in the absence or presence of CCK-8 and NF-κB binding activity was analyzed by electrophoretic mobility shift assay (EMSA) 1h after stimulation. The protein levels of IκB-αand p-IκB-αin the cytoplasma were detected by Western blot 30 min after stimulation. Data were presented as x±s and analyzed by one way ANOVA and least significant difference test using SPSS 11.5 statistical program. A level of P<0.05 was considered statistically significant.
Results: (1). Involvememt of NF-κB in CCK-8 regulation of B7.1 and B7.2 expression in mouse macrophages
①Gliotoxin, a NF-κB inhibitor, inhibited B7.1 and B7.2 expression and costimulatory activity in LPS-activated macrophages in a dose-dependant manner, which shows that the NF-κB binding activity was related to the B7.1 and B7.2 expression in macrophage, and NF-κB was involved in regulating the costimulation of macrophage.②The NF-κB binding activity was significantly higher in mouse peritoneal macrophages stimulated with 5mg/L LPS for 1h in comparison with unstimulated cells (P<0.01), and additional treatment with CCK-8 (10~(-6) mol/L, 10~(-8)mol/L, 10~(-10)mol/L) markedly reduced the binding activity in a dose-dependent manner, the inhibition ratio was 56.58%、37.26%、15.59% respectively (P<0.05 or P<0.01). CCK-8 alone markedly increased the NF-κB binding activity at the concentrations of 10~(-6) mol/L and 10~(-8) mol/L (P<0.05), while CCK-8 had no effect on it at the concentrations of 10~(-10) mol/L (P>0.05). The binding specificity was confirmed by using homologous (NF-κB) and nonhomologous (AP-2) oligonucleotides as competitors.③The IκB-αprotein level in mouse peritoneal macrophages was markedly decreased while p-IκB-αprotein level increased 30 min after incubation with 5mg/L LPS (P<0.01). When added with different concentrations of CCK-8 (10~(-10) mol/L~10~(-6) mol/L) in macrophages stimulated by LPS, IκB-αprotein level was increased obviously and p-IκB-αprotein level was decreased (P<0.05), leading to decrease the ratio of p-IκB-αprotein level/IκB-αprotein level in a dose-dependent manner (P<0.05). CCK-8 alone increased p-IκB-αprotein level and decreased IκB-αprotein level at the concentrations of 10~(-6)mol/L and 10~(-8)mol/L, then leading to increase the ratio of p-IκB-α/IκB-αprotein level (P<0.05), but with no effect on the IκB-αand p-IκB-αprotein level at the concentrations of 10~(-10)mol/L (P>0.05). These results showed that NF-κB had correlated with the macrophage B7.1 and B7.2 expression and costimulation, CCK-8 induced IκB-αphosphorylation and enhanced NF-κB activity in unstimulated macrophage, and at the same time, inhibited NF-κB activity and IκB-αdegradation in LPS-activated mouse peritoneal macrophage, which might be the upstream mechanism of the regulative effect of CCK-8 on B7.1 and B7.2 expressions.
(2). CCK-8 regulated NF-κB binding activity and B7.1 and B7.2 expression on mouse macrophages through cAMP-PKA signaling pathway
①Stimulating mouse peritoneal macrophages with 5mg·L~(-1) LPS resulted in an obvious increase in cAMP content and PKA activity (P<0.01). CCK-8 didn’t affect cAMP content and PKA activity at low concentration (10~(-10)mol·L~(-1)) (P>0.05), but led to a significant increase of them at high concentration (10~(-8)mol·L~(-1)~10~(-6)mol·L~(-1)) (P<0.05) in LPS-activated macrophage when compared with that of LPS group, suggesting that CCK-8 affect cAMP content and PKA activity in a dose-dependent manner. CCK-8 alone (10~(-10)mol·L~(-1)、10~(-8)mol·L~(-1) and 10~(-6)mol·L~(-1)) increased cAMP content and PKA activity in a dose-dependent manner in unstimulated macrophages. These results demonstrated that CCK-8 activated cAMP-PKA signaling pathway at high concentration, and had a synergistical effect with LPS at high concentration on cAMP content and PKA activity in mouse peritoneal macrophages, which might be one of the immunomodulatory molecular mechanisms activated by CCK-8.②Similar to CCK-8, Fsk could increase cAMP content and PKA activity in unstimulated macrophages when compared with control group (P<0.05). Both CCK-8 and Fsk could markedly increase cAMP content and PKA activity compared with CCK-8 group (P<0.05). Stimulating mouse peritoneal macrophages with 5mg·L~(-1) LPS resulted in an obvious increase in cAMP content and PKA activity (P<0.01), and with additional CCK-8 or Fsk, also increase cAMP content and PKA activity (P<0.05, P<0.01). However, there was no difference in cAMP content (P>0.05), and the PKA activity was decreased (P<0.05) when LPS+H89 group, CCK-8+H89 group and LPS+CCK-8+H89 group compared respectively with LPS group, CCK-8 group, LPS+CCK-8 group. These results showed that Fsk activated cAMP and increased PKA activity, while H89 had no influence on cAMP content and inhibited PKA activity.③The NF-κB binding activity and p-IκB-αprotein level were significantly higher in mouse peritoneal macrophages stimulated with 5mg·L~(-1) LPS in comparison with unstimulated cells (P<0.01), of which were too little to be detected, and additional treatment with CCK-8 or Fsk markedly reduce the binding activity and p-IκB-αprotein level (P<0.05). But CCK-8 alone increased the NF-κB binding activity and p-IκB-αprotein level at the concention of 10~(-6) mol/L and 10-8mol/L (P<0.05). Fsk alone could increase the NF-κB binding activity and p-IκB-αprotein level (P<0.05) which was similar to CCK-8 alone group. Compared with control group, LPS+Fsk group and CCK-8+Fsk group also increase the NF-κB binding activity and p-IκB-αprotein level (P<0.05). When CCK-8 incubated with LPS, the NF-κB binding activity and p-IκB-αprotein level were lower than LPS group (P<0.05). H89, a PKA inhibitor, co-incubating with CCK-8 and LPS, resulted in obvious increase of NF-κB binding activity and p-IκB-αprotein level (P<0.01) when compared with LPS+CCK-8 group. These results showed that CCK-8 could further activated cAMP-PKA and inhibited the NF-κB binding activity in LPS-induced macrophages.④Fsk (forskolin, a cAMP-inducing agent) had similar effects of CCK-8 with the maximal effect at the dose of 10~(-6)mol/L, which increasing B7.2 expression but having no effect on B7.1 expression in resting macrophages, and inhibiting B7.1 and B7.2 expression in LPS-induced macrophages. In compared with CCK-8 alone group, H89 (a PKA inhibitor) inhibited the B7.2 expression in macrophages activated by CCK-8 in a dose-dependent manner (P<0.05), on the other hand, it could reverse the effects of CCK-8, enhancing macrophage B7.1 and B7.2 expression in a dose-dependent manner (P<0.05), and had the highest effect at the dose of 10-8mol/L on LPS-activated macrophages (P<0.05). The results showed that CCK-8 modulated B7.1 and B7.2 expression through cAMP-PKA signaling pathway. All in all, the results of this part revealed that CCK-8 increased B7.1 and B7.2 expression and costimulation by increasing NF-κB binding activity and IκB-αdegradation in unstimulated macrophages and activating cAMP-PKA pathway. However, CCK-8 down-regulated B7.1 and B7.2 expression and costimulation by inhibiting NF-κB binding activity and decreasing IκB-αdegradation in macrophages stimulated by LPS and cAMP-PKA signaling pathway being involved in the regulation of NF-κB binding activity by CCK-8 at least in part, which might be one of immunomodulatory mechanisms activated by CCK-8.
(3). Effects of CCK-8 were mediated through CCK receptors
①CCK-8 increased resting macrophage costimulatory activity and inhibited that of LPS-induced macrophage by mainly up-regulating B7.2 expression and down-regulating B7.1 and B7.2 expression respectively, which was mediated by CCK1R and CCK2R. However, CCK1R and CCK2R reversed the effects of CCK-8. CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.②CR1409 and CR2945 could increase NF-κB binding activity and IκB-αdegradation in dose-dependent manner, which reversed the effects of CCK-8 inhibiting NF-κB activity and IκB-αdegradation in LPS-induced mouse peritoneal macrophage. The effect of CR2945 was a little lighter than CR1409. The results showed that both CCK1R and CCK2R were involved in CCK-8 regulating NF-κB activity in LPS-induced macrophages, and CCK1R might be the major receptor responsible for the modulation of CCK-8.③The effect of CCK-8 on cAMP content was abrogated to different degree by CR1409 and CR2945 accompanying with the increase of their concentration. CR1409 and CR2945 did not affect the CCK-8-resulted increase of cAMP content and PKA activity at low concentration (10~(-9) mol·L~(-1)), but decreased them significantly when the concentration reached to 10~(-7) mol·L~(-1)～10~(-5) mol·L~(-1) compared with that of LPS+CCK-8 group (P<0.05), but the effect of CR1409 was more powerful than CR2945 (P<0.05). The inhibitory effects of CR1409 and CR2945 on the CCK-8-resulted increase of cAMP content and PKA activity were dose-dependent. The effects of CCK-8 on cAMP content and PKA activities were attenuated by CR1409 and CR2945 significantly at 10~(-5) mol·L~(-1) compare with that of CCK+LPS group (P<0.05 or P<0.01) and the order of potency of different antagonist was as follows: CR1409>CR2945. The results showed that both CCK1R and CCK2R were involved in CCK-8 regulating cAMP content and PKA activities in LPS-induced macrophages, and CCK1R might be the major receptor responsible for the modulation of CCK-8.
Conclusions: All the upper results suggested that CCK-8 enhanced B7.1 and B7.2 expression and costimulation by increasing NF-κB binding activity and IκB-αdegradation in unstimulated macrophages, then leading to activate cAMP-PKA signaling pathway, which were mediated through CCK receptors. However, CCK-8 decreased B7.1 and B7.2 expression and costimulation by inhibiting NF-κB binding activity and IκB-αdegradation in LPS-induced macrophages, then leading to further activate cAMP-PKA signaling pathway, , which were mediated through CCK receptors, CCK1R and CCK2R, but CCK1R might play a main role in this process.
3 CCK-8 regulated macrophage B7.1/B7.2 expression and costimulatory function in vivo
Aim: To investigate in vivo effects of CCK-8 on the expressions of B7.1 and B7.2 and the costimulatory activity for T lymphocytes in unstimulated and LPS-activated macrophages. Methods: Several BALB/c mice were randomly assigned to 8 groups (n=4) injected i.p. with N.S. alone, or with LPS (100μg/mouse), in the presence or absence of CCK-8 (5 nmol/mouse) and CR1409 (100μg/mouse), CR2945 (100μg/mouse). After 12h, macrophages were purified from the peritoneal exudate as indicated above. The B7.1/B7.2 expression on purified macrophages was analyzed by flow cytometry and, alternatively, purified macrophages were assayed for macrophage costimulatory activity as described above. ConA was added into the culture medium to stimulate CD4~+T cell proliferation. The proliferation was determined by measuring [~3H]-TdR incorporation in aβ-scintillation counter.
Results and Conclusions: The in vivo administration of CCK-8 resulted in increase of B7.2 expression, but without any influence on B7.1 expression. Peritoneal macrophages harvested from CCK-8-injected mice also exhibited increased costimulatory activity for CD4~+T cells. We concluded that, similar to the in vitro experiments, the in vivo administration of CCK-8 stimulated B7.2 expression and induced the costimulatory activity of peritoneal macrophages. Second, we assessed the in vivo effects of CCK-8 and CCKR antagonists (CR1409 and CR2945) in LPS-injected mice. BALB/c mice were injected i.p. with LPS (100μg/mouse), with or without CCK-8 (5nmol/mouse), CR1409 (100μg/mouse), CR2945 (100μg/mouse). Macrophages harvested 12h later were analyzed in terms of B7.1/B7.2 expression and costimulatory activity. In vivo CCK-8 administration reduced both B7.1 and B7.2 expression. Also, CCK-8 completely abolished the costimulatory activity. These effects are consistent with the in vitro effects of CCK-8 on LPS-stimulated macrophages. In vivo modulation of CCK-8 on costimulation was mediated by CCK receptors and CCK1R might be the major receptor, which was in concordance with the in vitro results.
CONCLUSIONS
In the present study, we first systematically investigated the modulatory effects of CCK-8 on B7.1 and B7.2 expression and the costimulatory activity in resting and LPS-activated mouse peritoneal macrophages, in vitro and in vivo, at multi-levels from receptors to cAMP-PKA signaling pathway and transcription factors. Then we first studied the intracellular signaling mechanisms of CCK-8 in the regulation of B7.1 and B7.2 expression and its correlation with the costimulatory function of macrophages.
1 The present studies for the first time demonstrated that, in unstimulated macrophages, CCK-8 up-regulated B7.1 and B7.2 expression, but mainly B7.2 expression at protein level in vitro and in vivo, and induced the costimulatory activity. In constrast, in LPS-activated macrophages, CCK-8 down-regulated the expression of both B7.1 and B7.2 expressions, and abolished the costimulatory activity. The effects of CCK-8 on the costimulatory activity for T cells correlated closely with the expression of B7.1/B7.2, and might be mediated through the modulation of B7 expression.
2 CCK-8 up-regulated B7.1 and B7.2 expression and increased costimulatory activity by activating cAMP-PKA-NF-κB signaling pathway in macrophages. CCK-8 down-regulated B7.1 and B7.2 expression and inhibited its costimulatory activity in LPS-activated macrophages by activating cAMP-PKA signaling pathway, and inhibiting IκB-αphosphorylation and the NF-κB binding activity through CCK receptors, and CCK1R might play a main role in this process.
In conclusion, this study firstly demonstrated that CCK-8 regulated the function of macrophages through the modulation of B7.1 and B7.2 expression, which might be one of the mechanisms of CCK-8 on regulating immunomodulatory functions.
