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

目的： 探讨八肽胆囊收缩素（CCK-8）对静息巨噬细胞B7.1和B7.2表达及其协同刺激功能的影响。方法：用CCK-8（10-12-10-6 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∶〖KG-*2〗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24 h）共同体外培养，同时加入ConA 5 mg/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。结果：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10-9-10-7 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，CR1409及CR2945均能逆转CCK-8的上述作用，且CR1409的作用较CR2945更明显。结论：CCK-8通过上调巨噬细胞B7.2表达而增强其协同刺激活性，该作用由CCK1R及CCK2R介导，其中CCK1R起主要介导作用。     

八肽胆囊收缩素对脂多糖诱导小鼠分泌IL-12的影响及其机制研究

目的：观察八肽胆囊收缩素（CCK-8）对脂多糖诱导小鼠分泌IL-12的影响，探讨核因子-κB（NF-κB）和p38丝裂原活化蛋白激酶（p38 MAPK）的信号转导作用。方法：雌性BALB/c小鼠经LPS诱导后分别给予CCK及CCK-A、B受体拮抗剂。ELISA法检测小鼠血清及肺、脾组织中IL-12p40、p70的表达；Western blotting法检测肺脏、脾脏IκB、p38 MAPK的表达；EMSA法检测肺、脾组织中NF-κB/DNA的结合活性。结果：CCK-8进一步提高了LPS诱导的小鼠血清及肺、脾组织中IL-12p40、p70的表达；抑制IκB磷酸化和NF-κB/DNA结合活性；促进p38 MAPK磷酸化。而CCK-A受体拮抗剂（CR-1409）及CCK-B受体拮抗剂（CR-2945）部分逆转了CCK-8的效应。结论：CCK-8可促进LPS诱导小鼠分泌IL-12，p38 MAPK可能参与了其信号转导机制，而NF-κB途径可能并未参与CCK-8促IL-12分泌这一过程。     

四妙勇安汤含药血清对LPS诱导的M1/M2巨噬细胞极化及NF-κB/NLRP3通路的影响

目的 探讨四妙勇安汤含药血清对LPS刺激的M1/M2巨噬细胞极化及NF-κB/NLRP3通路的影响。方法 SD大鼠分别给予四妙勇安汤水煎液、生理盐水灌胃制备含药血清及正常对照血清。细胞分组为正常对照组、模型组、四妙勇安汤组、JSH-23组,除正常对照组外,其余组均用LPS诱导RAW264.7巨噬细胞。CCK8法检测细胞活性;ELISA检测细胞上清IL-4、IL-18水平;qRT-PCR法检测细胞i NOS、CD197、CD206、NLRP3 mRNA表达;免疫荧光标记测i NOS、CD197、CD206、NF-κB表达;Western blot检测NF-κB磷酸化、NLRP3炎症小体蛋白表达。结果 CCK8显示30%浓度以下的含药血清干预LPS诱导的巨噬细胞OD值无明显变化（P>0.05）。与正常对照组比较,模型组细胞iNOS、CD197、IL-18、NF-κB、NLRP3表达升高,CD206、IL-4表达降低（P<0.01）。与模型组比较,四妙勇安汤组及NF-κB抑制剂JSH-23组细胞iNOS、CD197、IL-18、NF-κB、NLRP3表达降低,CD206、IL-4...     

