【作者】 张风华；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2007， 博士

【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/L¹0^-6mol/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^-7mol/L～10^-9mol/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^-8mol/L、10^-10mol/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^-6mol/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^-6mol/L和10^-8mol/L浓度的CCK-8使细胞NF-κB活性升高（P<0.05）,而10^-10mol/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^-6mol/L和10^-8mol/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^-10mol/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^-10mol/L、10^-8mol/L和10^-6mol/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^-8mol/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^-6mol/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^-7mol·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。更多还原

【Abstract】 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 macrophageAim: 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 pathwayDocuments 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^-12mol·L^-110-6mol·L^-1), Fsk and/or H89, or with CCK receptor antagonists (10^-9 mol·L^-110^-5mol·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 macrophages①Gliotoxin, 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^-8mol/L, 10^-10mol/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/L¹0^-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^-6mol/L and 10^-8mol/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^-10mol/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 pathway①Stimulating 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^-10mol·L^-1) （P>0.05）, but led to a significant increase of them at high concentration (10^-8mol·L^-110^-6mol·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^-10mol·L^-1、10^-8mol·L^-1 and 10^-6mol·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^-6mol/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 receptors①CCK-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 vivoAim: 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.CONCLUSIONSIn 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.更多还原