CD4~+T细胞在抗原刺激下,可分化为Th1和Th2两种亚群,它们各自产生特征性细胞因子,介导不同的生理过程。Th1细胞主要分泌IL-2、IFN-γ、TNF-β、IL-12等,介导细胞免疫、细胞毒性T细胞和巨噬细胞活化及迟发型超敏反应,刺激IgG2a抗体产生。Th2细胞主要分泌IL-4、IL-5、IL-6、IL-10、IL-13等Th2型细胞因子,介导体液免疫、B细胞和嗜酸性粒细胞活化以及IgG1和IgE抗体产生,抑制巨噬细胞活化和抗原呈递。Th1/Th2细胞平衡对维持机体的免疫稳态十分重要。近年来研究发现,Th1/Th2细胞失衡与多种疾病有关,参与感染性疾病(内毒素血症、SIRS、结核、寄生虫感染等)、过敏性疾病(哮喘等)、自身免疫性疾病(类风湿性关节炎、葡萄膜炎等)及肿瘤等的发生、发展和预后。我们的系列研究发现,CCK-8具有一定的抗内毒素休克及缓解类风湿性关节炎的作用,但该作用是否与调节Th1/Th2细胞失衡有关尚属未知。研究CCK对Th1/Th2细胞平衡的调节作用及其机制,将有助于阐明神经免疫网络的生理及病理作用,为Th1/Th2失衡相关性疾病的临床治疗提供实验依据,具有深远的理论意义和广阔的应用前景。
初始CD4~+T细胞被激活、增殖和分化为Th0细胞。Th0细胞具有分化为Th1或Th2细胞的潜能,局部微环境中存在的细胞因子种类是调控Th0细胞分化的关键因素。IL-12可促进Th0细胞分化为Th1细胞;IL-4可促进Th0细胞分化为Th2细胞。T细胞活化及活化后产生有效的免疫应答不仅需要由TCR介导的抗原特异性信号刺激,还需要协同刺激分子介导的抗原非特异性信号刺激。在参与T细胞激活的诸多协同刺激分子中,最重要的是T细胞表面CD28与APC表面相应配体B7.1(CD80)和B7.2(CD86)的结合。B7.1和B7.2刺激T细胞增生的功能一致,但诱导T细胞分化的方向不同,B7.2优先作为Th2细胞极化的协同刺激分子,促进Th2细胞极化;B7.1则优先作为Th1细胞的协同刺激分子,因此,阻断B7.1抑制了Th1细胞极化,促进Th2细胞的极化,而阻断B7.2则引起相反的结果。因此,本课题拟观察CCK-8对静息及活化的巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响,以探讨CCK-8调节Th1/Th2细胞平衡的作用是否与巨噬细胞协同刺激分子B7的表达有关。
另有研究证实:NF-κB可以正向调控协同刺激分子B7.1(CD80)和B7.2(CD86)的表达。结合我们前一项国家自然基金的研究结果:CCK受体作为一种G蛋白偶联受体,可介导CCK-8激活大鼠肺间质巨噬细胞内cAMP-PKA信号通路,进而抑制NF-κB活性,实现CCK-8抗炎作用。本课题将以小鼠腹腔巨噬细胞为研究对象,首先从体外和体内实验分别研究CCK-8对静息和LPS活化的巨噬细胞B7.1、B7.2表达和协同刺激活性的影响,并从受体、cAMP-PKA通路、转录因子NF-κB等多个层面,系统研究CCK-8对静息及LPS作用下小鼠腹腔巨噬细胞内CCKR―cAMP―PKA―NF-κB―B7.1和B7.2表达的这一信号转导通路的影响。深入探讨了CCK-8通过干预巨噬细胞协同刺激分子的表达进而间接调节Th1/Th2平衡的机制。
1 CCK-8对巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响
体外应用CCK-8(10~(-12) mol/L~10~(-6)mol/L)孵育小鼠腹腔巨噬细胞一定时间,采用流式细胞术分析细胞表面B7.1和B7.2含量的变化。用免疫磁珠从小鼠脾细胞分离CD4~+T细胞,按4:1数量比与腹腔巨噬细胞(预先用CCK-8和/或抗B7.1抗体、抗B7.2抗体、CCK1R拮抗剂CR1409、CCK2R拮抗剂CR2945孵育24h)共同体外培养,同时加入ConA 5mg/L,采用~3H掺入法测定CD4~+T细胞增殖程度以反映巨噬细胞的协同刺激活性。结果发现:CCK-8可上调静息巨噬细胞B7.1及B7.2的表达,并增强巨噬细胞的协同刺激活性。CCK-8的作用呈剂量依赖性,最大效应剂量是在10~(-7)mol/L～10~(-9)mol/L之间。抗B7.2抗体可减轻CCK-8增强巨噬细胞协同刺激活性的作用,CCK-8主要是通过上调巨噬细胞B7.2表达而增强其协同刺激活性。
体外应用CCK-8(10~(-12)～10~(-6))mol/L和(或)脂多糖(LPS)孵育小鼠腹腔巨噬细胞,采用流式细胞术分析细胞表面B7.1和B7.2含量的变化,用免疫磁珠从小鼠脾细胞分离CD4~+T细胞,按4:1数量比与腹腔巨噬细胞(预先用LPS、CCK-8和/或抗B7.1抗体、抗B7.2抗体孵育24h)共同体外培养,同时加入ConA 5mg/L,采用3H掺入法测定CD4~+T细胞增殖程度。发现:CCK-8可以下调LPS诱导的巨噬细胞的B7.1和B7.2表达,抑制LPS活化的巨噬细胞的协同刺激活性。CCK-8的作用呈剂量依赖性,最大效应剂量是在(10~(-7)～10~(-9) )mol/L之间。抗B7.1抗体和抗B7.2抗体可减轻LPS活化的腹腔巨噬细胞协同刺激活性。CCK-8通过下调LPS诱导的腹腔巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性。
2 CCK-8对巨噬细胞B7.1和B7.2表达的信号转导通路的研究
NF-κB可以诱导协同刺激分子B7.1(CD80)和B7.2(CD86)的表达。巨噬细胞是体内重要的炎性和免疫效应细胞,而脂多糖(lipopolysaccharide, LPS)是诱导巨噬细胞产生和释放炎症介质最强烈、最有效的激活物。研究表明,LPS可激活巨噬细胞cAMP-PKA信号通路并介导了一个抑制性信号,它可能抑制巨噬细胞表达一系列细胞因子,下调TNF-α和IL-1β的释放量。我们的研究证实CCK-8作用于表达CCK受体的巨噬细胞,通过激活cAMP-PKA、抑制PKC活性而抑制NF-κB活性,从而抑制脂多糖诱生促炎细胞因子TNF-α、IL-1β、IL-6。CCK-8是否通过CCKR-cAMP-PKA-NF-κB通路调节巨噬细胞B7.1和B7.2的表达,目前尚未见报道。据此,本部分实验将主要观察CCK-8调节静息巨噬细胞和LPS诱导的巨噬细胞B7.1和B7.2表达的细胞内信号转导通路。
本部分采用FACS检测巨噬细胞B7.1和B7.2的表达,[~3H]掺入法测定CD4~+T细胞增殖程度以反映巨噬细胞的协同刺激活性。ELISA法检测细胞内cAMP含量和PKA活性,EMSA检测小鼠腹腔巨噬细胞内NF-κB活性,Western blot检测小鼠腹腔巨噬细胞内IκB-α和p-IκB-α蛋白水平的变化。
结果:(一)、NF-κB参与CCK-8对巨噬细胞B7.1和B7.2表达的调节作用。
(1)NF-κB抑制剂Gliotoxin组剂量依赖性地抑制了LPS诱导的巨噬细胞B7.1和B7.2表达和LPS诱导的巨噬细胞协同刺激活性,表明NF-κB活性与LPS诱导的巨噬细胞B7.1、B7.2的表达有关,NF-κB参与调节巨噬细胞的协同刺激活性。
(2)在溶剂对照组,小鼠腹腔巨噬细胞核内几乎检测不到与特异性寡核苷酸探针结合的NF-κB。用LPS孵育小鼠腹腔巨噬细胞1h,细胞内NF-κB活性明显高于溶剂对照组(P<0.01),而用不同浓度的CCK-8(10~(-6) mol/L、10~(-8)mol/L、10~(-10)mol/L)处理后,则剂量依赖性地显著地抑制了LPS诱导的NF-κB活性增高,抑制率分别为56.58%、37.26%、15.59%(P<0.05),以LPS+CCK~(-8)(10~(-6)mol/L)组的抑制作用最为显著。不同浓度的CCK-8单独孵育小鼠腹腔巨噬细胞,10~(-6)mol/L和10~(-8)mol/L浓度的CCK-8使细胞NF-κB活性升高(P<0.05),而10~(-10)mol/L的CCK-8对细胞NF-κB活性无明显影响(P>0.05)。在反应体系中加入同源性寡核苷酸(含NF-κB结合位点)及异源性寡核苷酸(含AP-2结合位点),分别作为特异性和非特异性竞争物,以证实该实验中DNA-蛋白质结合的特异性。
(3)用LPS孵育小鼠腹腔巨噬细胞30min,与溶剂对照组比较,可使胞浆中IκB-α蛋白水平明显降低而p-IκB-α蛋白水平明显增加,p-IκB-α蛋白水平/IκB-α蛋白水平比值明显增加(P<0.05);在上述各组反应体系内加入CCK-8后,可使胞浆中IκB-α蛋白水平明显升高而p-IκB-α蛋白水平明显降低,p-IκB-α/IκB-α比值明显降低(P<0.05);不同浓度的CCK-8单独孵育小鼠腹腔巨噬细胞,10~(-6)mol/L和10~(-8)mol/L的CCK-8使胞浆中IκB-α蛋白水平降低,p-IκB-α蛋白水平增加,导致p-IκB-α蛋白水平/IκB-α蛋白水平比值明显增加(P<0.05);而10~(-10)mol/L的CCK-8对细胞内IκB-α和p-IκB-α蛋白水平均无明显影响(P>0.05)。上述结果表明:CCK-8诱导静息巨噬细胞IκB磷酸化及NF-κB活化,而抑制LPS诱导的巨噬细胞IκB磷酸化及NF-κB活化,参与巨噬细胞B7.1和B7.2的表达及其协同刺激活性。
(二)、CCK-8通过激活巨噬细胞内cAMP-PKA通路调节NF-κB活性,进而调节B7.1和B7.2表达
(1)用LPS孵育小鼠腹腔巨噬细胞15min或30min,细胞内cAMP含量及PKA活性升高,与对照组相比有显著性差异(P<0.01)。10~(-10)mol/L、10~(-8)mol/L和10~(-6)mol/LCCK-8预处理后加入LPS孵育15 min,CCK-8剂量依赖性地促进LPS引起的cAMP含量及PKA活性增加。不同浓度的CCK-8(10~(-10) mol/L、10~(-8) mol/L、10~(-6) mol/L)剂量依赖性地增加静息小鼠腹腔巨噬细胞内cAMP含量及PKA活性,表明CCK-8可以激活cAMP-PKA。
(2)在静息状态下,与对照组比较,Fsk(forskolin, cAMP激动剂毛喉素)组可以增加巨噬细胞内cAMP含量及PKA活性(P<0.05)。与CCK-8组比较,Fsk+CCK-8组cAMP含量及PKA活性升高(P<0.05)。在LPS活化的巨噬细胞内cAMP含量及PKA活性显著增高(P<0.01), Fsk,CCK-8分别与LPS合用均可使LPS诱导的巨噬细胞内cAMP含量及PKA活性进一步明显升高(P<0.05,P<0.01);但LPS+H89组,CCK-8+H89组,LPS+CCK-8+H89组cAMP含量分别与LPS组,CCK-8组,LPS+CCK-8组相比,cAMP含量均无明显改变,而PKA活性均下降(P<0.05),表明Fsk可激活cAMP,升高PKA活性,而H89对cAMP含量无明显影响,却抑制PKA活性。
(3)用不同刺激因素孵育小鼠腹腔巨噬细胞60min,与control组相比,LPS可以可以升高细胞内p-IκB-α蛋白表达及增强NF-κB活性(P<0.05)。单独应用CCK-8孵育小鼠腹腔巨噬细胞,在10~(-6) mol/L和10~(-8)mol/L时,胞浆中p-IκB-α蛋白表达及NF-κB活性升高(P<0.05)。Fsk单独孵育小鼠腹腔巨噬细胞,可以升高细胞内p-IκB-α蛋白表达及NF-κB活性(P<0.05)。与control组相比,LPS+Fsk组,CCK-8+Fsk组细胞内p-IκB-α蛋白表达及NF-κB活性均升高(P<0.05)。用LPS+CCK-8共同孵育巨噬细胞,细胞内p-IκB-α蛋白表达及NF-κB活性低于LPS组(P<0.05)。H89与LPS和CCK-8共同孵育,p-IκB-α蛋白表达及NF-κB活性高于LPS+CCK-8组,且有显著性差异(P<0.01),低于LPS组(P<0.05)。表明CCK-8通过进一步激活cAMP-PKA抑制LPS诱导的巨噬细胞NF-κB活化。
(4)cAMP激动剂Fsk与CCK-8的作用相似,对静息巨噬细胞B7.1的表达无明显影响,而升高B7.2的表达;剂量依赖性地抑制LPS诱导的小鼠腹腔巨噬细胞B7.1和B7.2的表达,以Fsk 10~(-6)mol/L的作用最强。PKA抑制剂H89可以剂量依赖性地抑制CCK-8诱导的巨噬细胞表面B7.2表达,对B7.1表达无影响(P>0.05)。H89可以逆转CCK-8对LPS活化的巨噬细胞的作用,即剂量依赖性地升高巨噬细胞B7.1和B7.2表达,以LPS+CCK-8+H89 10-8mol/L的作用最强,有显著性差异(P<0.05)。表明CCK-8通过激活腹腔巨噬细胞cAMP-PKA信号通路调节B7.1和B7.2表达。
以上结果表明:CCK-8通过激活cAMP-PKA-NF-κB信号通路上调B7.1和B7.2表达,增强巨噬细胞的协同刺激活性;并通过进一步激活LPS诱导的巨噬细胞cAMP-PKA-NF-κB信号通路,抑制IκB蛋白降解及NF-κB活性,下调B7.1和B7.2表达,抑制巨噬细胞的协同刺激活性。
(三)、CCK-8通过CCK受体调节cAMP―PKA―NF-κB―B7信号通路
(1)体外实验中,CCK1R拮抗剂CR1409及CCK2R拮抗剂CR2945均可逆转CCK-8作用下增强的巨噬细胞协同刺激活性和逆转CCK-8对LPS活化的巨噬细胞协同刺激活性的抑制作用,而且相同浓度的CR1409和CR2945相比较,CR1409的作用强于CR2945,表明CCK1R、CCK2R共同介导了CCK-8调节静息及LPS活化的巨噬细胞协同刺激活性的作用,并且CCK1R起主导作用。
(2)CCK-8显著抑制了LPS诱导的巨噬细胞中IκB磷酸化及NF-κB活性增高,对NF-κB活性的抑制率达37.26%(P<0.05)。CCK1R拮抗剂CR1409及CCK2R拮抗剂CR2945均可逆转CCK-8抑制的LPS诱导的巨噬细胞内IκB磷酸化及NF-κB活性增高,即剂量依赖性地升高巨噬细胞核内p-IκB-α蛋白表达及NF-κB活性(P<0.05),CR1409的作用强于CR2945。表明CCK1R、CCK2R共同介导了CCK-8调节LPS活化的巨噬细胞内IκB磷酸化及NF-κB活性,并且CCK1R起主导作用。
(3)两种拮抗剂均能不同程度地抑制CCK-8诱导的巨噬细胞内cAMP含量增高及PKA活性升高。CR1409和CR2945在10~(-9) mol·L~(-1)时对CCK-8的抑制作用不明显(P>0.05),当二者浓度达到10~(-7)mol·L~(-1)以上时,细胞内cAMP含量及PKA活性较LPS+CCK-8组明显降低(P<0.01),且有显著性差异(P<0.05)。两种拮抗剂对CCK-8的抑制效应均有剂量依赖性,CR1409的作用较CR2945强,说明两种CCK受体拮抗剂均能影响cAMP含量及PKA活性,并且CCK1R起主导作用。以上结果表明: CCK-8主要是通过上调静息巨噬细胞B7.2表达而增强其协同刺激活性,下调LPS诱导的巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性,该作用由CCK1R及CCK2R介导,其中CCK1R起主要介导作用。
综上,CCK-8通过激活静息巨噬细胞cAMP-PKA-NF-κB信号通路增强NF-κB活性,上调B7.1和B7.2表达,增强巨噬细胞的协同刺激活性;并通过进一步激活LPS诱导的巨噬细胞cAMP-PKA信号通路,抑制IκB蛋白降解及NF-κB活性,下调B7.1和B7.2表达,抑制巨噬细胞的协同刺激活性。CCK1R及CCK2R共同参与介导作用,其中CCK1R起主要介导作用。
3体内实验CCK-8对静息和LPS活化的巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响
第一部分的体外实验证实了CCK-8对静息及LPS诱导的巨噬细胞B7.1和B7.2表达及其协同刺激活性的影响,本部分将从整体实验进一步验证上述结果。实验将小鼠(n=4)分为:Control组、CCK-8组、CCK-8+CR1409/CR2945组、LPS组、LPS+CCK-8组、LPS+CCK-8+CR1409/ CR2945组,分别腹腔注射(i.p.)一定剂量的N.S.,LPS和/或CCK-8及CCK1R、CCK2R,12h后按收集并纯化腹腔巨噬细胞。采用流式细胞术分析细胞表面B7.1和B7.2含量的变化;用免疫磁珠从小鼠脾细胞分离CD4~+T细胞,按4:1数量比与上述各组腹腔巨噬细胞共同体外培养,同时加入ConA 5mg/L,采用~3H掺入法测定CD4~+T细胞增殖反映巨噬细胞的协同刺激活性。整体应用CCK-8作用小鼠腹腔巨噬细胞12h,与对照组相比,CCK-8可使小鼠腹腔巨噬细胞B7.2的表达增加,而对B7.1的表达则无影响。并且CCK-8还可使CD4~+T细胞[~3H]-TdR的掺入率升高,即促进其增殖,与对照组相比,有显著性差异(P<0.05)。小鼠腹腔注射CCK-8和/或CCKR拮抗剂,然后观察他们对于LPS i.p.(100μg/mouse)小鼠的作用,12h后收集小鼠腹腔巨噬细胞,流式细胞术检测B7的表达。与LPS组相比,CCK-8可以降低LPS诱导的小鼠腹腔巨噬细胞B7.1和B7.2的表达(P<0.05);并降低CD4~+T细胞的[~3H]-TdR掺入率,即抑制其增殖,说明CCK-8可降低小鼠腹腔巨噬细胞的协同刺激活性。整体应用CCKR拮抗剂可部分逆转CCK-8对巨噬细胞和LPS刺激的巨噬细胞的B7.1和B7.2表达及其协同刺激活性,以CCK1R的作用为著。综上,体内实验表明CCK-8通过上调小鼠腹腔巨噬细胞B7.2的表达而增强巨噬细胞的协同刺激活性;通过下调LPS诱导的腹腔巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性,该作用由CCK1R及CCK2R介导,其中CCK1R起主要介导作用。
结论
本研究分别从体内实验和体外实验入手,首次系统研究了CCK-8对静息及LPS活化的小鼠腹腔巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响,并从受体、cAMP-PKA、转录因子NF-κB等多个层次系统研究了CCK-8调节小鼠腹腔巨噬细胞B7.1和B7.2表达的信号转导机制,深入揭示了CCK-8发挥免疫调节作用的机制。
1、体内及体外实验证实,CCK-8主要通过上调小鼠腹腔巨噬细胞B7.2表达而增强其协同刺激活性;通过下调LPS诱导的巨噬细胞B7.1和B7.2表达而抑制其协同刺激活性。CCK-8调节巨噬细胞的协同刺激活性与B7.1和B7.2表达密切相关。
2、CCK-8通过激活静息巨噬细胞cAMP-PKA-NF-κB信号通路增强NF-κB活性,上调B7.1和B7.2表达,增强巨噬细胞的协同刺激活性;并通过进一步激活LPS诱导的巨噬细胞cAMP-PKA-NF-κB信号通路,抑制IκB蛋白磷酸化及NF-κB活性,下调B7.1和B7.2表达,而抑制巨噬细胞的协同刺激活性。CCK1R及CCK2R共同参与介导作用,其中CCK1R起主要介导作用。
因此,CCK-8通过干预巨噬细胞协同刺激分子的表达影响Th细胞的功能,这可能是CCK-8发挥免疫调节作用的机制之一。
Cholecystokinin (CCK) being a kind of neuropeptide presents extensively in many systems of the body, with a broad spectrum of biological functions, such as regulating ingestion, anxiety, memory and so on. Our research have found that CCK-8 has some immunomodulatory and antiinflammatory functions in one of our National Nature Scinece Foundation (CCK receptor and anti-inflammatory intracellular signaling mechanisms activated by CCK-8, No. 30270529), CCK-8 inhibits activity of NF-κB by activiting cAMP-PKA and inhibiting PKC activity, and then inhibits the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, which shows that CCK-8 can modulate natural (or innate) immunity. But there are still no reports about how CCK-8 modulating acquired immunity. It has been reported that CCK2 receptor and mRNA express on human Jurkat T lymphocytes and CCK mRNA expresses on human T lymphocytes. CCK inhibits the proliferation of human peripheral blood lymphocytes induced by PHA (phytohemagglutinin) and so do the murine lymphocytes. Furthermore, it also inhibits the mobility of murine lymphocytes, but enhances its spontaneous hyperplasia and adhension. The existence and expression of specific CCK receptor on lymphocytes indicates CCK functioning as modulators of T lymphocytes by autocrine or paracrine secretion and affecting acquired immunity.