淋巴细胞缺陷对内毒素血症小鼠腹腔巨噬细胞活化的影响

目的观察淋巴细胞缺陷对内毒素血症小鼠腹腔巨噬细胞活化的影响。方法采用腹腔注射脂多糖(LPS)建立Balb/c小鼠和T、B细胞缺陷的重症联合免疫缺陷(SCID)小鼠内毒素血症模型,ELISA检测2种小鼠腹腔灌洗液TNF-α和IL-10水平,实时荧光定量PCR检测腹腔巨噬细胞(peritoneal macrophage,PMa)TNF-α、IL-10及丝裂原蛋白激酶磷酸酶-1(mito-gen-activated protein kinase phosphatase-1,MKP-1)mRNA表达;ELISA检测Balb/c及SCID小鼠PMa体外刺激后细胞因子分泌情况。结果 LPS注射后1 h,SCID小鼠腹腔灌洗液TNF-α水平高于Balb/c小鼠,注射后3 h,IL-10水平低于Balb/c小鼠;LPS注射前及注射后,SCID小鼠PMa TNF-αmRNA表达高于Balb/c小鼠PMa,IL-10 mRNA表达低于Balb/c小鼠PMa;体外实验LPS刺激下,SCID小鼠PMa较Balb/c小鼠PMa分泌更多的TNF-α,IL-10的分泌却偏少。LPS注射前及注射后,Balb/c小鼠PMa MKP-1 mRNA表达均明显高于SCID小鼠。结论淋巴细胞缺陷导致内毒素血症小鼠腹腔巨噬细胞活性的增加,淋巴细胞抑制巨噬细胞的活化并可能调控其发育及成熟;MKP-1表达的减少可能是淋巴细胞缺陷导致腹腔巨噬细胞活性增加的分子机制之一。     

CCK-8抑制LPS诱导大鼠肺间质巨噬细胞TNF-α转录及NF-κB活性

目的:观察硫酸化八肽胆囊收缩素(CCK-8)对体外脂多糖(LPS)诱导大鼠肺间质巨噬细胞(PIMs)TNF-α基因表达的影响,探讨核因子κB(NF-κB)是否参与这一过程,以揭示CCK-8抗炎作用的信号转导机制。方法:分离大鼠肺PIMs,经LPS、CCK-8、CCK受体拮抗剂丙谷胺及溶剂单独或联合应用孵育3h,用RT-PCR技术检测细胞TNF-αmRNA的表达,孵育1h,用电泳迁移率改变分析方法检测NF-κB活性,孵育30min,用Westernblot技术检测胞浆IκBα蛋白表达情况。结果:CCK-8(10^-8-10^-6mol·L^-1)明显降低了LPS诱导的TNF-αmRNA表达及NF-κB活性,增加了胞浆中IκBα蛋白水平,呈剂量依赖性,并可被丙谷胺所拮抗。结论:对LPS激活的肺PIMs,CCK-8通过抑制NF-κB活性而抑制其TNF-αmRNA表达,该作用由CCK受体介导,并与CCK-8减少IκBα蛋白降解有关,此为CCK-8抗炎作用的机制之一。     

MicroRNA-126基因敲减小鼠腹腔巨噬细胞功能的变化及意义

研究microRNA-126(miR-126)基因敲减小鼠腹腔巨噬细胞的功能变化,初步探讨其意义。采用real-time PCR检测LPS刺激前后野生型(wild type,WT)小鼠腹腔巨噬细胞miR-126表达变化,CCK8法检测其增殖情况;观察miR-126基因敲减(knock down,KD)小鼠腹腔巨噬细胞的形态变化,并用real-time PCR检测miR-126表达变化;CCK8法检测LPS刺激下miR-126KD小鼠腹腔巨噬细胞增殖变化;FACS检测巨噬细胞表面MHCⅡ类分子和CD86分子的表达变化;最后,real-time PCR检测巨噬细胞表达炎症因子IL-6、TGF-β和Ⅰ型精氨酸酶(arginase 1,Arg-1)等的变化情况。结果显示,WT小鼠腹腔巨噬细胞在LPS刺激后miR-126表达水平下调,显著低于未刺激组(P0.05),而增殖能力明显增强(P0.05);与WT小鼠相比,miR-126KD小鼠腹腔巨噬细胞的miR-126表达量明显下调(P0.05);形态上,WT小鼠腹腔巨噬细胞有较长伪足,多呈梭形,而miR-126KD小鼠腹腔巨噬细胞多呈圆形,细胞边缘光滑较少见伪足;LPS作用48h后,miR-126KD小鼠腹腔巨噬细胞的增殖能力明显强于WT小鼠(P0.05);且其膜MHCⅡ类分子和CD86分子表达也较WT小鼠显著上调(P0.05);LPS刺激下,miR-126KD小鼠腹腔巨噬细胞CCL-1表达显著增加,而IL-6、TGF-β和Arg-1的表达水平显著减少(P0.05)。这些结果提示,miR-126基因敲减可显著影响小鼠腹腔巨噬细胞的增殖能力和功能相关分子的表达,提示miR-126对小鼠腹腔巨噬细胞的功能具有重要的调控作用。     