Upon antigenic stimulation, CD4~+Th cells can differentiate into two distinct types of effector cells, Th1 and Th2 subsets, each producing its own set of cytokines and mediating separate functions. Th1 cells secrete cytokines (IL-2, IFN-γ, and TNF-β, IL-12) critical for the generation of a cellular immune response, thereby activating CTL (cytotoxic T lymphocytes) and macrophages, inducing delayed-type hypersensitivity (DTH) responses, and stimulating IgG2a Ab production in mice. Th2 cells produce IL-4, IL-5, IL-13 and IL-10, which are critical for IgG1 and IgE Ab production and immunity against helminthic parasites, and inhibit macrophage, B cell and eosinophilic granulocyte activation and Ag presentation, thereby down-regulating the cellular immune responses. It is very important that Th1/Th2 balance maintain the immunostasis of the body. In the current research it has been found that Th1/Th2 balance relates with many diseases and participates in the initiation, development and prognosis of infective diseases (endotoxemia, SIRS, tuberculosis, parasitic infection et al.), hypersensitivity diseases (asthma et al.), the autoimmune diseases (RA, uveitis et al.). A series of studies in our laboratory has been focused on the effect of CCK-8 against endotoxin shock (ES) and releasing rheumatoid arthritis, but we still do not know whether this effect is related to the regulation of CCK-8 on Th1/Th2 balance? Focus on the modulating fuctions and its mechanism of CCK-8 on Th1/Th2 balance will conduce to interpreting the physiological and pathology functions of neuroimmune network, and presenting theory for clinical therapies of Th1/Th2 unbalance diseases.
Na?ve CD4~+T lymphocytes become activated and undergo clonal expasion, then differentiate into Th0 lymphocytes. Th0 cells have the potential ability of differentiating into Th1 or Th2 phenotype cells. Determining factors include the nature of the APC, the nature and amount of Ag, and the genetic background of the host, with the cytokine microenvironment as the dominant factor. Cytokine microenvironment is the dominant factor on regulating the differentiation of precursor Th cells (Th0). Increasing evidence demonstrates that IL-12 and, to a lesser extent IFN-γ, direct CD4~+T cells to differentiate into Th1. In contrast, IL-4 is necessary to induce the development of Th2. When both IL-4 and IL-12 are added to in vitro cultures, IL-4 dominates over IL-12, driving naive CD4~+T cells toward the Th2 phenotype. The activation of naive CD4~+T lymphocytes requires two signals delivered by APCs, leading to enhanced cytokine production and proliferation and produce effective immune response. The first signal, which confers specificity, is provided by the interaction of the antigenic peptide/MHC complex with the T cell receptor. The second signal is provided by costimulatory molecules expressed on APCs. Among the accessory molecules, the B7 family appears to be the most potent. The B7 costimulatory pathway involves at least two molecules, B7.1 (CD80) and B7.2 (CD86), both of which can interact with their counterreceptors, CD28 and CTLA-4, on T cells. B7.1 and B7.2 appear to be equivalent in the costimulation of T cell proliferation. However, B7.1 and B7.2 may differ in the signals provided for T cell differentiation, with B7.2 favoring Th2 development. As a general rule, B7.2 is induced earlier during the activation process and at higher levels than B7.1. Therefore, we will investigate the role of CCK-8 on macrophage B7.1 and B7.2 expression and costimulatory function for Ag-primed CD4~+T cells, and on the macrophage-induced regulation of Th1/Th2 differation in vitro and in vivo.
Some researchers have proved that the activation of Toll/NF-κB results in induction of cytokines and costimulatory molecules― B7.1(CD80) and B7.2(CD86). It is well known that CCK exerts a variety of physiological actions through its cell surface receptors, which have been pharmacologically classified into two subtypes CCK1R and CCK2R according to their affinity to the peptide agonist sulfated carboxyl-terminal octapeptide (sCCK-8) and gastrin. CCK receptors belong to G protein-coupled receptor (GPCR) superfamily and distribute extensively in mammalian body. Our current research suggested that CCK-8 inhibited activity of NF-κB by activiting cAMP-PKA and inhibiting PKC activity through its receptors in rat PIMs. To elucidate the intracellular signaling mechanism of CCK-8 regulating macrophage B7.1 and B7.2 expression, we performed a series of studies on mouse peritoneal macrophages, from the effect of CCK-8 on cAMP-PKA pathway, and transcription factors NF-κB which involved in regulating B7.1 and B7.2 expression, and the costimulatory activity on macrophages induced by LPS, and on the macrophage-induced regulation of Th1/Th2 differation in vitro and in vivo.
1 Effects of CCK-8 on B7.1 and B7.2 expression and costimulatory activity of macrophage
Aim: To investigate in vitro effects of CCK-8 on the expressions of B7.1 and B7.2 and on the costimulatory activity for T lymphocytes in unstimulated and LPS-activated macrophages. Methods: Mouse peritoneal macrophages were isolated and incubated with CCK-8(10~(-12) mol/L ~10~(-6) mol/L), or with LPS and/or CCK-8(10~(-12)～10~(-6))mol/L for indicated times. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry. CD4~+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, or with LPS, CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, for 24h. ConA was added into the culture medium to stimulate CD4~+T cell proliferation. The proliferation was determined by measuring [~3H]-TdR incorporation in aβ-scintillation counter. Results: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations of 10~(-7) mol/L to 10~(-9) mol/L. Anti-B7.2 antibody, but not anti-B7.1 antibody, reduced the modulatory role of CCK-8 on costimulatory activity. LPS-induced B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were inhibited by CCK-8 in a dose-dependent manner, with the maximal effects occurred at the concentrations of 10~(-7) mol/L to 10~(-9) mol/L. Anti-B7.1 antibody and anti-B7.2 antibody inhibited the modulatory role of LPS on costimulatory activity. Conclusions: CCK-8 enhanced macrophage costimulatory activity by upregulating B7.2 expression, and inhibited LPS-induced macrophage costimulatory activity by down-regulating B7.1 and B7.2 expressions.
2 CCK-8 regulated macrophage B7.1 and B7.2 expression by cAMP-PKA-NF-κB signaling pathway
Documents reported that NF-κB induces the expression of costimulatory molecule, B7.1 (CD80) and B7.2 (CD86). Macrophages are the most important inflammatory and immune effector cells, while LPS is the most effective and intensive activator inducing macrophages to produce and release mediators of inflammation. Some studies proved that cAMP-PKA pathway and an inhibitory signaling are involved in activation of macrophages by LPS. This signaling can down-regulate cytokine production of macrophages, such as TNF-αand IL-1β. Our current studies demonstrated that CCK-8 inhibited LPS-induced NF-κB binding activity via activation of cAMP-PKA signaling pathway and inhibition of PKC activity, and then inhibited the production of proinflammatory cytokines such as TNF-α, IL-1β, IL-6. However, there are still no reports about whether CCK-8 regulates B7.1 and B7.2 expression through CCKR-cAMP-PKA-NF-κB signaling pathway? Thus, we will focus on the signaling pathway relating to the B7.1 and B7.2 expression in resting and LPS-inducecd macrophage.
Methods:①Mouse peritoneal macrophages were isolated and incubated with LPS and NF-κB inhibitor Gliotoxin, or with CCK-8 at different concentration (10~(-12)mol·L~(-1)~10-6mol·L~(-1)), Fsk and/or H89, or with CCK receptor antagonists (10~(-9) mol·L~(-1)~10~(-5)mol·L~(-1)) for 10 min prior to addition of LPS (5mg·L~(-1)) and CCK-8 (10-8mol·L~(-1)) for indicated time. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry (FACS). CD4~+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages, which were pretreated with the superior reagents. ConA was added into the culture medium to stimulate CD4~+T cell proliferation. The proliferation was determined by measuring [~3H]-TdR incorporation in aβ-scintillation counter representing the assay of macrophage costimulatory activity.②Mouse peritoneal macrophages were isolated and stimulated with LPS in the absence or presence of CCK-8 and NF-κB binding activity was analyzed by electrophoretic mobility shift assay (EMSA) 1h after stimulation. The protein levels of IκB-αand p-IκB-αin the cytoplasma were detected by Western blot 30 min after stimulation. Data were presented as x±s and analyzed by one way ANOVA and least significant difference test using SPSS 11.5 statistical program. A level of P<0.05 was considered statistically significant.
Results: (1). Involvememt of NF-κB in CCK-8 regulation of B7.1 and B7.2 expression in mouse macrophages
①Gliotoxin, a NF-κB inhibitor, inhibited B7.1 and B7.2 expression and costimulatory activity in LPS-activated macrophages in a dose-dependant manner, which shows that the NF-κB binding activity was related to the B7.1 and B7.2 expression in macrophage, and NF-κB was involved in regulating the costimulation of macrophage.②The NF-κB binding activity was significantly higher in mouse peritoneal macrophages stimulated with 5mg/L LPS for 1h in comparison with unstimulated cells (P<0.01), and additional treatment with CCK-8 (10~(-6) mol/L, 10~(-8)mol/L, 10~(-10)mol/L) markedly reduced the binding activity in a dose-dependent manner, the inhibition ratio was 56.58%、37.26%、15.59% respectively (P<0.05 or P<0.01). CCK-8 alone markedly increased the NF-κB binding activity at the concentrations of 10~(-6) mol/L and 10~(-8) mol/L (P<0.05), while CCK-8 had no effect on it at the concentrations of 10~(-10) mol/L (P>0.05). The binding specificity was confirmed by using homologous (NF-κB) and nonhomologous (AP-2) oligonucleotides as competitors.③The IκB-αprotein level in mouse peritoneal macrophages was markedly decreased while p-IκB-αprotein level increased 30 min after incubation with 5mg/L LPS (P<0.01). When added with different concentrations of CCK-8 (10~(-10) mol/L~10~(-6) mol/L) in macrophages stimulated by LPS, IκB-αprotein level was increased obviously and p-IκB-αprotein level was decreased (P<0.05), leading to decrease the ratio of p-IκB-αprotein level/IκB-αprotein level in a dose-dependent manner (P<0.05). CCK-8 alone increased p-IκB-αprotein level and decreased IκB-αprotein level at the concentrations of 10~(-6)mol/L and 10~(-8)mol/L, then leading to increase the ratio of p-IκB-α/IκB-αprotein level (P<0.05), but with no effect on the IκB-αand p-IκB-αprotein level at the concentrations of 10~(-10)mol/L (P>0.05). These results showed that NF-κB had correlated with the macrophage B7.1 and B7.2 expression and costimulation, CCK-8 induced IκB-αphosphorylation and enhanced NF-κB activity in unstimulated macrophage, and at the same time, inhibited NF-κB activity and IκB-αdegradation in LPS-activated mouse peritoneal macrophage, which might be the upstream mechanism of the regulative effect of CCK-8 on B7.1 and B7.2 expressions.
(2). CCK-8 regulated NF-κB binding activity and B7.1 and B7.2 expression on mouse macrophages through cAMP-PKA signaling pathway
①Stimulating mouse peritoneal macrophages with 5mg·L~(-1) LPS resulted in an obvious increase in cAMP content and PKA activity (P<0.01). CCK-8 didn’t affect cAMP content and PKA activity at low concentration (10~(-10)mol·L~(-1)) (P>0.05), but led to a significant increase of them at high concentration (10~(-8)mol·L~(-1)~10~(-6)mol·L~(-1)) (P<0.05) in LPS-activated macrophage when compared with that of LPS group, suggesting that CCK-8 affect cAMP content and PKA activity in a dose-dependent manner. CCK-8 alone (10~(-10)mol·L~(-1)、10~(-8)mol·L~(-1) and 10~(-6)mol·L~(-1)) increased cAMP content and PKA activity in a dose-dependent manner in unstimulated macrophages. These results demonstrated that CCK-8 activated cAMP-PKA signaling pathway at high concentration, and had a synergistical effect with LPS at high concentration on cAMP content and PKA activity in mouse peritoneal macrophages, which might be one of the immunomodulatory molecular mechanisms activated by CCK-8.②Similar to CCK-8, Fsk could increase cAMP content and PKA activity in unstimulated macrophages when compared with control group (P<0.05). Both CCK-8 and Fsk could markedly increase cAMP content and PKA activity compared with CCK-8 group (P<0.05). Stimulating mouse peritoneal macrophages with 5mg·L~(-1) LPS resulted in an obvious increase in cAMP content and PKA activity (P<0.01), and with additional CCK-8 or Fsk, also increase cAMP content and PKA activity (P<0.05, P<0.01). However, there was no difference in cAMP content (P>0.05), and the PKA activity was decreased (P<0.05) when LPS+H89 group, CCK-8+H89 group and LPS+CCK-8+H89 group compared respectively with LPS group, CCK-8 group, LPS+CCK-8 group. These results showed that Fsk activated cAMP and increased PKA activity, while H89 had no influence on cAMP content and inhibited PKA activity.③The NF-κB binding activity and p-IκB-αprotein level were significantly higher in mouse peritoneal macrophages stimulated with 5mg·L~(-1) LPS in comparison with unstimulated cells (P<0.01), of which were too little to be detected, and additional treatment with CCK-8 or Fsk markedly reduce the binding activity and p-IκB-αprotein level (P<0.05). But CCK-8 alone increased the NF-κB binding activity and p-IκB-αprotein level at the concention of 10~(-6) mol/L and 10-8mol/L (P<0.05). Fsk alone could increase the NF-κB binding activity and p-IκB-αprotein level (P<0.05) which was similar to CCK-8 alone group. Compared with control group, LPS+Fsk group and CCK-8+Fsk group also increase the NF-κB binding activity and p-IκB-αprotein level (P<0.05). When CCK-8 incubated with LPS, the NF-κB binding activity and p-IκB-αprotein level were lower than LPS group (P<0.05). H89, a PKA inhibitor, co-incubating with CCK-8 and LPS, resulted in obvious increase of NF-κB binding activity and p-IκB-αprotein level (P<0.01) when compared with LPS+CCK-8 group. These results showed that CCK-8 could further activated cAMP-PKA and inhibited the NF-κB binding activity in LPS-induced macrophages.④Fsk (forskolin, a cAMP-inducing agent) had similar effects of CCK-8 with the maximal effect at the dose of 10~(-6)mol/L, which increasing B7.2 expression but having no effect on B7.1 expression in resting macrophages, and inhibiting B7.1 and B7.2 expression in LPS-induced macrophages. In compared with CCK-8 alone group, H89 (a PKA inhibitor) inhibited the B7.2 expression in macrophages activated by CCK-8 in a dose-dependent manner (P<0.05), on the other hand, it could reverse the effects of CCK-8, enhancing macrophage B7.1 and B7.2 expression in a dose-dependent manner (P<0.05), and had the highest effect at the dose of 10-8mol/L on LPS-activated macrophages (P<0.05). The results showed that CCK-8 modulated B7.1 and B7.2 expression through cAMP-PKA signaling pathway. All in all, the results of this part revealed that CCK-8 increased B7.1 and B7.2 expression and costimulation by increasing NF-κB binding activity and IκB-αdegradation in unstimulated macrophages and activating cAMP-PKA pathway. However, CCK-8 down-regulated B7.1 and B7.2 expression and costimulation by inhibiting NF-κB binding activity and decreasing IκB-αdegradation in macrophages stimulated by LPS and cAMP-PKA signaling pathway being involved in the regulation of NF-κB binding activity by CCK-8 at least in part, which might be one of immunomodulatory mechanisms activated by CCK-8.
(3). Effects of CCK-8 were mediated through CCK receptors
①CCK-8 increased resting macrophage costimulatory activity and inhibited that of LPS-induced macrophage by mainly up-regulating B7.2 expression and down-regulating B7.1 and B7.2 expression respectively, which was mediated by CCK1R and CCK2R. However, CCK1R and CCK2R reversed the effects of CCK-8. CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.②CR1409 and CR2945 could increase NF-κB binding activity and IκB-αdegradation in dose-dependent manner, which reversed the effects of CCK-8 inhibiting NF-κB activity and IκB-αdegradation in LPS-induced mouse peritoneal macrophage. The effect of CR2945 was a little lighter than CR1409. The results showed that both CCK1R and CCK2R were involved in CCK-8 regulating NF-κB activity in LPS-induced macrophages, and CCK1R might be the major receptor responsible for the modulation of CCK-8.③The effect of CCK-8 on cAMP content was abrogated to different degree by CR1409 and CR2945 accompanying with the increase of their concentration. CR1409 and CR2945 did not affect the CCK-8-resulted increase of cAMP content and PKA activity at low concentration (10~(-9) mol·L~(-1)), but decreased them significantly when the concentration reached to 10~(-7) mol·L~(-1)～10~(-5) mol·L~(-1) compared with that of LPS+CCK-8 group (P<0.05), but the effect of CR1409 was more powerful than CR2945 (P<0.05). The inhibitory effects of CR1409 and CR2945 on the CCK-8-resulted increase of cAMP content and PKA activity were dose-dependent. The effects of CCK-8 on cAMP content and PKA activities were attenuated by CR1409 and CR2945 significantly at 10~(-5) mol·L~(-1) compare with that of CCK+LPS group (P<0.05 or P<0.01) and the order of potency of different antagonist was as follows: CR1409>CR2945. The results showed that both CCK1R and CCK2R were involved in CCK-8 regulating cAMP content and PKA activities in LPS-induced macrophages, and CCK1R might be the major receptor responsible for the modulation of CCK-8.
Conclusions: All the upper results suggested that CCK-8 enhanced B7.1 and B7.2 expression and costimulation by increasing NF-κB binding activity and IκB-αdegradation in unstimulated macrophages, then leading to activate cAMP-PKA signaling pathway, which were mediated through CCK receptors. However, CCK-8 decreased B7.1 and B7.2 expression and costimulation by inhibiting NF-κB binding activity and IκB-αdegradation in LPS-induced macrophages, then leading to further activate cAMP-PKA signaling pathway, , which were mediated through CCK receptors, CCK1R and CCK2R, but CCK1R might play a main role in this process.
3 CCK-8 regulated macrophage B7.1/B7.2 expression and costimulatory function in vivo
Aim: To investigate in vivo effects of CCK-8 on the expressions of B7.1 and B7.2 and the costimulatory activity for T lymphocytes in unstimulated and LPS-activated macrophages. Methods: Several BALB/c mice were randomly assigned to 8 groups (n=4) injected i.p. with N.S. alone, or with LPS (100μg/mouse), in the presence or absence of CCK-8 (5 nmol/mouse) and CR1409 (100μg/mouse), CR2945 (100μg/mouse). After 12h, macrophages were purified from the peritoneal exudate as indicated above. The B7.1/B7.2 expression on purified macrophages was analyzed by flow cytometry and, alternatively, purified macrophages were assayed for macrophage costimulatory activity as described above. ConA was added into the culture medium to stimulate CD4~+T cell proliferation. The proliferation was determined by measuring [~3H]-TdR incorporation in aβ-scintillation counter.
Results and Conclusions: The in vivo administration of CCK-8 resulted in increase of B7.2 expression, but without any influence on B7.1 expression. Peritoneal macrophages harvested from CCK-8-injected mice also exhibited increased costimulatory activity for CD4~+T cells. We concluded that, similar to the in vitro experiments, the in vivo administration of CCK-8 stimulated B7.2 expression and induced the costimulatory activity of peritoneal macrophages. Second, we assessed the in vivo effects of CCK-8 and CCKR antagonists (CR1409 and CR2945) in LPS-injected mice. BALB/c mice were injected i.p. with LPS (100μg/mouse), with or without CCK-8 (5nmol/mouse), CR1409 (100μg/mouse), CR2945 (100μg/mouse). Macrophages harvested 12h later were analyzed in terms of B7.1/B7.2 expression and costimulatory activity. In vivo CCK-8 administration reduced both B7.1 and B7.2 expression. Also, CCK-8 completely abolished the costimulatory activity. These effects are consistent with the in vitro effects of CCK-8 on LPS-stimulated macrophages. In vivo modulation of CCK-8 on costimulation was mediated by CCK receptors and CCK1R might be the major receptor, which was in concordance with the in vitro results.
CONCLUSIONS
In the present study, we first systematically investigated the modulatory effects of CCK-8 on B7.1 and B7.2 expression and the costimulatory activity in resting and LPS-activated mouse peritoneal macrophages, in vitro and in vivo, at multi-levels from receptors to cAMP-PKA signaling pathway and transcription factors. Then we first studied the intracellular signaling mechanisms of CCK-8 in the regulation of B7.1 and B7.2 expression and its correlation with the costimulatory function of macrophages.
1 The present studies for the first time demonstrated that, in unstimulated macrophages, CCK-8 up-regulated B7.1 and B7.2 expression, but mainly B7.2 expression at protein level in vitro and in vivo, and induced the costimulatory activity. In constrast, in LPS-activated macrophages, CCK-8 down-regulated the expression of both B7.1 and B7.2 expressions, and abolished the costimulatory activity. The effects of CCK-8 on the costimulatory activity for T cells correlated closely with the expression of B7.1/B7.2, and might be mediated through the modulation of B7 expression.
2 CCK-8 up-regulated B7.1 and B7.2 expression and increased costimulatory activity by activating cAMP-PKA-NF-κB signaling pathway in macrophages. CCK-8 down-regulated B7.1 and B7.2 expression and inhibited its costimulatory activity in LPS-activated macrophages by activating cAMP-PKA signaling pathway, and inhibiting IκB-αphosphorylation and the NF-κB binding activity through CCK receptors, and CCK1R might play a main role in this process.
In conclusion, this study firstly demonstrated that CCK-8 regulated the function of macrophages through the modulation of B7.1 and B7.2 expression, which might be one of the mechanisms of CCK-8 on regulating immunomodulatory functions.
引文
1 Cong B, Li SJ, Yan YL, et al. Effect of cholecystokinin octapeptide on tumor necrosis factor α transcription and nuclear factor-κB activity induced by lipopolysaccharide in rat pulmonary interstitial macrophages. World J Gastroenterol 2002; 8: 718-723. (SCIE 期刊).
2 Meng AH, Ling YL, Zhang XP, et al. CCK-8 inhibits expression of TNF-α in the spleen of endotoxic shock rats and signal transduction mechanism of P38 MAPK. World J Gastroenterol 2002; 8: 139-143.
3 Ling YL, Meng AH, Zhao XY, et al. Effects of cholecystokinin on cytokines during endotoxic shock in rats. World J Gastroenterol 2001; 7: 667-671.
4 Cuq P, Gross A, Terraza A, et al. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurkat lymphoblastoid cells. Life Sci 1997; 61:543-555.
5 Kimoto Y. A possibility of all mRNA expression in a human singlelymphocyte. Hum Cell 1996; 9:367-370.
6 Carrasco M, Hernanz A, De La Fuente M. Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2. Regul Pept. 1997; 70:135-142.
7 Medina S, Rio MD, Cuadra BD, et al. Age-related changes in the modulatory action of gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide in the proliferation of murine lymphocytes. Neuropeptides. 1999; 33: 173-179.
8 Xu SJ, Gao WJ, Cong B, et al. Effect of lipopolysaccharide on expression and characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages. Acta Pharmacol Sin. 2004; 25: 1347-1353.
9 Lauw FN, ten Hove T, Dekkers PE, et al. Reduced Th1, but not Th2, cytokine production by lymphocytes after in vivo exposure of healthy subjects to endotoxin. Infect Immun. 2000; 68:1014-1018.
10 Iwasaka H, Noguchi T. Th1/Th2 balance in systemic inflammatory response syndrome (SIRS). Nippon Rinsho. 200; 62:2237-2243.
11 Yudoh K, Matsuno H , Nakazawa F , et al . Reduced expression of theregulatory CD4+ T cell subset is related to Th1/ Th2 balance and diseaseseverity in rheumatoid arthritis . Arthritis Rheuma 2000; 43 : 617- 627.
12 凌亦凌,黄善生,王乐丰,等. 八肽胆囊收缩素抗内毒素休克的实验研究. 生理学报 1996;48:390-394.
13 金玉怀,赵占胜,徐锦荣,等. 八肽胆囊收缩素对大鼠滑膜细胞株RSC-364 增生的影响及其可能的分子机制. 中华风湿病学杂志, 2003; 7: 167-171.
14 龚非力. 医学免疫学. 科学出版社, 2000.
15 Kuchroo, V. K., Das M. P., Brown J. A., Ranger A. M., Zamvil S. S., Sobel R. A., Weiner H. L., Nabavi N., and Glimcher L.H. B7.1 and B7.2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell. 1995,80:707-807
16 Paola Massari, Philipp Henneke, Yu Ho, Eicke Latz, Douglas T. Golenbock, and Lee M. Wetzler. Immune Stimulation by Neisserial Porins Is Toll-Like Receptor 2 and MyD88 Dependent. J Immunol, 2002, 168: 1533–1537.
17 Ruslan Medzhitov and Charles A. Janeway, Jr. Innate Immunity: The Virtues of a Nonclonaln System of Recognition. Cell, 1997; 91, 295–298.
18 June, C.H., Bluestone, J.A., Nadler, L.M., and Thompson, C.B. The B7 and CD28 receptor families. ImmunolToday. 1994, 15:321.
19 Lenschow D.J., Walunas, T.L., and Bluestone J.A. CD28/B7 system of T cell costimulation. Annu.Rev.Immunol.1996, 14:233.
20 Lenschow, DJ., Ho SC., Sattar H., Rhee L., Gray G., Nabavi N., Herold K. C, and Bluestone J. A.. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J. Exp. Med. 1995. 181:1145.
21 Baskar, S., Glimcher L., Nabavi N., Jones R. T., and Ostrand-Rosenberg. S. Major histocompatibility complex class II+B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice. J. Exp. Med. 1995, 181:619.
22 Wu, T. C., Huang A. Y. C., Jaffee E. M., Levitsky H. I., and Pardoll D. M.. A reassessment of the role of B7-1 expression in tumor rejection. J. Exp. Med. 1995, 182:1415.
23 Freeman, G. J., Boussiotis V. A., Anumanthan A., Bernstein G. M., Ke X. Y., Rennert P. D., Gray G. S., Gribben J. G., and Nadler L. M.. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 1995, 2:523.
24 Corry, D. B., Reiner S. L., Linsley P. S., and Locksley T. M. Differential effects of blockade of CD28–B7 on the development of Th1 or Th2 effector cells in experimental Leishmaniasis. J. Immunol. 1994, 153:4142.
25 Thompson, C. B. Distinct roles for the costimulatory ligands B7.1 andB7.2 in T helper cell differentiation? Cell 1995, 81:979.
26 Hathcock. K. S., Laszlo G., Pucillo C., et al. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J. Exp. Med. 1994, 180(8):631-640.
27 Zhang P, Martin M, Yang QB, et al. Role of B7 costimulatory molecules in immune responses and T-helper cell differentiation in response to recombinant HagB from Porphyromonas gingivalis. Infect Immun. 2004 ,72(2):637-644
28 Manickasingham. SP, Anderton. SM, Burkhart C, et al. Qualitative and quantitative effects of CD28/B7-mediated costimulation on na?ve T cells in vitro. J Immunol. 1998, 161(8): 3827-3835.
29 Thorsten W. Orlikowsky, Gunther E. Dannecker, Barael Spring, et al. Effect of examethasone on B7 Regulation and T Cell Activation in Neonates and Adults. Pediatr Res 2005; 57(5):656–661.
30 Lewis L. Lanier, Shawn O’Fallon, Chamorro Somoza, et al. CD80 (B7) and CD86 (B70) Provide Similar Costimulatory Signals for T Cell Proliferation, Cytokine Production, and Generation of CTL. The Journal of Immunology, 1995, 154: 97-105.
31 倪志宇,闫玉仙,丛斌等.CCK-8 对 LPS 攻击小鼠 IL-1β、IL-6、IL-4、IL-10表达的影响.中国病理生理杂志,2007,23(2):331~335.
32 孟爱宏, 凌亦凌, 赵晓云, 等. 丝裂素活化蛋白激酶在胆囊收缩素对脂多糖刺激小鼠脾细胞增殖中的作用. 河北医科大学学报, 2002, 23(1):27~28
33 宋宁,李淑瑾,丛斌等. 八肽胆囊收缩素对经钥孔戚血蓝蛋白免疫小鼠 T 淋巴细胞亚群的影响. 中国病理生理杂志,2007,23(23):423-427
34 De la Fuente M, Campos M, Del Rio M, et al. Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide. Regul Pept, 1995, 55: 47-56.
35 De la Fuente M, Carrasco M, Del Rio M, et al. Modulation of murine lymphocyte functions by sulfated cholecystokinin octapeptide. Neuropeptides,1998, 32: 225-233
36 Ganea D, Delgado M. Neuropeptides as modulators of macrophage functions. Regulation of cytokine production and antigen presentation by VIP and PACAP. Arch Immunol Ther Exp (Warsz). 2001, 49(2):101-110.
37 Cong, Y, Weaver CT, and Elson CO. The mucosal adjuvanticity of cholera toxin involves enhancement of costimulatory activity by selective upregulation of B7.2 expression. J. Immunol. 1997. 159(11):5301-5308.
38 Kalechman, Y, and B. Sredni. Differential effect of the immunomodulator AS101 on B7-1 and B7-2 costimulatory molecules: role in the antitumoral effects of AS101. J. Immunol. 1996.157(2):589-597.
39 Schmittel, A., C. Scheibenbogen, and U. Keilholz. Lipopolysaccharide effectively up-regulates B7-1 (CD80) expression and costimulatory function of human monocytes. Scand. J. Immunol. 1995. 42(6):701-704.
40 Razi-wolf. Z., Freeman G.J., Galvin F, et al. Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells. Proc. Natl. Acad. Sci. USA, 1992, 89(9): 4210-4214.
41 Linsley, P. S., W. Brady, M. Urnes, L. S. Grosmaire, N. K. Damle, and J. A. Ledbetter. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 1991.173(3):721-730.
42 Galvin, F, Freeman GJ, Razi-Wolf Z, et al. Murine B7 antigen provides a sufficient costimulatory signal for antigen-specific and MHC-restricted T cell activation. J. Immunol. 1992.149(12):3802-3808.
43 Freeman, GJ, Gribben JG, Boussioris VA, et al. Cloning of B7.2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 1993. 262(5135):909-911
44 Freeman, G. J., Borriello F., Hodes R. J., et al. Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice. Science 1993.262(5135):907-909.
45 Fields, PE, Finch RJ, Gray GS, et al. B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of naive CD8+TCR- transgenic Tcells. J. Immunol. 1998.161(10):5268-5675.
46 Delgado M, Wei Sun, Javier Leceta, and Doina Ganea. VIP and PACAP differentially regulate the costimulatory activity of resting and activated macrophages through the modulation of B7.1 and B7.2 Expression. J. Immunol. 1999, 163:4213-4223.
47 张风华,李淑瑾,丛斌等. CCK-8 上调小鼠腹腔巨噬细胞 B7.1 和 B7.2表达并增强其协同刺激活性. 中国病理生理杂志,2007,23(4):776-779.
1 Paola Massari, Philipp Henneke, Yu Ho, Eicke Latz, Douglas T. Golenbock, and Lee M. Wetzler. Immune Stimulation by Neisserial Porins Is Toll-Like Receptor 2 and MyD88 Dependent. J Immunol, 2002, 168: 1533–1537.
2 Ruslan Medzhitov and Charles A. Janeway, Jr. Innate Immunity: The Virtues of a Nonclonaln System of Recognition. Cell, 1997; 91, 295–298.
3 Zhong WW, Burke PA, Drotar ME, et al. Effects of prostaglandin E2, cholera toxin and 8-bromo-cycli AMP on lipopolysaccharide induced gene expression of cytokines in nhman macrophages. Immunol, 1995, 84: 446~452
4 Lin K, Abraham KM. Targets of p561ck activity in immature thymoblasts: stimulation of the Ras/Raf/MAPK pathway. Int Immunol, 1997, 9: 291~306
5 Uotila P. The role of cyclic AMP and oxygen intermediates in the inhibition of cellular immunity in cancer. Cancer Immunol Immunother, 1996, 43: 1~9
6 Cong B, Li SJ, Yan YL, et al. Effect of cholecystokinin octapeptide on tumor necrosis factor α transcription and nuclear factor-κB activity induced by lipopolysaccharide in rat pulmonary interstitial macrophages. World J Gastroenterol 2002; 8: 718-723. (SCIE 期刊).
7 Meng AH, Ling YL, Zhang XP, et al. CCK-8 inhibits expression of TNF-α in the spleen of endotoxic shock rats and signal transduction mechanism of P38 MAPK. World J Gastroenterol 2002; 8: 139-143.
8 Ling YL, Meng AH, Zhao XY, et al. Effects of cholecystokinin on cytokines during endotoxic shock in rats. World J Gastroenterol 2001; 7: 667-671.