C1632通过MAPK和NF-κB抑制巨噬细胞M1型极化

目的 探讨C1632抑制巨噬细胞炎症和M1型极化的作用及机制。方法 RAW264.7（小鼠单核巨噬细胞系）经LPS(10 ng/ml)和IFN-γ（20 ng/ml）处理24 h，诱导M1型极化后，分为对照组（DMSO 0.1%）、M1型巨噬细胞组（LPS和IFN-γ处理）、M1型巨噬细胞+C1632组（C1632质量浓度10μg/ml、25μg/ml和50μg/ml）。CCK8和流式细胞术检测C1632对M1型巨噬细胞活性和凋亡的影响。显微观察巨噬细胞极化形态。定量PCR检测炎性分子（IL-1β、IL-6、TNF-α、CXCL10、iNOS）和Lin28以及let7家族水平。流式细胞术检测CD80、CD86和MHC2水平。Western blot检测p38和NF-κB的磷酸化水平。LPS腹腔注射构建脓毒症小鼠模型。结果 相较于对照组的M1型巨噬细胞，C1632处理后能明显抑制其活性和形态学改变，并且显著下调IL-1β、IL-6、TNF-α、iNOS和CXCL10等促炎因子的转录水平，抑制CD80、CD86和MHC2的蛋白水平。小鼠巨噬细胞中低表达或不表达Lin28，且C1632处理后...     

HSP72过度表达对脂多糖刺激的巨噬细胞中NF-κB活性的影响

目的研究过度表达的热休克蛋白72(heat shock protein,HSP72)对脂多糖(lipopolysaccha-ride,LPS)刺激的巨噬细胞中NF-κB活性的影响.方法构建pCD-hsp72重组质粒,转染小鼠巨噬细胞RAW264.7,G418筛选稳定转染hsp72基因的细胞,Western印迹检测转染细胞HSP72的表达水平及LPS刺激后RAW264.7细胞内HSP72的表达水平、LPS刺激后转染细胞中IκBα的含量和胞核中NF-κB的含量的变化.结果构建pCD-hsp72重组质粒并转入巨噬细胞,获得稳定转染hsp72基因的细胞株,LPS刺激细胞内HSP72表达增加;HSP72可通过减少LPS刺激的巨噬细胞中IκBα的降解而抑制NF-κB活性.结论HSP72能抑制LPS激活的巨噬细胞中NF-κB的活化.     

CD305分子在小鼠巨噬细胞的表达

目的:研究CD305分子在不同小鼠巨噬细胞上的表达规律.方法:对小鼠巨噬细胞系、免疫组织中的巨噬细胞和脂多糖刺激的腹腔巨噬细胞进行免疫荧光染色, 流式细胞术分析小鼠CD305在各种巨噬细胞上的表达规律.结果:小鼠CD305高表达于小鼠巨噬细胞系和组织中的巨噬细胞.此外, LPS刺激后6 h内腹腔巨噬细胞上小鼠CD305的表达显著下降, 与此同时上清中IL-6的水平显著升高.结论:小鼠CD305分子高表达于小鼠巨噬细胞, LPS刺激后表达明显下降(P<0.05).     