9 Downey JS, Han J. Cellular activation mechanism in septic shock. Frontiers in Bioscience, 1998, 3: d468~476
10 Huxford T, Huang DB, Maiek S, et al. The crystal structure of the IκBα/NF-κB complex reveals mechanisms of NF-κB inactivation. Cell, 1998, 95:759~770
11 Jacobs MD, Harrison SC. Structure of an IκBα/ NF-κB complex. Cell, 1998, 95:749~758
12 Chen F, Castranova V, Shi X, et al. New insights into the role of Nuclear Factor-κB, an ubiquitous transcription factor in the initiation of diseases. Clin Chem, 1999, 45:1~17
13 Karin M, Ben NY. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol, 2000, 18:621~663
14 Baeuerle PA. IκB- NF-κB structures: at the interface of inflammation control. Cell, 1998, 95: 729~731
15 陈丹英,翟中和,舒红兵.NF-κB 激活的调节机理.科学通报,2003,48(18):1893~1911
16 De la Fuente M, Campos M, Del Rio M, et al. Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide. Regul Pept, 1995, 55: 47~56
17 Carrasco M, Del Rio M, Hernanz A, et al. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides, 1997, 18: 415~422
18 Carrasco M, Hernanz A, De La Fuente M. Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2. Regul Pept, 1997, 70: 135~142
19 高维娟,许顺江,丛斌等. CCK-8 对大鼠肺间质巨噬细胞 cAMP-PKA信号通路的激活作用. 中国药理学通报,2007, 23(3): 321~326
20 Seldon PM, Barnes PJ, Giembycz MA. Interleukin-10 does not mediate the inhibitory effect of PDE-4 inhibitors and other cAMsP-elevating drugs on lipopolysaccharide-induced tumors necrosis factor-alpha generation from human peripheral blood monocytes. Cell Biochem Biophys, 1998, 29: 179~201
21 Vial D, Arbibe L, Havet N, et al. Down-regulation by prostaglandins of type-II phospholipase A2 expression in guinea-pig alveolar macrophages:a possible involvement of cAMP. Biochem J, 1998, 330: 89~94
22 Baldwin AS. The NF-κB and IκB proteins: New discoveries and insights. Annu Rev Immunol, 1996, 14: 649~681
23 Marino CR, Leach SD, Schaefer JF, et al. Characterization of cAMP- dependent protein kinase activation by CCK in rat pancreas. FEBS, 1993, 316(1): 48~52
24 Yule DI, Tseng MJ, Williams JA, et al. A cloned CCK-A receptor transduces multiple signals in response to full and partial agonists. Am J Physiol, 1993, 267:G999~G1004
25 丛斌, 凌亦凌, 谷振勇, 等. 八肽胆囊收缩素对脂多糖诱导的大鼠肺组织NF-κB 活性增高的调节作用. 中国病理生理杂志, 2002, 18:615~618
26 Sweet MJ, Hume DA. Endotoxin signal transduction in macrophages. J Leukoc Biol, 1996, 60: 8~26
27 Ulmer AJ, Rietschel E Th, Zahringer U. Lipopolysaccharide: structure, bioactive, receptors and signal transduction. Trends in Glycoscience and Glycotechnology, 2002, 14(76): 53~66
28 Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol, 2000, 12: 13~19
29 O’Neill LA, Greene C. Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J Leukoc Biol, 1998, 63: 650~657
30 Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol, 2000, 18: 621~663
31 Krupinski J, Coussen F, Bakalyar HA, et al. Adenylyl cyclasebamino acid sequence: possible channel-or transporter like structure. Science, 1989, 244: 1558~1564
32 林明群,张宗梁.蛋白激酶 A(PKA)介导的信号通路:正调节还是负调节?科学通报,1999,44(17):1793~1803
33 Corbin JD, Sugden PH, West L, et al. Studies on the properties and mode of action of the purified regulatory subunit of bovine heart adenosine 3’,5’-monophosphate dependent protein kinase. J Biol Chem, 1978, 253: 3997~4003
34 Pieroni J P, Harry A, Chen J, et al. Distinct characteristics of the basal activities of adenylyl cyclases 2 and 6. J Biol Chem, 1995, 270: 21368~21373
35 Meyer Franke A, Kaplan MR, Pfrieger FW, et al. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron, 1995, 15: 805~819
36 O’Shaughnessy C, Poat JA, Turnbull MJ. Cyclic AMP efflux from rat striatal slices is enhabced by CCK. Biochem Pharmacol, 1987, 36(6):976~979
37 项鹏, 陈曼玲, 吴兆锋. 胆囊收缩素 8 对大鼠大脑皮质细胞钙调素和蛋白激酶 C 活性的影响. 中国生物化学与分子生物学报, 2001, 17(4):510~514
38 Wank SA. Cholecystokinin receptors. Am J Physiol, 1995, 269:G628-646
39 Noble F, Wank SA, Crawley JN, et al. International Union of Pharma- cology. XXI. Structure, distribution, and functions of cholecystokinin receptors. Pharmacol Rev, 1999, 51:745-781
40 Xu SJ, Gao WJ, Cong B, et al. Effect of lipopolysaccharide on expression and characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages. Acta Pharmacol Sin, 2004; 25: 1347-1353.
41 高维娟,许顺江,丛斌,李淑瑾,凌亦凌,姚玉霞,杨世方,孟爱宏,谷振勇.大鼠肺间质巨噬细胞 CCK 受体的结合特性.中国病理生理杂志,2004,20(3): 295-299
42 许顺江,高维娟,姚玉霞,谷振勇,丛斌.脂多糖对大鼠肺间质巨噬细胞 CCK 受体 mRNA 表达的影响.第二军医大学学报,2003,24(11): 1208-1211
43 Wank SA, Harkins R, Jensen RT, et al. Purification, molecular cloning, and funcional expression of the cholecystokinin receptor from rat pancreas. Proc Natl Acad Sci USA, 1992, 82: 3125-3129
44 Wank SA, Pisegna JR, Weerth AD. Brain and gastrointestinalcholecystokinin receptor family: Structure and functional expression. Proc Natl Acad Sci USA, 1992, 89(11): 8691-8695
45 帅建.胆囊收缩素受体分子生物学研究进展.上海第二医科大学学报,1998,18(6):518-521
46 Sacerdote P, Wiedermann CJ, Wahl LM, et al. Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen. Peptides, 1991, 12: 167-176
47 Lignon MF, Bernad N, Martinez J. Pharmacological characterization of type B cholecystokinin binding sites on the human JURKAT T lymphocyte cell line. Mol Pharmacol, 1991, 39: 615-620
48 Dornand J, Serge R, Francoise M, et al. Gastrin-CCK-B type receptors on human T lymphoblastoid Jurkat cells. Am J Physiol, 1995, 268: G522-529
49 Cuq P, Gross A, Terraza A, et al. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurkat lymphoblastoid cells. Life Sci, 1997, 61: 543-555
50 Cong B, Li SJ, Ling YL, et al. Expression and cell-specific localization of cholecystokinin receptors in rat lung. World J Gastroenterol, 2003, 9: 1273-1277
51 Hansen TVO. Cholecystokinin gene transcription: promoter elements, transcription factors and signaling pathways. Peptides, 2001, 22: 1201-1211
1 李淑瑾,凌亦凌,王殿华,谷振勇,孟爱宏,朱铁年. 一氧化氮在八肽胆囊收缩素抗脂多糖致离体兔胸主动脉低血管反应性中的作用. 中国病理生理杂志,2002,18(7):770~773.
2 Cong B, Li SJ, Yan YL, et al. Effect of cholecystokinin octapeptide on tumor necrosis factor α transcription and nuclear factor-κB activity induced by lipopolysaccharide in rat pulmonary interstitial macrophages. World J Gastroenterol, 2002; 8: 718-723.
3 Meng AH, Ling YL, Zhang XP, et al. CCK-8 inhibits expression of TNF-α in the spleen of endotoxic shock rats and signal transduction mechanism of P38 MAPK. World J Gastroenterol, 2002; 8: 139-143.
4 Ling YL, Meng AH, Zhao XY, et al. Effects of cholecystokinin on cytokines during endotoxic shock in rats. World J Gastroenterol, 2001; 7: 667-671.
5 倪志宇,闫玉仙,丛斌等.CCK-8 对 LPS 攻击小鼠 IL-1β、IL-6、IL-4、IL-10表达的影响.中国病理生理杂志,2007,23(2):331~335.
6 Li Shujin, Ni Zhiyu, Cong Bin, Gao Weijuan, Xu Shunjiang, Wang ChunYan, Yao Yuxia, Ma Chunling, Ling Yiling. CCK-8 inhibits LPS-induced IL-1β production in pulmonary interstitial macrophages by PKA, P38, NF-κB pathway. Shock, 2007, in press.
7 倪志宇.河北医科大学博士研究生论文.NO.200201029.2005.
8 Fields, P., Finch, R. J., Gray G. S., et al. B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of na?ve CD28+TCR- transgenic T cells. J. Immunol. 1998, 161:5268-5275.
9 Gause, W.C., Urban J.F., Linsley P. & LU P.. Role of B7 signaling in the differentiation of na?ve CD4+ T cells to effector IL-4-producing T helper cells. Immunol. Res. 1995, 14: 176–188.
10 Manickasingham, S., Anderton S.M., Burkhart C. & Wraith D. C.. Qualitative and quantitative effects of CD28/B7-mediated costimulation on na?ve T cells in vitro. J. Immunol. 1998, 161: 3827–3835.
11 Harris N.L., Peach R.J. & Ronchese F.. CTLA4-Ig inhibits optimal Th2 cell development but not protective immunity or memory response to Nippostrongylus rasiliensis. Eur. J. Immunol. 1999, 29: 311–316.
12 Levine, B. L., Ueda Y., Craighead N., et al. CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro. Int. Immunol. 1995, 7(6): 891-904.
13 Lanier, L.L., O’Fallon S., Somoza C., et al. CD80 (B7-1) and CD86 (B7-2) provide similar costimulatory signals for T cell proliferation, cytokine production and generation of CTL. J. Immunol. 1995, 154(1):97-105.
14 Freeman, G. J., Boussiotis V. A., Anumanthan A., Bernstein G. M., Ke X. Y., Rennert P. D., Gray G. S., Gribben J. G., and Nadler L. M.. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 1995, 2:523.
15 Ranger, A. M., Prabbu Das M., Kuchroo V. K., and Glimcher L. H.. B7-2 (CD86) is essential for the development of interleukin-4 producing T cells. Int. Immunol. 1996,8:1549-1560.
16 Nakajima, A., Watanabe N., Yoshino S., Okumura K., and Azuma N.. Requirement of CD28-CD86 costimulation in the interaction between antigenprimed T helper type 2 and B cells. Int. Immunol. 1997,9:637-644.
17 Palmer, E. M. and van Seventer G. A.. Human T helper cell differentiation is regulated by the combined action of cytokines and accessory cell-dependent costimulatory signals.J. Immunol. 1997, 158(6):2654-2662.
18 Lenschow, D. J., Ho S.C., Sattar H., et al. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J. Exp.Med.1995,181(3): 1145-1155
19 Kuchroo, V.K., Das M.P., Brown J.A., et al. B7.1 and B7.2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell, 1995,80(5): 707-718
20 Thompson, C. B. Distinct roles for the costimulatory ligands B7.1 and B7.2 in T helper cell differentiation? Cell 1995,81:979-982
21 Bluestone, J. A. New perspectives of CD28–B7 mediated T cell costimulation. Immunity 1995,2:555-559
22 De la Fuente M, Medina S, Del Rio M, et al. Effect of aging on the modulation of macrophage functions by neuropeptides. Life Sci. 2000, 67(17):2125-2135
23 Cuq P, Gross A, Terraza A, et al. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurkat lymphoblastoid cells. Life Sci 1997; 61:543-555
24 Carrasco M, Hernanz A, De La Fuente M, et al. Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2. Regul Pept. 1997; 70:135-142
25 Medina S, Rio MD, Cuadra BD, et al. Age-related changes in the modulatory action of gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide in the proliferation of murine lymphocytes. Neuropeptides. 1999; 33: 173-179
26 Carrasco M, Monica DR, Angel H, et al. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides, 1997, 18:415-422
1 Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat. Rev. Immunol. 2002. 2:116–126
2 Khoury SJ, Sayegh MH. The roles of the new negative T cell costimulatory pathways in regulating autoimmunity. Immunity.2004.20:529–538
3 Chen LP. Co-inhibitory molecules of the B7-CD28 family in the control of T cell immunity. Nat. Rev. Immunol. 2004. 4:336–347
4 Abbas AK. The control of T cell activation vs. tolerance. Autoimmun. Rev. 2003. 2:115–118
5 Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu. Rev. Immunol. 2002. 20:29–53
6 Chikuma S, Bluestone JA. CTLA-4 and tolerance: the biochemical point of view. Immunol. Res. 2003.28:241–253
7 Coyle AJ, Gutierrez-Ramos JC. More negative feedback? Nat. Immunol. 2003.4:647–648
8 Greenwald RJ, Latchman YE, Sharpe AH. Negative co-receptors on lymphocytes. Curr. Opin. Immunol. 2002.14:391–396
9 Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, et al. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 1999. 397:263–266
10 Swallow MM, Wallin JJ, Sha WC. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNF-α. Immunity. 1999.11:423–432
11 Ling V, Wu PW, Finnerty HF, Bean KM, Spaulding V, et al. Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor. J. Immunol. 2000. 164:1653–1657
12 Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J, et al. T-cell costimulation through B7RP-1 and ICOS. Nature 1999. 402:827–832
13 Brodie D, Collins AV, Iaboni A, Fennelly JA, Sparks LM, et al. LICOS, a primordial costimulatory ligand? Curr. Biol. 2000.10:333–336
14 Wang SD, Zhu GF, Chapoval AI, Dong H, Tamada K, et al. Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS. Blood 2000. 96:2808–2813
15 Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992.11:3887–3895
16 Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 2000.192:1027–1034
17 Dong HD, Zhu GF, Tamada K, Chen LP. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 1999. 5:1365–1369
18 Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, et al. 2001. PDL2 is a second ligand for PD-1 and inhibits T cell activation. Nat. Immunol. 2:261–268
19 Tseng SY, Otsuji M, Gorski K, Huang X, Slansky JE, et al. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J.Exp. Med. 2001.193:839–846
20 Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, et al. B7-H3: a costimulatory molecule for T cell activation and IFN-γ production. Nat. Immunol. 2001. 2:269–274
21 Sica GL, Choi IH, Zhu GF, Tamada K, Wang SD, et al. B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 2003. 18:849–861
22 Zang XX, Loke P, Kim J, Murphy K, Waitz R, Allison JP. B7x: a widely expressed B7 family member that inhibits T cell activation. Proc. Natl. Acad. Sci. USA 2003. 100:10388–10392
23 Prasad DVR, Richards S, Mai XM, Dong C. 2003. B7S1, a novelB7 family member that negatively regulates T cell activation. Immunity 18:863–873
24 Sansom D, Manzotti C, Zheng Y. What’s the difference between CD80and CD86.Trends in Immunology. 2003. 24(6):313-318
25 Shin T, Kennedy G, Gorski K, et a1. Cooerative B7-1/2(CD80/CD86) and B7-DC costimulation of CD4+T cells independent of the PD-1 receptor.Journal of Experimental Medicine. 2003,198(1):31-38
26 Wang SD, Bajorath J, Dallas B. Molecular modeling and functional mapping of B7-H1 and B7-DC uncouple costimulatory function from PD-1 interaction. Journal of Experimental Medicine. 2003. 197(9):1083-1091
27 Ito T, Ueon T, Clarkson MR, et a1. Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo. Journal of Immunology. 2005. 174(3):6648-6656
28 Taylor PA, Lees CJ, Fournier S, Allison JP, Sharpe AH, Blazar BR. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J. Immunol. 2004.172:34–39
29 Paust S, Lu LR, McCarty N, Cantor H. Engagement of B7 on effector T cells by regulatory T cells prevents autoimmune disease. Proc. Natl. Acad. Sci USA .2004.101:10398–10403
30 Tang QZ, Henriksen KJ, Boden EK, Tooley AJ, Ye JQ, et al. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J. Immunol. 2003.171:3348–3352
31 Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 2002. 3:1097–10101
32 Short JJ, Pereboev AV, Kawakami Y, Vasu C, Holterman MJ, Curiel DT. Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. Virology 2004.322:349–359
33 Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, et al. Induction of B7-1 in podocytes is associated with nephritic syndrome. J. Clin. Invest. 2004. 113:1390–1397
34 Lohr J, Knoechel B, Jiang S, Sharpe AH, Abbas AK. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nat. Immunol. 2003. 4:664–669
35 Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 2003.4:1206–1212
36 Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J. Immunol. 2004.172:4100–4110
37 Podojil JR, Kin NW, Sanders VM. CD86 and β2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells. J. Biol. Chem. 2004. 279:23394–23404
38 Podojil JR, Sanders VM. Selective regulation of mature IgG1 transcription by CD86 and β2-adrenergic receptor stimulation. J. Immunol. 2003. 170:5143–5151
39 Ling V,Wu PW,Miyashiro JS,et a1.Cutting edge:identification of GL50,a novel B7-like protein that functionally binds to ICOS receptor. J Immunol. 2000. 164:1653-1657
40 LingV,Wu PW,Miyashim JS,et al.Differential expression of inducible costimulator-ligand splice variants:lymphoid regulation of mouse GL50-B and human GL50 molecules. J Immunol. 2001, 166:7300-7308
41 Wang S,Zhu G,Chapoval AI,et a1.Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS . Blood, 2000, 96: 2808-2813
42 Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000.13:95–105
43 Okamoto N, Tezuka K, Kato M, Abe R, Tsuji T. PI3-kinase and MAPkinase signaling cascades in AILIM/ICOS- and CD28-costimulated T-cells have distinct functions between cell proliferation and IL-10 production. Biochem. Biophys. Res. Commun. 2003.310:691–702