E3泛素连接酶RNF121对脂多糖诱导的巨噬细胞表达促炎性细胞因子的影响

目的探讨E3泛素连接酶RNF121对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞表达促炎性细胞因子的调控作用。方法 LPS刺激小鼠腹腔巨噬细胞,Western blot检测RNF121的表达。用RNA干扰(si-RNF121)降低RNF121的表达,小鼠腹腔巨噬细胞经LPS刺激后,实时荧光定量PCR法检测TNF-α和IL-6的mRNA水平,ELISA检测细胞培养上清中TNF-α和IL-6的浓度,Western blot检测小鼠腹腔巨噬细胞中P65及磷酸化P65(p-P65)的表达水平,利用双荧光素酶报告基因法检测NF-κB的活性。结果 LPS诱导小鼠腹腔巨噬细胞后,RNF121的蛋白水平显著降低(P0.05),蛋白酶抑制剂MG132能够显著增加RNF121的蛋白水平。si-RNF121降低RNF121的表达后,给予LPS刺激小鼠腹腔巨噬细胞,IL-6和TNF-α的表达水平均显著下降(P0.05),转录因子P65的磷酸化(p-P65)水平及NF-κB的活性均显著降低(P0.05)。结论 E3泛素连接酶RNF121通过调控NF-κB的活性从而影响小鼠巨噬细胞促炎性细胞因子TNF-α及IL-6的表达。     

Antagonism between MyD88- and TRIF-dependent signals in B7RP-1 up-regulation

Type I interferons (IFN) play a critical role in the Toll-like receptor (TLR)-mediated expression of B7 costimulatory family members. For example, LPS-induced up-regulation of CD80 (B7.1) and CD86 (B7.2) is abrogated in antigen-presenting cells (APC) deficient in TRIF or TRAM, two adaptors that are responsible for TLR4-mediated production of Type I IFN. In this report, we demonstrate that LPS-induced up-regulation of B7-related protein 1 (B7RP-1), a ligand for ICOS, is dependent primarily upon the MyD88-dependent signaling pathway. Signaling via the TRIF pathway sharply limits MyD88-dependent B7RP-1 up-regulation. Hence, LPS induces significantly higher B7RP-1 expression on TRIF- or TRAM-deficient mouse peritoneal macrophages and on TRIF-deficient mouse splenic B cells as compared to wild-type cells. Further studies reveal that Type I IFN are general suppressors of TLR-mediated up-regulation of B7RP-1. These data indicate that Type I IFN play a dual role in the TLR-mediated expression of B7 costimulatory family members and suggest that they may act to limit B7RP-1 expression and thus limit signals derived from B7RP-1-ICOS interaction.     

M-CSF和staurosporine促进小鼠腹腔巨噬细胞巨涎蛋白摄取ox-LDL

目的：探讨巨噬细胞集落刺激因子（M-CSF）和蛋白激酶C抑制剂(STA)对小鼠腹腔巨噬细胞（MPM）巨涎蛋白摄取氧化低密度脂蛋白(ox-LDL)的影响。 方法： M-CSF与蛋白激酶C抑制剂STA预处理MPM后制备膜蛋白，开展SDS-PAGE电泳及配体印迹试验，测定不同条件下巨涎蛋白结合［125I］ox-LDL的值。 结果： MPM膜蛋白经唾液酸酶预处理后，巨涎蛋白与［125I］ox-LDL的结合值为(2.45±0.46)μg/g细胞蛋白，显著低于对照组的(58.38±1.78)μg/g细胞蛋白。用M-CSF与STA预处理MPM后，处理组与对照组巨涎蛋白结合配体［125I］ox-LDL呈现一趋向饱和的浓度曲线，经线性回归得两类似平行的直线，M-CSF组Bmax(453.59±15.39)μg/g蛋白，显著高于对照组的Bmax(322.77±12.54)μg/g蛋白，而Kd值变化不大。STA处理组Bmax=(362.40±15.31)μg/g蛋白，Kd值为(15.10±2.67)mg/L，对照组Bmax为(264.76±11.29)μg/g蛋白，Kd值为(17.43±2.98)mg/L。 结论： 巨涎蛋白接受M-CSF和STA的上调作用，通过增加其在MPM表面数目而促进对ox-LDL的摄取，促进泡沫细胞的形成。     