44 Harada Y, Tanabe E, Watanabe R, Weiss BD, Matsumoto A, et al. Novel role of phosphatidylinositol 3-kinase in CD28-mediated costimulation. J. Biol. Chem. 2001.276:9003–9008
45 Parry RV, Rumbley CA, Vandenberghe LH, June CH, Riley JL. CD28 and inducible costimulatory protein Src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase, Bcl-xL, and IL-2 expression in primary human CD4 T lymphocytes. J. Immunol. 2003.171:166–174
46 McAdam AJ, Chang TT, Lumelsky AE, Greenfield EA, BoussiotisVA, et al. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells. J. Immunol. 2000.165:5035–5040
47 Wassink L, Vieira PL, Smits HH, Kingsbury GA, Coyle AJ, et al. ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells. J. Immunol. 2004. 173:1779–1786
48 Beier KC, Hutloff A, Dittrich AM, Heuck C, Rauch A, et al. Induction, binding specificity and function of human ICOS. Eur. J. Immunol. 2000. 30:3707–3717
49 Mages HW, Hutloff A, Heuck C, Buchner K, Himmelbauer H, et al. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. Eur. J. Immunol. 2000. 30:1040–4107
50 Richter G, Hayden-Ledbetter M, Irgang M, Ledbetter JA, Westermann J, et al. Tumor necrosis factor- α regulates the expression of inducible costimulator receptor ligand onCD34+ progenitor cells during differentiation into antigen presenting cells. J. Biol. Chem. 2001.276:45686–45693
51 Yoshinaga SK, Zhang M, Pistillo J, Horan T, Khare SD, et al. Characterization of a newhuman B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS. Int. Immunol. 2000.12:1439–1447
52 Aicher A, Hayden-Ledbetter M, Brady WA, Pezzutto A, Richter G, et al. Characterization of human inducible costimulator ligand expression and function. J. Immunol. 2000.164:4689–4696
53 Wiendl H, Mitsdoerffer M, Schneider D, Melms A, Lochmuller H, et al.Muscle fibres and cultured muscle cells express the B7.1/2-related inducible costimulatory molecule, ICOSL: implications for the pathogenesis of inflammatory myopathies. Brain 2003.126:1026–1035
54 Liang L, Porter EM, Sha WC. Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling. J. Exp. Med. 2002.196:97–108
55 Kopf M, Coyle AJ, Schmitz N, Barner M, Oxenius A, et al. Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection. J. Exp. Med. 2000.192:53–61
56 Villegas EN, Lieberman LA, Mason N, Blass SL, Zediak VP, et al. A role for inducible costimulator protein in the CD28-independent mechanism of resistance to Toxoplasma gondii. J. Immunol. 2002. 169:937–943
57 Kanai T, Totsuka T, Tezuka K, Watanabe M. 2002. ICOS costimulation in inflammatory bowel disease. J. Gastroenterol. 37(Suppl. 14):78–81
58 Akbari O, Freeman GJ,Meyer EH, Greenfield EA, Chang TT, et al. 2002. Antigenspecific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat. Med. 8:1024–1032
59 Bonhagen K, Liesenfeld O, Stadecker MJ, Hutloff A, Erb K, et al. 2003. ICOS+ Th cells produce distinct cytokines in different mucosal immune responses. Eur. J. Immunol. 33:392–401
60 Rudd CE, Schneider H. 2003. Unifying concepts in CD28, ICOS and CTLA4 coreceptor signalling. Nat. Rev. Immunol. 3: 544–556
61 Nurieva RI, Duong J, Kishikawa H, Dianzani U, Rojo JM, et al. 2003. Transcriptional regulation of th2 differentiation by inducible costimulator. Immunity 18:801–811
62 Nurieva RI, Mai XM, Forbush K, Bevan MJ, Dong C. 2003. B7h is required for T cell activation, differentiation, and effector function. Proc. Natl. Acad. Sci. USA 100:14163–14168
63 Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, et al. B7-H3: acostimulatory molecule for T cell activation and IFN-γ production. Nat. Immunol. 2001.2:269–274
64 Sporici RA, Beswick RL, von Allmen C, Rumbley CA, Hayden-Ledbetter M, et al. ICOS ligand costimulation is required for T-cell encephalitogenicity. Clin. Immunol. 2001.100:277–288
65 Rottman JB, Smith T, Tonra JR, Ganley K, Bloom T, et al. The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE. Nat. Immunol. 2001.2:605–611
66 Dong C, Nurieva RI. Regulation of immune and autoimmune responses by ICOS. J. Autoimmun. 2003. 21:255–260
67 Iwai H, Kozono Y, Hirose S, Akiba H, Yagita H, et al. Amelioration of collagen-induced arthritis by blockade of inducible costimulator-B7 homologous protein costimulation. J. Immunol. 2002.169:4332–4339
68 Nurieva RI, Treuting P, Duong J, Flavell RA, Dong C. Inducible costimulator is essential for collagen-induced arthritis. J. Clin. Invest. 2003. 111:701–706
69 Herman AE, Freeman GJ, Mathis D, Benoist C. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J. Exp. Med. 2004. 199:1479–1489
70 Coyle AJ, Gutierrez-Ramos JC. The role of ICOS and other costimulatory molecules in allergy and asthma. Springer Semin. Immunopathol. 2004.25:349–359
71 Dong C, Juedes AE, Temann UA, Shresta S, Allison JP, et al. ICOS costimulatory receptor is essential for T-cell activation and function. Nature 2001. 409:97–101
72 Gonzalo JA, Tian J, Delaney T, Corcoran J, Rottman JB, et al. ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. Nat. Immunol. 2001.2:597–604
73 Taylor PA,Panoskaltsis-Mortari A,Freeman GJ,et al.Targeting of inducible costimulator(ICOS) expressed on alloreative T-cells down regulates graft-versus-host disease(GVHD) and facilitates engraftment ofallogeneic bone marrow(BM). Blood. 2005, 105:3372-3380
74 Schreiner B,Mitsdoerffer M,Kieseier BC,et a1.Interferon-beta enhances monocyte and dendritic cell expression of B7-H1(PD-L1),a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis . J Neuroimmunol. 2004. 155(1-2):172-182
75 Wiendl H,Mitsdoerffer M,Schneider D,et a1.Human muscle cells express a B7-related molecule,B7-H1,with strong negative immune regulatory potential:a novel mechanism of counterbalancing the immune attack in idiopathic inflammatory myopathies.FASEB J. 2003, 17(13): 1892-1894
76 Petroff MG,Chen L,Phillips TA,et a1. B7 family molecules are favorably positioned at the human maternal-fetal interface. Bid Reprod. 2003. 68(5): 1496-1504
77 Can Y,Zhou H,Tao J,et a1.Keratinocytes induce local tolerance to skin graft by activating interleukin-10-secreting T cells in the context of costimulation molecule B7-H1. Transplantation. 2003. 75(8):1390-l396
78 Freeman GJ,Long AJ,Lwei Y,et a1.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.J Exp Med. 2000. 192(7):1027-1034
79 Dong HD,Stroke SE,Salomao DR,et a1.Tumor-associated B7-H1 promotes T-cell apoptosis:A potential mechanism of immune evasion. Nat Med. 2002. 8(8):793-800
80 Konishi J,Yarnazaki K,Azuma M,et a1. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res. 2004, 10(15): 5094-5100
81 Kanai T,Totsuka K,Uraushihara K,et a1.Blocked of B7-H1 suppresses the development of chronic intestinal inflammation. J lmmunol. 2003. 171(8):4156-4163
82 Ishida Y,Agate Y,sahib Sahara,et a1.induced expression of PD-1, a novel member of the immunoglobulin gene super family,upon programmed celldeath.J Ambo J. 1992. 11:3887-3895
83 Ravetch JV,Lanier LL,Immune inhibitory receptors,Science,2000, 290:84-89
84 Sharpe AH,Freeman GJ,The B7-CD28 Super family. J.Nat Revlmmunjol. 2002. 2(2):116-126.
85 Tamura H,Dong HD,Zhu GF,et al,B7HI costimulation preferentially enhances CD28-independent T-helper cell function. J. Blood, 2001, 97 (60):1809-1816,
86 Ngulyen LT, Radhakrishnan S, Iric B, et a1. Cross-linking the B7 family molecule B7-DC directly activates immune functions of dendritic cells. Journal of Experimental Medcine, 2002, 196(10):1393-1398
87 Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur. J. Immunol. 2003. 33:2706–2716
88 Loke P, Allison JP. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells. Proc. Natl. Acad. Sci. USA 2003. 100:5336–5341
89 Zhang X, Schwartz JC, Gun X, et a1. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity, 2004, 20(3):337-347
90 Ozkuynak E, Wang L, Goodearl A, et a1. Programmed death-1 targeting can promote allograft survival. Journal of Immunology, 2002, 169(11):6546-6553
91 Rietz C, Chen L.New B7 family member's with positive and negative costimulatory function. Am J Transplant, 2004. 4(1):8-14
92 Salarna AD, Imitola TC, Akiba H, et a1. Critical role of programmed death-1(PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. Journal of Experimental Medcine. 2003. 198(1):71-78
93 Dong H, Strome SE, Matesin EL, et a1. Costimulating aberrant T cell responses by b7-h1 autoantibedies in Rheumatoid arthritis. Journal of Clinical Investigation. 2003. 111(3):363-370
94 Bennett F, Luxenberg D, Ling V,et a1.Program death-1 engagement upontcr activation has distince effects on costimulation and cytokin-driven proliferation: attenuation of ICOS, IL-4 and IL-21, but not CD8, IL-7 and IL-15 responses. Joumal of Immunology. 2003. 170(2):711-718
95 Shin T, Kennedy G, Gorski K, et a1. Cooperative B7-1/2(CD80/86) and B7-DC costimulation of CD4+T cells independent of the PD-1 receptor. Journal of Experimental Medicine, 2003. 198(1):31-38
96 Loke P, Allison JP. PD-L1 and PD-L2 are differentially related by Th1 and Th2 cells. Proc Natl Aad Sci, 2003, 100(9): 5336-5341
97 Liu X,Gao J,Wen J,et a1.B7-DC/PD-12 promotes tumor immunity by a PD-1-independent mechanism. Journal of Experimental Medicine. 2008. l97(2): 1721-1730.
98 Prasad DVR, Nguyen T, Li ZX, et a1. Murine B7-H3 is a negative regulator of T cells. Journal of Immunology. 2004. 173:2500-2506
99 Suh WK, Gajewska BU, Okada H, Gronski MA, Bertram EM, et al. The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses. Nat. Immunol. 2003.4:899-906
100 Zang X, Loke P, Kim J, et a1. B7x, a widely expressed B7 family member that inhibits T cell activation. Proc Nail Aad Sci, 2003, 1130(8):10388-10392
101 Liqing Wang,Christopher C,Fraser,et a1. Eur J Immuno1.2005,35(2): 428-438
102 Prasad DVR, Richards S, Mai X, et a1. B7S1, a novel B7 family member that negatively regulates T cell activation. Immunity, 2003.18(6):863-873
103 Watanabe N, Gavrieli M, Sedy JR, et a1. BTLA is a lymphocyte inhibitory receptor with similarities to TLA-4 and PD-1. Nature Immunology. 2003. 4(1):670-679
104 Steinberger P, Majdic O, Derdak SV, Pfistershammer K, Kirchberger S, et al. 2004. Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains. J. Immunol. 172:2352–59
105 Sica GL, Choi IN,Zhu G, et a1. Immunity. 2003, 18:849-86
106 Sedy JR, Gavrieli M, Potter KG, et a1. Nat Immuno1.2005. 6(1):90-98
107 Gonzalez LC, Loyet KM, Calemine-Fenaux J, et a1. Proc Natl Acad Sci USA. 2005. 102(4) :1116-1121
108 Sica GL, Choi IH, Zhu G, et al. Immunity. 2003. 18(6):849-861
2 Meng AH, Ling YL, Zhang XP, et al. CCK-8 inhibits expression of TNF-α in the spleen of endotoxic shock rats and signal transduction mechanism of P38 MAPK. World J Gastroenterol 2002; 8: 139-143.
3 Ling YL, Meng AH, Zhao XY, et al. Effects of cholecystokinin on cytokines during endotoxic shock in rats. World J Gastroenterol 2001; 7: 667-671.
4 Cuq P, Gross A, Terraza A, et al. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurkat lymphoblastoid cells. Life Sci 1997; 61:543-555.
5 Kimoto Y. A possibility of all mRNA expression in a human singlelymphocyte. Hum Cell 1996; 9:367-370.
6 Carrasco M, Hernanz A, De La Fuente M. Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2. Regul Pept. 1997; 70:135-142.
7 Medina S, Rio MD, Cuadra BD, et al. Age-related changes in the modulatory action of gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide in the proliferation of murine lymphocytes. Neuropeptides. 1999; 33: 173-179.
8 Xu SJ, Gao WJ, Cong B, et al. Effect of lipopolysaccharide on expression and characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages. Acta Pharmacol Sin. 2004; 25: 1347-1353.
9 Lauw FN, ten Hove T, Dekkers PE, et al. Reduced Th1, but not Th2, cytokine production by lymphocytes after in vivo exposure of healthy subjects to endotoxin. Infect Immun. 2000; 68:1014-1018.
10 Iwasaka H, Noguchi T. Th1/Th2 balance in systemic inflammatory response syndrome (SIRS). Nippon Rinsho. 200; 62:2237-2243.
11 Yudoh K, Matsuno H , Nakazawa F , et al . Reduced expression of theregulatory CD4+ T cell subset is related to Th1/ Th2 balance and diseaseseverity in rheumatoid arthritis . Arthritis Rheuma 2000; 43 : 617- 627.
12 凌亦凌,黄善生,王乐丰,等. 八肽胆囊收缩素抗内毒素休克的实验研究. 生理学报 1996;48:390-394.
13 金玉怀,赵占胜,徐锦荣,等. 八肽胆囊收缩素对大鼠滑膜细胞株RSC-364 增生的影响及其可能的分子机制. 中华风湿病学杂志, 2003; 7: 167-171.
14 龚非力. 医学免疫学. 科学出版社, 2000.
15 Kuchroo, V. K., Das M. P., Brown J. A., Ranger A. M., Zamvil S. S., Sobel R. A., Weiner H. L., Nabavi N., and Glimcher L.H. B7.1 and B7.2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell. 1995,80:707-807
16 Paola Massari, Philipp Henneke, Yu Ho, Eicke Latz, Douglas T. Golenbock, and Lee M. Wetzler. Immune Stimulation by Neisserial Porins Is Toll-Like Receptor 2 and MyD88 Dependent. J Immunol, 2002, 168: 1533–1537.
17 Ruslan Medzhitov and Charles A. Janeway, Jr. Innate Immunity: The Virtues of a Nonclonaln System of Recognition. Cell, 1997; 91, 295–298.
18 June, C.H., Bluestone, J.A., Nadler, L.M., and Thompson, C.B. The B7 and CD28 receptor families. ImmunolToday. 1994, 15:321.
19 Lenschow D.J., Walunas, T.L., and Bluestone J.A. CD28/B7 system of T cell costimulation. Annu.Rev.Immunol.1996, 14:233.
20 Lenschow, DJ., Ho SC., Sattar H., Rhee L., Gray G., Nabavi N., Herold K. C, and Bluestone J. A.. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J. Exp. Med. 1995. 181:1145.
21 Baskar, S., Glimcher L., Nabavi N., Jones R. T., and Ostrand-Rosenberg. S. Major histocompatibility complex class II+B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice. J. Exp. Med. 1995, 181:619.
22 Wu, T. C., Huang A. Y. C., Jaffee E. M., Levitsky H. I., and Pardoll D. M.. A reassessment of the role of B7-1 expression in tumor rejection. J. Exp. Med. 1995, 182:1415.
23 Freeman, G. J., Boussiotis V. A., Anumanthan A., Bernstein G. M., Ke X. Y., Rennert P. D., Gray G. S., Gribben J. G., and Nadler L. M.. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 1995, 2:523.
24 Corry, D. B., Reiner S. L., Linsley P. S., and Locksley T. M. Differential effects of blockade of CD28–B7 on the development of Th1 or Th2 effector cells in experimental Leishmaniasis. J. Immunol. 1994, 153:4142.
25 Thompson, C. B. Distinct roles for the costimulatory ligands B7.1 andB7.2 in T helper cell differentiation? Cell 1995, 81:979.
26 Hathcock. K. S., Laszlo G., Pucillo C., et al. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J. Exp. Med. 1994, 180(8):631-640.
27 Zhang P, Martin M, Yang QB, et al. Role of B7 costimulatory molecules in immune responses and T-helper cell differentiation in response to recombinant HagB from Porphyromonas gingivalis. Infect Immun. 2004 ,72(2):637-644
28 Manickasingham. SP, Anderton. SM, Burkhart C, et al. Qualitative and quantitative effects of CD28/B7-mediated costimulation on na?ve T cells in vitro. J Immunol. 1998, 161(8): 3827-3835.
29 Thorsten W. Orlikowsky, Gunther E. Dannecker, Barael Spring, et al. Effect of examethasone on B7 Regulation and T Cell Activation in Neonates and Adults. Pediatr Res 2005; 57(5):656–661.
30 Lewis L. Lanier, Shawn O’Fallon, Chamorro Somoza, et al. CD80 (B7) and CD86 (B70) Provide Similar Costimulatory Signals for T Cell Proliferation, Cytokine Production, and Generation of CTL. The Journal of Immunology, 1995, 154: 97-105.
31 倪志宇,闫玉仙,丛斌等.CCK-8 对 LPS 攻击小鼠 IL-1β、IL-6、IL-4、IL-10表达的影响.中国病理生理杂志,2007,23(2):331~335.
32 孟爱宏, 凌亦凌, 赵晓云, 等. 丝裂素活化蛋白激酶在胆囊收缩素对脂多糖刺激小鼠脾细胞增殖中的作用. 河北医科大学学报, 2002, 23(1):27~28
33 宋宁,李淑瑾,丛斌等. 八肽胆囊收缩素对经钥孔戚血蓝蛋白免疫小鼠 T 淋巴细胞亚群的影响. 中国病理生理杂志,2007,23(23):423-427
34 De la Fuente M, Campos M, Del Rio M, et al. Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide. Regul Pept, 1995, 55: 47-56.
35 De la Fuente M, Carrasco M, Del Rio M, et al. Modulation of murine lymphocyte functions by sulfated cholecystokinin octapeptide. Neuropeptides,1998, 32: 225-233
36 Ganea D, Delgado M. Neuropeptides as modulators of macrophage functions. Regulation of cytokine production and antigen presentation by VIP and PACAP. Arch Immunol Ther Exp (Warsz). 2001, 49(2):101-110.
37 Cong, Y, Weaver CT, and Elson CO. The mucosal adjuvanticity of cholera toxin involves enhancement of costimulatory activity by selective upregulation of B7.2 expression. J. Immunol. 1997. 159(11):5301-5308.
38 Kalechman, Y, and B. Sredni. Differential effect of the immunomodulator AS101 on B7-1 and B7-2 costimulatory molecules: role in the antitumoral effects of AS101. J. Immunol. 1996.157(2):589-597.
39 Schmittel, A., C. Scheibenbogen, and U. Keilholz. Lipopolysaccharide effectively up-regulates B7-1 (CD80) expression and costimulatory function of human monocytes. Scand. J. Immunol. 1995. 42(6):701-704.
40 Razi-wolf. Z., Freeman G.J., Galvin F, et al. Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells. Proc. Natl. Acad. Sci. USA, 1992, 89(9): 4210-4214.
41 Linsley, P. S., W. Brady, M. Urnes, L. S. Grosmaire, N. K. Damle, and J. A. Ledbetter. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 1991.173(3):721-730.
42 Galvin, F, Freeman GJ, Razi-Wolf Z, et al. Murine B7 antigen provides a sufficient costimulatory signal for antigen-specific and MHC-restricted T cell activation. J. Immunol. 1992.149(12):3802-3808.
43 Freeman, GJ, Gribben JG, Boussioris VA, et al. Cloning of B7.2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 1993. 262(5135):909-911
44 Freeman, G. J., Borriello F., Hodes R. J., et al. Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice. Science 1993.262(5135):907-909.
45 Fields, PE, Finch RJ, Gray GS, et al. B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of naive CD8+TCR- transgenic Tcells. J. Immunol. 1998.161(10):5268-5675.
46 Delgado M, Wei Sun, Javier Leceta, and Doina Ganea. VIP and PACAP differentially regulate the costimulatory activity of resting and activated macrophages through the modulation of B7.1 and B7.2 Expression. J. Immunol. 1999, 163:4213-4223.
47 张风华,李淑瑾,丛斌等. CCK-8 上调小鼠腹腔巨噬细胞 B7.1 和 B7.2表达并增强其协同刺激活性. 中国病理生理杂志,2007,23(4):776-779.
1 Paola Massari, Philipp Henneke, Yu Ho, Eicke Latz, Douglas T. Golenbock, and Lee M. Wetzler. Immune Stimulation by Neisserial Porins Is Toll-Like Receptor 2 and MyD88 Dependent. J Immunol, 2002, 168: 1533–1537.
2 Ruslan Medzhitov and Charles A. Janeway, Jr. Innate Immunity: The Virtues of a Nonclonaln System of Recognition. Cell, 1997; 91, 295–298.
3 Zhong WW, Burke PA, Drotar ME, et al. Effects of prostaglandin E2, cholera toxin and 8-bromo-cycli AMP on lipopolysaccharide induced gene expression of cytokines in nhman macrophages. Immunol, 1995, 84: 446~452
4 Lin K, Abraham KM. Targets of p561ck activity in immature thymoblasts: stimulation of the Ras/Raf/MAPK pathway. Int Immunol, 1997, 9: 291~306
5 Uotila P. The role of cyclic AMP and oxygen intermediates in the inhibition of cellular immunity in cancer. Cancer Immunol Immunother, 1996, 43: 1~9
6 Cong B, Li SJ, Yan YL, et al. Effect of cholecystokinin octapeptide on tumor necrosis factor α transcription and nuclear factor-κB activity induced by lipopolysaccharide in rat pulmonary interstitial macrophages. World J Gastroenterol 2002; 8: 718-723. (SCIE 期刊).
7 Meng AH, Ling YL, Zhang XP, et al. CCK-8 inhibits expression of TNF-α in the spleen of endotoxic shock rats and signal transduction mechanism of P38 MAPK. World J Gastroenterol 2002; 8: 139-143.
8 Ling YL, Meng AH, Zhao XY, et al. Effects of cholecystokinin on cytokines during endotoxic shock in rats. World J Gastroenterol 2001; 7: 667-671.
9 Downey JS, Han J. Cellular activation mechanism in septic shock. Frontiers in Bioscience, 1998, 3: d468~476
10 Huxford T, Huang DB, Maiek S, et al. The crystal structure of the IκBα/NF-κB complex reveals mechanisms of NF-κB inactivation. Cell, 1998, 95:759~770
11 Jacobs MD, Harrison SC. Structure of an IκBα/ NF-κB complex. Cell, 1998, 95:749~758
12 Chen F, Castranova V, Shi X, et al. New insights into the role of Nuclear Factor-κB, an ubiquitous transcription factor in the initiation of diseases. Clin Chem, 1999, 45:1~17
13 Karin M, Ben NY. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol, 2000, 18:621~663
14 Baeuerle PA. IκB- NF-κB structures: at the interface of inflammation control. Cell, 1998, 95: 729~731
15 陈丹英,翟中和,舒红兵.NF-κB 激活的调节机理.科学通报,2003,48(18):1893~1911
16 De la Fuente M, Campos M, Del Rio M, et al. Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide. Regul Pept, 1995, 55: 47~56
17 Carrasco M, Del Rio M, Hernanz A, et al. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides, 1997, 18: 415~422
18 Carrasco M, Hernanz A, De La Fuente M. Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2. Regul Pept, 1997, 70: 135~142
19 高维娟,许顺江,丛斌等. CCK-8 对大鼠肺间质巨噬细胞 cAMP-PKA信号通路的激活作用. 中国药理学通报,2007, 23(3): 321~326
20 Seldon PM, Barnes PJ, Giembycz MA. Interleukin-10 does not mediate the inhibitory effect of PDE-4 inhibitors and other cAMsP-elevating drugs on lipopolysaccharide-induced tumors necrosis factor-alpha generation from human peripheral blood monocytes. Cell Biochem Biophys, 1998, 29: 179~201
21 Vial D, Arbibe L, Havet N, et al. Down-regulation by prostaglandins of type-II phospholipase A2 expression in guinea-pig alveolar macrophages:a possible involvement of cAMP. Biochem J, 1998, 330: 89~94
22 Baldwin AS. The NF-κB and IκB proteins: New discoveries and insights. Annu Rev Immunol, 1996, 14: 649~681
23 Marino CR, Leach SD, Schaefer JF, et al. Characterization of cAMP- dependent protein kinase activation by CCK in rat pancreas. FEBS, 1993, 316(1): 48~52
24 Yule DI, Tseng MJ, Williams JA, et al. A cloned CCK-A receptor transduces multiple signals in response to full and partial agonists. Am J Physiol, 1993, 267:G999~G1004
25 丛斌, 凌亦凌, 谷振勇, 等. 八肽胆囊收缩素对脂多糖诱导的大鼠肺组织NF-κB 活性增高的调节作用. 中国病理生理杂志, 2002, 18:615~618
26 Sweet MJ, Hume DA. Endotoxin signal transduction in macrophages. J Leukoc Biol, 1996, 60: 8~26
27 Ulmer AJ, Rietschel E Th, Zahringer U. Lipopolysaccharide: structure, bioactive, receptors and signal transduction. Trends in Glycoscience and Glycotechnology, 2002, 14(76): 53~66
28 Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol, 2000, 12: 13~19
29 O’Neill LA, Greene C. Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J Leukoc Biol, 1998, 63: 650~657
30 Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol, 2000, 18: 621~663
31 Krupinski J, Coussen F, Bakalyar HA, et al. Adenylyl cyclasebamino acid sequence: possible channel-or transporter like structure. Science, 1989, 244: 1558~1564
32 林明群,张宗梁.蛋白激酶 A(PKA)介导的信号通路:正调节还是负调节?科学通报,1999,44(17):1793~1803
33 Corbin JD, Sugden PH, West L, et al. Studies on the properties and mode of action of the purified regulatory subunit of bovine heart adenosine 3’,5’-monophosphate dependent protein kinase. J Biol Chem, 1978, 253: 3997~4003
34 Pieroni J P, Harry A, Chen J, et al. Distinct characteristics of the basal activities of adenylyl cyclases 2 and 6. J Biol Chem, 1995, 270: 21368~21373
35 Meyer Franke A, Kaplan MR, Pfrieger FW, et al. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron, 1995, 15: 805~819
36 O’Shaughnessy C, Poat JA, Turnbull MJ. Cyclic AMP efflux from rat striatal slices is enhabced by CCK. Biochem Pharmacol, 1987, 36(6):976~979
37 项鹏, 陈曼玲, 吴兆锋. 胆囊收缩素 8 对大鼠大脑皮质细胞钙调素和蛋白激酶 C 活性的影响. 中国生物化学与分子生物学报, 2001, 17(4):510~514
38 Wank SA. Cholecystokinin receptors. Am J Physiol, 1995, 269:G628-646
39 Noble F, Wank SA, Crawley JN, et al. International Union of Pharma- cology. XXI. Structure, distribution, and functions of cholecystokinin receptors. Pharmacol Rev, 1999, 51:745-781
40 Xu SJ, Gao WJ, Cong B, et al. Effect of lipopolysaccharide on expression and characterization of cholecystokinin receptors in rat pulmonary interstitial macrophages. Acta Pharmacol Sin, 2004; 25: 1347-1353.
41 高维娟,许顺江,丛斌,李淑瑾,凌亦凌,姚玉霞,杨世方,孟爱宏,谷振勇.大鼠肺间质巨噬细胞 CCK 受体的结合特性.中国病理生理杂志,2004,20(3): 295-299
42 许顺江,高维娟,姚玉霞,谷振勇,丛斌.脂多糖对大鼠肺间质巨噬细胞 CCK 受体 mRNA 表达的影响.第二军医大学学报,2003,24(11): 1208-1211
43 Wank SA, Harkins R, Jensen RT, et al. Purification, molecular cloning, and funcional expression of the cholecystokinin receptor from rat pancreas. Proc Natl Acad Sci USA, 1992, 82: 3125-3129
44 Wank SA, Pisegna JR, Weerth AD. Brain and gastrointestinalcholecystokinin receptor family: Structure and functional expression. Proc Natl Acad Sci USA, 1992, 89(11): 8691-8695
45 帅建.胆囊收缩素受体分子生物学研究进展.上海第二医科大学学报,1998,18(6):518-521
46 Sacerdote P, Wiedermann CJ, Wahl LM, et al. Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen. Peptides, 1991, 12: 167-176
47 Lignon MF, Bernad N, Martinez J. Pharmacological characterization of type B cholecystokinin binding sites on the human JURKAT T lymphocyte cell line. Mol Pharmacol, 1991, 39: 615-620
48 Dornand J, Serge R, Francoise M, et al. Gastrin-CCK-B type receptors on human T lymphoblastoid Jurkat cells. Am J Physiol, 1995, 268: G522-529
49 Cuq P, Gross A, Terraza A, et al. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurkat lymphoblastoid cells. Life Sci, 1997, 61: 543-555
50 Cong B, Li SJ, Ling YL, et al. Expression and cell-specific localization of cholecystokinin receptors in rat lung. World J Gastroenterol, 2003, 9: 1273-1277
51 Hansen TVO. Cholecystokinin gene transcription: promoter elements, transcription factors and signaling pathways. Peptides, 2001, 22: 1201-1211
1 李淑瑾,凌亦凌,王殿华,谷振勇,孟爱宏,朱铁年. 一氧化氮在八肽胆囊收缩素抗脂多糖致离体兔胸主动脉低血管反应性中的作用. 中国病理生理杂志,2002,18(7):770~773.
2 Cong B, Li SJ, Yan YL, et al. Effect of cholecystokinin octapeptide on tumor necrosis factor α transcription and nuclear factor-κB activity induced by lipopolysaccharide in rat pulmonary interstitial macrophages. World J Gastroenterol, 2002; 8: 718-723.
3 Meng AH, Ling YL, Zhang XP, et al. CCK-8 inhibits expression of TNF-α in the spleen of endotoxic shock rats and signal transduction mechanism of P38 MAPK. World J Gastroenterol, 2002; 8: 139-143.
4 Ling YL, Meng AH, Zhao XY, et al. Effects of cholecystokinin on cytokines during endotoxic shock in rats. World J Gastroenterol, 2001; 7: 667-671.
5 倪志宇,闫玉仙,丛斌等.CCK-8 对 LPS 攻击小鼠 IL-1β、IL-6、IL-4、IL-10表达的影响.中国病理生理杂志,2007,23(2):331~335.
6 Li Shujin, Ni Zhiyu, Cong Bin, Gao Weijuan, Xu Shunjiang, Wang ChunYan, Yao Yuxia, Ma Chunling, Ling Yiling. CCK-8 inhibits LPS-induced IL-1β production in pulmonary interstitial macrophages by PKA, P38, NF-κB pathway. Shock, 2007, in press.
7 倪志宇.河北医科大学博士研究生论文.NO.200201029.2005.
8 Fields, P., Finch, R. J., Gray G. S., et al. B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of na?ve CD28+TCR- transgenic T cells. J. Immunol. 1998, 161:5268-5275.
9 Gause, W.C., Urban J.F., Linsley P. & LU P.. Role of B7 signaling in the differentiation of na?ve CD4+ T cells to effector IL-4-producing T helper cells. Immunol. Res. 1995, 14: 176–188.
10 Manickasingham, S., Anderton S.M., Burkhart C. & Wraith D. C.. Qualitative and quantitative effects of CD28/B7-mediated costimulation on na?ve T cells in vitro. J. Immunol. 1998, 161: 3827–3835.
11 Harris N.L., Peach R.J. & Ronchese F.. CTLA4-Ig inhibits optimal Th2 cell development but not protective immunity or memory response to Nippostrongylus rasiliensis. Eur. J. Immunol. 1999, 29: 311–316.
12 Levine, B. L., Ueda Y., Craighead N., et al. CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro. Int. Immunol. 1995, 7(6): 891-904.
13 Lanier, L.L., O’Fallon S., Somoza C., et al. CD80 (B7-1) and CD86 (B7-2) provide similar costimulatory signals for T cell proliferation, cytokine production and generation of CTL. J. Immunol. 1995, 154(1):97-105.
14 Freeman, G. J., Boussiotis V. A., Anumanthan A., Bernstein G. M., Ke X. Y., Rennert P. D., Gray G. S., Gribben J. G., and Nadler L. M.. B7-1 and B7-2 do not deliver identical costimulatory signals, since B7-2 but not B7-1 preferentially costimulates the initial production of IL-4. Immunity 1995, 2:523.
15 Ranger, A. M., Prabbu Das M., Kuchroo V. K., and Glimcher L. H.. B7-2 (CD86) is essential for the development of interleukin-4 producing T cells. Int. Immunol. 1996,8:1549-1560.
16 Nakajima, A., Watanabe N., Yoshino S., Okumura K., and Azuma N.. Requirement of CD28-CD86 costimulation in the interaction between antigenprimed T helper type 2 and B cells. Int. Immunol. 1997,9:637-644.
17 Palmer, E. M. and van Seventer G. A.. Human T helper cell differentiation is regulated by the combined action of cytokines and accessory cell-dependent costimulatory signals.J. Immunol. 1997, 158(6):2654-2662.