丹酚酸B对缺血小鼠脑能量代谢和脑水肿的影响

目的： 通过探讨丹酚酸B（SalB）对缺血小鼠脑能量代谢的影响，研究其对脑水肿的作用。方法：将NIH小鼠分为假手术组、缺血组、SalB治疗组和尼莫地平（Nim）治疗组，测定缺血30 min时脑组织能荷（EC）、磷酸肌酸（PCr）、ATP酶活性、兴奋性氨基酸（EAA）含量以及脑含水量。结果：SalB治疗组EC（0.520±0.034）和PCr［（98.344±13.249）μmol/g］的含量、Na⁺-K⁺-ATPase［（0.593±0.013）×10³ U/g］和Ca²⁺-ATPase［（0.484±0.053）×10³ U/g］的活性明显高于缺血组EC（0.465±0.037）、PCr［（81.614±9.919）μmol/g］的含量、Na+-K+-ATPase［（0.244±0.065）×10³ U/g］和Ca²⁺-ATPase［（0.321±0.086）×10³ U/g］ 的活性，2者相比显著差异（P<0.01）；而SalB治疗组Glu［（0.405±0.110）μmol/g］和Asp［（0.141±0.020）μmol/g］的含量和脑含水量［（38.1±0.1）%］ 则明显低于缺血组Glu［（0.550±0.140）μmol/g］、Asp［（0.287±0.050）μmol/g］的含量和脑含水量［（44.1±0.1）%］，2者比较亦有显著差异（P<0.05,P<0.01）。结论：增强脑组织能量代谢和ATP酶活性，并降低脑组织中兴奋性氨基酸的含量，可能是SalB减轻小鼠缺血性脑水肿的作用机制。     

表皮生长因子对传代培养的大鼠生长板软骨细胞增殖与胶原合成的影响

目的：探讨不同浓度的表皮生长因子（EGF）对传代培养的大鼠肋生长板软骨细胞（RGC）增殖和胶原合成的影响。 方法： 分离、培养RGC，分别用Western blot和阿尔新蓝（Alcine blue）染色检测各代RGC中Ⅱ型胶原和蛋白多糖的表达；用［³H］-TdR和［³H］-proline掺入分别检测1、10和100 μg/L EGF对第1、3和5代RGC增殖和胶原合成的影响。 结果： 原代培养的RGC表达Ⅱ型胶原和蛋白多糖，从第4代起Ⅱ型胶原和蛋白多糖的表达迅速降低，RGC发生了去分化。［³H］-TdR掺入表明，EGF促进第1代RGC的增殖（P<0.01），3个浓度的作用依次为：1 μg/L>10 μg/L>100 μg/L，差异显著（P<0.01）；3个浓度的EGF对第3代RGC保持了相近的促增殖作用（P<0.01）；对第5代RGC均无促增殖作用(P>0.05)。与促增殖作用不同，不同浓度EGF促不同传代的RGC的［³H］-proline掺入率比对照组均高20%左右（P<0.01）。 结论： EGF具有促进培养RGC增殖和胶原合成的作用，连续传代引起的去分化降低了RGC对EGF促增殖作用的反应，但对EGF的促胶原合成作用没有产生影响。     

大鼠肺间质巨噬细胞CCK受体的结合特性

目的:观察大鼠肺间质巨噬细胞(PIMs)胆囊收缩素(CCK)受体的表达亚型和结合特性。方法:用酶消化法结合肺泡耗竭灌洗和肺循环灌洗技术分离纯化大鼠PIMs,超速离心法提取细胞膜,与标记的硫酸化CCK-8(-CCK-8S)进行放射配基结合实验,用非标记的CCK-8S、CCK-A受体(CCK-AR)特异性拮抗剂CR1409及CCK-B受体(CCK-BR)特异性拮抗剂CR2945进行竞争抑制实验,观察配体受体结合的特异性及CCK受体表达亚型,观察孵育时间和温度对特异性结合的影响。结果:正常大鼠PIMs未能检出特异性结合,静脉注射脂多糖(LPS)48h出现特异性结合,且对孵育时间与温度有依赖性。经Scatchard分析,平衡解离常数(Kd)值为:(0.68±0.28)nmol·L^-1,最大结合容量(Bmax)值为(32.50±2.70)pmol·g^-1蛋白。通过竞争抑制实验,-CCK-8S与膜的结合可被CCK-8S、CR1409、CR2945所抑制,其IC₅₀值分别为:(3.20±1.13)nmol·L^-1,(0.19±0.06)μmol·L^-1和(2.30±0.80)nmol·L^-1。结论:大鼠PIMs存在CCK-A和CCK-B两种受体亚型,为CCK对生理及病理条件下巨噬细胞发挥效应提供了直接的结构依据。     

Comparison of CD28-B7.1 and B7.2 functional interaction in resting human T cells: Phosphatidylinositol 3-kinase association to CD28 and cytokine production

CD28 is a 44-kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these costimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC₅₀ of wortmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.     