18 Lenschow, D. J., Ho S.C., Sattar H., et al. Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J. Exp.Med.1995,181(3): 1145-1155
19 Kuchroo, V.K., Das M.P., Brown J.A., et al. B7.1 and B7.2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell, 1995,80(5): 707-718
20 Thompson, C. B. Distinct roles for the costimulatory ligands B7.1 and B7.2 in T helper cell differentiation? Cell 1995,81:979-982
21 Bluestone, J. A. New perspectives of CD28–B7 mediated T cell costimulation. Immunity 1995,2:555-559
22 De la Fuente M, Medina S, Del Rio M, et al. Effect of aging on the modulation of macrophage functions by neuropeptides. Life Sci. 2000, 67(17):2125-2135
23 Cuq P, Gross A, Terraza A, et al. mRNAs encoding CCKB but not CCKA receptors are expressed in human T lymphocytes and Jurkat lymphoblastoid cells. Life Sci 1997; 61:543-555
24 Carrasco M, Hernanz A, De La Fuente M, et al. Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2. Regul Pept. 1997; 70:135-142
25 Medina S, Rio MD, Cuadra BD, et al. Age-related changes in the modulatory action of gastrin-releasing peptide, neuropeptide Y and sulfated cholecystokinin octapeptide in the proliferation of murine lymphocytes. Neuropeptides. 1999; 33: 173-179
26 Carrasco M, Monica DR, Angel H, et al. Inhibition of human neutrophil functions by sulfated and nonsulfated cholecystokinin octapeptides. Peptides, 1997, 18:415-422
1 Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat. Rev. Immunol. 2002. 2:116–126
2 Khoury SJ, Sayegh MH. The roles of the new negative T cell costimulatory pathways in regulating autoimmunity. Immunity.2004.20:529–538
3 Chen LP. Co-inhibitory molecules of the B7-CD28 family in the control of T cell immunity. Nat. Rev. Immunol. 2004. 4:336–347
4 Abbas AK. The control of T cell activation vs. tolerance. Autoimmun. Rev. 2003. 2:115–118
5 Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu. Rev. Immunol. 2002. 20:29–53
6 Chikuma S, Bluestone JA. CTLA-4 and tolerance: the biochemical point of view. Immunol. Res. 2003.28:241–253
7 Coyle AJ, Gutierrez-Ramos JC. More negative feedback? Nat. Immunol. 2003.4:647–648
8 Greenwald RJ, Latchman YE, Sharpe AH. Negative co-receptors on lymphocytes. Curr. Opin. Immunol. 2002.14:391–396
9 Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, et al. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature 1999. 397:263–266
10 Swallow MM, Wallin JJ, Sha WC. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNF-α. Immunity. 1999.11:423–432
11 Ling V, Wu PW, Finnerty HF, Bean KM, Spaulding V, et al. Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor. J. Immunol. 2000. 164:1653–1657
12 Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J, et al. T-cell costimulation through B7RP-1 and ICOS. Nature 1999. 402:827–832
13 Brodie D, Collins AV, Iaboni A, Fennelly JA, Sparks LM, et al. LICOS, a primordial costimulatory ligand? Curr. Biol. 2000.10:333–336
14 Wang SD, Zhu GF, Chapoval AI, Dong H, Tamada K, et al. Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS. Blood 2000. 96:2808–2813
15 Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992.11:3887–3895
16 Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 2000.192:1027–1034
17 Dong HD, Zhu GF, Tamada K, Chen LP. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 1999. 5:1365–1369
18 Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, et al. 2001. PDL2 is a second ligand for PD-1 and inhibits T cell activation. Nat. Immunol. 2:261–268
19 Tseng SY, Otsuji M, Gorski K, Huang X, Slansky JE, et al. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J.Exp. Med. 2001.193:839–846
20 Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, et al. B7-H3: a costimulatory molecule for T cell activation and IFN-γ production. Nat. Immunol. 2001. 2:269–274
21 Sica GL, Choi IH, Zhu GF, Tamada K, Wang SD, et al. B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 2003. 18:849–861
22 Zang XX, Loke P, Kim J, Murphy K, Waitz R, Allison JP. B7x: a widely expressed B7 family member that inhibits T cell activation. Proc. Natl. Acad. Sci. USA 2003. 100:10388–10392
23 Prasad DVR, Richards S, Mai XM, Dong C. 2003. B7S1, a novelB7 family member that negatively regulates T cell activation. Immunity 18:863–873
24 Sansom D, Manzotti C, Zheng Y. What’s the difference between CD80and CD86.Trends in Immunology. 2003. 24(6):313-318
25 Shin T, Kennedy G, Gorski K, et a1. Cooerative B7-1/2(CD80/CD86) and B7-DC costimulation of CD4+T cells independent of the PD-1 receptor.Journal of Experimental Medicine. 2003,198(1):31-38
26 Wang SD, Bajorath J, Dallas B. Molecular modeling and functional mapping of B7-H1 and B7-DC uncouple costimulatory function from PD-1 interaction. Journal of Experimental Medicine. 2003. 197(9):1083-1091
27 Ito T, Ueon T, Clarkson MR, et a1. Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo. Journal of Immunology. 2005. 174(3):6648-6656
28 Taylor PA, Lees CJ, Fournier S, Allison JP, Sharpe AH, Blazar BR. B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J. Immunol. 2004.172:34–39
29 Paust S, Lu LR, McCarty N, Cantor H. Engagement of B7 on effector T cells by regulatory T cells prevents autoimmune disease. Proc. Natl. Acad. Sci USA .2004.101:10398–10403
30 Tang QZ, Henriksen KJ, Boden EK, Tooley AJ, Ye JQ, et al. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J. Immunol. 2003.171:3348–3352
31 Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 2002. 3:1097–10101
32 Short JJ, Pereboev AV, Kawakami Y, Vasu C, Holterman MJ, Curiel DT. Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. Virology 2004.322:349–359
33 Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, et al. Induction of B7-1 in podocytes is associated with nephritic syndrome. J. Clin. Invest. 2004. 113:1390–1397
34 Lohr J, Knoechel B, Jiang S, Sharpe AH, Abbas AK. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nat. Immunol. 2003. 4:664–669
35 Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 2003.4:1206–1212
36 Munn DH, Sharma MD, Mellor AL. Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells. J. Immunol. 2004.172:4100–4110
37 Podojil JR, Kin NW, Sanders VM. CD86 and β2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells. J. Biol. Chem. 2004. 279:23394–23404
38 Podojil JR, Sanders VM. Selective regulation of mature IgG1 transcription by CD86 and β2-adrenergic receptor stimulation. J. Immunol. 2003. 170:5143–5151
39 Ling V,Wu PW,Miyashiro JS,et a1.Cutting edge:identification of GL50,a novel B7-like protein that functionally binds to ICOS receptor. J Immunol. 2000. 164:1653-1657
40 LingV,Wu PW,Miyashim JS,et al.Differential expression of inducible costimulator-ligand splice variants:lymphoid regulation of mouse GL50-B and human GL50 molecules. J Immunol. 2001, 166:7300-7308
41 Wang S,Zhu G,Chapoval AI,et a1.Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS . Blood, 2000, 96: 2808-2813
42 Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000.13:95–105
43 Okamoto N, Tezuka K, Kato M, Abe R, Tsuji T. PI3-kinase and MAPkinase signaling cascades in AILIM/ICOS- and CD28-costimulated T-cells have distinct functions between cell proliferation and IL-10 production. Biochem. Biophys. Res. Commun. 2003.310:691–702
44 Harada Y, Tanabe E, Watanabe R, Weiss BD, Matsumoto A, et al. Novel role of phosphatidylinositol 3-kinase in CD28-mediated costimulation. J. Biol. Chem. 2001.276:9003–9008
45 Parry RV, Rumbley CA, Vandenberghe LH, June CH, Riley JL. CD28 and inducible costimulatory protein Src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase, Bcl-xL, and IL-2 expression in primary human CD4 T lymphocytes. J. Immunol. 2003.171:166–174
46 McAdam AJ, Chang TT, Lumelsky AE, Greenfield EA, BoussiotisVA, et al. Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells. J. Immunol. 2000.165:5035–5040
47 Wassink L, Vieira PL, Smits HH, Kingsbury GA, Coyle AJ, et al. ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells. J. Immunol. 2004. 173:1779–1786
48 Beier KC, Hutloff A, Dittrich AM, Heuck C, Rauch A, et al. Induction, binding specificity and function of human ICOS. Eur. J. Immunol. 2000. 30:3707–3717
49 Mages HW, Hutloff A, Heuck C, Buchner K, Himmelbauer H, et al. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. Eur. J. Immunol. 2000. 30:1040–4107
50 Richter G, Hayden-Ledbetter M, Irgang M, Ledbetter JA, Westermann J, et al. Tumor necrosis factor- α regulates the expression of inducible costimulator receptor ligand onCD34+ progenitor cells during differentiation into antigen presenting cells. J. Biol. Chem. 2001.276:45686–45693
51 Yoshinaga SK, Zhang M, Pistillo J, Horan T, Khare SD, et al. Characterization of a newhuman B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS. Int. Immunol. 2000.12:1439–1447
52 Aicher A, Hayden-Ledbetter M, Brady WA, Pezzutto A, Richter G, et al. Characterization of human inducible costimulator ligand expression and function. J. Immunol. 2000.164:4689–4696
53 Wiendl H, Mitsdoerffer M, Schneider D, Melms A, Lochmuller H, et al.Muscle fibres and cultured muscle cells express the B7.1/2-related inducible costimulatory molecule, ICOSL: implications for the pathogenesis of inflammatory myopathies. Brain 2003.126:1026–1035
54 Liang L, Porter EM, Sha WC. Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling. J. Exp. Med. 2002.196:97–108
55 Kopf M, Coyle AJ, Schmitz N, Barner M, Oxenius A, et al. Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection. J. Exp. Med. 2000.192:53–61
56 Villegas EN, Lieberman LA, Mason N, Blass SL, Zediak VP, et al. A role for inducible costimulator protein in the CD28-independent mechanism of resistance to Toxoplasma gondii. J. Immunol. 2002. 169:937–943
57 Kanai T, Totsuka T, Tezuka K, Watanabe M. 2002. ICOS costimulation in inflammatory bowel disease. J. Gastroenterol. 37(Suppl. 14):78–81
58 Akbari O, Freeman GJ,Meyer EH, Greenfield EA, Chang TT, et al. 2002. Antigenspecific regulatory T cells develop via the ICOS-ICOS-ligand pathway and inhibit allergen-induced airway hyperreactivity. Nat. Med. 8:1024–1032
59 Bonhagen K, Liesenfeld O, Stadecker MJ, Hutloff A, Erb K, et al. 2003. ICOS+ Th cells produce distinct cytokines in different mucosal immune responses. Eur. J. Immunol. 33:392–401
60 Rudd CE, Schneider H. 2003. Unifying concepts in CD28, ICOS and CTLA4 coreceptor signalling. Nat. Rev. Immunol. 3: 544–556
61 Nurieva RI, Duong J, Kishikawa H, Dianzani U, Rojo JM, et al. 2003. Transcriptional regulation of th2 differentiation by inducible costimulator. Immunity 18:801–811
62 Nurieva RI, Mai XM, Forbush K, Bevan MJ, Dong C. 2003. B7h is required for T cell activation, differentiation, and effector function. Proc. Natl. Acad. Sci. USA 100:14163–14168
63 Chapoval AI, Ni J, Lau JS, Wilcox RA, Flies DB, et al. B7-H3: acostimulatory molecule for T cell activation and IFN-γ production. Nat. Immunol. 2001.2:269–274
64 Sporici RA, Beswick RL, von Allmen C, Rumbley CA, Hayden-Ledbetter M, et al. ICOS ligand costimulation is required for T-cell encephalitogenicity. Clin. Immunol. 2001.100:277–288
65 Rottman JB, Smith T, Tonra JR, Ganley K, Bloom T, et al. The costimulatory molecule ICOS plays an important role in the immunopathogenesis of EAE. Nat. Immunol. 2001.2:605–611
66 Dong C, Nurieva RI. Regulation of immune and autoimmune responses by ICOS. J. Autoimmun. 2003. 21:255–260
67 Iwai H, Kozono Y, Hirose S, Akiba H, Yagita H, et al. Amelioration of collagen-induced arthritis by blockade of inducible costimulator-B7 homologous protein costimulation. J. Immunol. 2002.169:4332–4339
68 Nurieva RI, Treuting P, Duong J, Flavell RA, Dong C. Inducible costimulator is essential for collagen-induced arthritis. J. Clin. Invest. 2003. 111:701–706
69 Herman AE, Freeman GJ, Mathis D, Benoist C. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J. Exp. Med. 2004. 199:1479–1489
70 Coyle AJ, Gutierrez-Ramos JC. The role of ICOS and other costimulatory molecules in allergy and asthma. Springer Semin. Immunopathol. 2004.25:349–359
71 Dong C, Juedes AE, Temann UA, Shresta S, Allison JP, et al. ICOS costimulatory receptor is essential for T-cell activation and function. Nature 2001. 409:97–101
72 Gonzalo JA, Tian J, Delaney T, Corcoran J, Rottman JB, et al. ICOS is critical for T helper cell-mediated lung mucosal inflammatory responses. Nat. Immunol. 2001.2:597–604
73 Taylor PA,Panoskaltsis-Mortari A,Freeman GJ,et al.Targeting of inducible costimulator(ICOS) expressed on alloreative T-cells down regulates graft-versus-host disease(GVHD) and facilitates engraftment ofallogeneic bone marrow(BM). Blood. 2005, 105:3372-3380
74 Schreiner B,Mitsdoerffer M,Kieseier BC,et a1.Interferon-beta enhances monocyte and dendritic cell expression of B7-H1(PD-L1),a strong inhibitor of autologous T-cell activation: relevance for the immune modulatory effect in multiple sclerosis . J Neuroimmunol. 2004. 155(1-2):172-182
75 Wiendl H,Mitsdoerffer M,Schneider D,et a1.Human muscle cells express a B7-related molecule,B7-H1,with strong negative immune regulatory potential:a novel mechanism of counterbalancing the immune attack in idiopathic inflammatory myopathies.FASEB J. 2003, 17(13): 1892-1894
76 Petroff MG,Chen L,Phillips TA,et a1. B7 family molecules are favorably positioned at the human maternal-fetal interface. Bid Reprod. 2003. 68(5): 1496-1504
77 Can Y,Zhou H,Tao J,et a1.Keratinocytes induce local tolerance to skin graft by activating interleukin-10-secreting T cells in the context of costimulation molecule B7-H1. Transplantation. 2003. 75(8):1390-l396
78 Freeman GJ,Long AJ,Lwei Y,et a1.Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.J Exp Med. 2000. 192(7):1027-1034
79 Dong HD,Stroke SE,Salomao DR,et a1.Tumor-associated B7-H1 promotes T-cell apoptosis:A potential mechanism of immune evasion. Nat Med. 2002. 8(8):793-800
80 Konishi J,Yarnazaki K,Azuma M,et a1. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res. 2004, 10(15): 5094-5100
81 Kanai T,Totsuka K,Uraushihara K,et a1.Blocked of B7-H1 suppresses the development of chronic intestinal inflammation. J lmmunol. 2003. 171(8):4156-4163
82 Ishida Y,Agate Y,sahib Sahara,et a1.induced expression of PD-1, a novel member of the immunoglobulin gene super family,upon programmed celldeath.J Ambo J. 1992. 11:3887-3895
83 Ravetch JV,Lanier LL,Immune inhibitory receptors,Science,2000, 290:84-89
84 Sharpe AH,Freeman GJ,The B7-CD28 Super family. J.Nat Revlmmunjol. 2002. 2(2):116-126.
85 Tamura H,Dong HD,Zhu GF,et al,B7HI costimulation preferentially enhances CD28-independent T-helper cell function. J. Blood, 2001, 97 (60):1809-1816,
86 Ngulyen LT, Radhakrishnan S, Iric B, et a1. Cross-linking the B7 family molecule B7-DC directly activates immune functions of dendritic cells. Journal of Experimental Medcine, 2002, 196(10):1393-1398
87 Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur. J. Immunol. 2003. 33:2706–2716
88 Loke P, Allison JP. PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells. Proc. Natl. Acad. Sci. USA 2003. 100:5336–5341
89 Zhang X, Schwartz JC, Gun X, et a1. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity, 2004, 20(3):337-347
90 Ozkuynak E, Wang L, Goodearl A, et a1. Programmed death-1 targeting can promote allograft survival. Journal of Immunology, 2002, 169(11):6546-6553
91 Rietz C, Chen L.New B7 family member's with positive and negative costimulatory function. Am J Transplant, 2004. 4(1):8-14
92 Salarna AD, Imitola TC, Akiba H, et a1. Critical role of programmed death-1(PD-1) pathway in regulation of experimental autoimmune encephalomyelitis. Journal of Experimental Medcine. 2003. 198(1):71-78
93 Dong H, Strome SE, Matesin EL, et a1. Costimulating aberrant T cell responses by b7-h1 autoantibedies in Rheumatoid arthritis. Journal of Clinical Investigation. 2003. 111(3):363-370
94 Bennett F, Luxenberg D, Ling V,et a1.Program death-1 engagement upontcr activation has distince effects on costimulation and cytokin-driven proliferation: attenuation of ICOS, IL-4 and IL-21, but not CD8, IL-7 and IL-15 responses. Joumal of Immunology. 2003. 170(2):711-718
95 Shin T, Kennedy G, Gorski K, et a1. Cooperative B7-1/2(CD80/86) and B7-DC costimulation of CD4+T cells independent of the PD-1 receptor. Journal of Experimental Medicine, 2003. 198(1):31-38
96 Loke P, Allison JP. PD-L1 and PD-L2 are differentially related by Th1 and Th2 cells. Proc Natl Aad Sci, 2003, 100(9): 5336-5341
97 Liu X,Gao J,Wen J,et a1.B7-DC/PD-12 promotes tumor immunity by a PD-1-independent mechanism. Journal of Experimental Medicine. 2008. l97(2): 1721-1730.
98 Prasad DVR, Nguyen T, Li ZX, et a1. Murine B7-H3 is a negative regulator of T cells. Journal of Immunology. 2004. 173:2500-2506
99 Suh WK, Gajewska BU, Okada H, Gronski MA, Bertram EM, et al. The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses. Nat. Immunol. 2003.4:899-906
100 Zang X, Loke P, Kim J, et a1. B7x, a widely expressed B7 family member that inhibits T cell activation. Proc Nail Aad Sci, 2003, 1130(8):10388-10392
101 Liqing Wang,Christopher C,Fraser,et a1. Eur J Immuno1.2005,35(2): 428-438
102 Prasad DVR, Richards S, Mai X, et a1. B7S1, a novel B7 family member that negatively regulates T cell activation. Immunity, 2003.18(6):863-873
103 Watanabe N, Gavrieli M, Sedy JR, et a1. BTLA is a lymphocyte inhibitory receptor with similarities to TLA-4 and PD-1. Nature Immunology. 2003. 4(1):670-679
104 Steinberger P, Majdic O, Derdak SV, Pfistershammer K, Kirchberger S, et al. 2004. Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains. J. Immunol. 172:2352–59
105 Sica GL, Choi IN,Zhu G, et a1. Immunity. 2003, 18:849-86
106 Sedy JR, Gavrieli M, Potter KG, et a1. Nat Immuno1.2005. 6(1):90-98
107 Gonzalez LC, Loyet KM, Calemine-Fenaux J, et a1. Proc Natl Acad Sci USA. 2005. 102(4) :1116-1121
108 Sica GL, Choi IH, Zhu G, et al. Immunity. 2003. 18(6):849-861