Down-Regulation of Th2 Responses by Brucella abortus, a Strong Th1 Stimulus, Correlates with Alterations in the B7.2-CD28 Pathway

Down-regulation of the Th2-like response induced by ovalbumin-alum (OVA/alum) immunization by heat-killed Brucella abortus was not reversed by anti-IL-12 antibody treatment or in gamma interferon (IFN-gamma) knockout mice, suggesting that induction of Th1 cytokines was not the only mechanism involved in the B. abortus-mediated inhibition of the Th2 response to OVA/alum. The focus of this study was to determine whether an alternative pathway involves alteration in expression of costimulatory molecules. First we show that the Th2-like response to OVA/alum is dependent on B7.2 interaction with ligand since it can be abrogated by anti-B7.2 treatment. Expression of costimulatory molecules was then studied in mice immunized with OVA/alum in the absence or presence of B. abortus. B7.2, but not B7.1, was up-regulated on mouse non-T and T cells following immunization with B. abortus. Surprisingly, B. abortus induced down-regulation of CD28 and up-regulation of B7.2 on murine CD4(+) and CD8(+) T cells. These effects on T cells were maximal for CD28 and B7.2 at 40 to 48 h and were not dependent on interleukin-12 (IL-12) or IFN-gamma. On the basis of these results, we propose that the IL-12/IFN-gamma-independent inhibition of Th2 responses to OVA/alum is secondary to the effects of B. abortus on expression of costimulatory molecules on T cells. We suggest that down-regulation of CD28 following activation inhibits subsequent differentiation of Th0 into Th2 cells. In addition, decreased expression of CD28 and increased expression of B7.2 on T cells would favor B7.2 interaction with CTLA-4 on T cells, and this could provide a negative signal to developing Th2 cells.     

Differential distribution of B7.1 (CD80) and B7.2 (CD86) costimulatory molecules on mucosal macrophage subsets in human inflammatory bowel disease (IBD)

The molecules B7.1 and B7.2 deliver costimulatory signals of critical importance to naive T cells, and may thus be involved in abrogation of oral tolerance in IBD. Functional disparity apparently exists among antigen-presenting cells in vivo. We wanted to examine if differential B7 expression occurs on mucosal macrophage subsets. Cryosections of bowel specimens from patients with IBD and normal controls were subjected to immunofluorescence and immunoperoxidase staining. In normal mucosa, selective subepithelial accumulation of B7.2⁺ cells was found. In inflamed IBD mucosa, however, subsets appeared consisting of both B7.2^hi and B7.1^hi cells as well as CD14^hi macrophages. Notably, outside lymphoid aggregates the prominent fraction of recently recruited CD14^hi macrophages comprised most (≈ 80%) of the B7.1^hi cells, whereas most (≈ 70%) B7.2^hi cells were identified as resident mucosal macrophages (CD14^lo or CD14⁻). Differential expression of B7.1 and B7.2 on two functionally different subsets of intestinal macrophages implies separate immunoregulatory roles for the two molecules. This finding is in keeping with recent experimental data demonstrating that monocyte-derived cells are crucial for immune responses at mucosal surfaces. Preferential B7.1 up-regulation might be critical in breaking the immunological tolerance to luminal antigens in IBD, but it cannot be excluded that it is a secondary pathogenic event.     

Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes.

The hormonal active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T-cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen-presenting cells (APC), when engaged to their counter-receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell-surface markers (CD14 and CD4) and antigen-presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose- and time-dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), was studied. The 1,25(OH)2D3-induced B7.2 down-regulation was still detectable when monocytes were activated by IL-10, IFN-gamma and TNF-alpha but not with LPS. Moreover, the induction of B7.1 by TNF-alpha was inhibited by addition of 1, 25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC-dependent T-cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.