hCAP-18／LL-37全长肽真核表达产物对树突状细胞分化成熟的影响

目的研究人源阳离子抗菌肽（hCAP）－18／LL-37全长肽真核表达产物对树突状细胞分化成熟的影响。方法将已构建的hCAP-18／LL-37全长肽真核表达载体pcDNA4／Myc－His-hCAP-18转染Hela细胞株,在转染后48h收获培养上清;人外周血单个核细胞分离,体外定向诱导分化成树突状细胞（Dc）;将转染后收获的培养上清与体外培养的Dc共同孵育,48h收获Dc,经流式细胞仪检测DC表面分子CD83、CD86、CIM0及mA．DR的表达。结果FCM结果显示：pcDNA4／Myc—His-hCAP-18转染Hela细胞培养上清与pcDNA4／Myc-His空载体转染细胞培养上清相比较,均可上调Dc表面分子CD86、CIMO及HLA—DR的表达。结论构建的hCAP-18／LL-37全长肽真核表达载体pcDNA4／Myc。His-hCAP-18转染Hela细胞株培养上清液均可上调Dc表面分子CD86、CIMO及HLA—DR的表达,促进Dc的分化成熟。     

人Regucalcin cDNA真核表达载体的构建及表达

目的构建人regucalcin（RGN）全长cDNA的真核表达载体，观察其在人肾皮质近曲小管上皮细胞（HK-2细胞）中表达。方法用RT-PCR技术扩增获得人HK-2细胞RGN全长cNDA，将扩增的产物连接入pMD18-T克隆载体上。将重绢质粒转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确，再用双酶切方法将RGN基因定向连接到真核表达载体DECFP-NI中，构成pEGFP-RGN，将pEGFP-RGN转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确。再用双酶切方法将RGN基因与EGFP定向连接到真核表达载体pcDNA3．1（＋）-zeocin中，构建真核表达pcDNA3．1-RGN-EGFP-zeo的重组体。将pcDNA3．1-RGN．EGFP-zeo转化人大肠杆菌DHSa内并提取质粒，双酶切鉴定、DNA测序鉴定准确，用脂质体转染入HK-2细胞。荧光显微镜、激光共聚焦显微镜观察pcDNA3．1-RGN-EGFP-zeo表达情况。结果RGNcDNA全长是897bp。以此构建的pcDNA3．1-RGN-EGFP-zeo真核表达载体，经DNA测序与GenBank中的人RGNcDNA序列一致。荧光显微镜、激光共聚焦显微镜观察到pcDNA3．1-RGN-EGFP-zeo转染的细胞中有绿色荧光蛋白的表达。结论本实验成功构建了pcDNA3．1-RGN．EGFP．zeO真核表达质粒，并在HK-2细胞中表达。     

流式细胞技术检测肾移植后外周血白细胞介素18受体的表达

背景:细胞因子常常在肾移植后排斥反应早期即被释放,因此可以作为排斥反应的早期诊断指标.目的:观察尿毒症患者肾移植前后外周血白细胞介素18 受体(IL-18R)表达的变化.方法:将2004-09/2005-09 在解放军济南军区总医院进行的肾移植尿毒症32 例患者分为肾功能稳定组24 例,急性排斥组8 例.另选择以往手术后移植肾功能正常,平均存活时间4 年的肾移植患者12 例为长期存活组.同期查体未发现异常健康自愿者7 例为健康对照组.提取受试者的外周血淋巴细胞,加入双荧光标记的鼠抗人CD4/IL-18Rα及CD8/IL-18Rα单克隆抗体,利用流式细胞仪进行测定.结果与结论:流式细胞技术检测CD4/IL-18Rα及CD8/IL-18Rα在尿毒症组的阳性率明显高于健康对照组(P=0.02,P=0.04).肾功能稳定组及长期存活组外周血CD4/IL-18Rα及CD8/IL-18Rα的表达明显低于急性排斥组(P ＜ 0.05).急性排斥反应组激素冲击治疗后其阳性率明显低于激素冲击治疗前(P ＜ 0.05).3 例耐激素排斥反应者IL-18R 表达值高于5 例激素治疗有效者.结果显示外周血CD4/IL-18Rα及CD8/IL-18Rα的变化可以较早预测肾移植后急性排斥反应,预测肾移植后恢复情况,评估排斥反应对激素治疗的效果.     

流式细胞技术检测肾移植后外周血白细胞介素18受体的表达

背景：细胞因子常常在肾移植后排斥反应早期即被释放,因此可以作为排斥反应的早期诊断指标。目的：观察尿毒症患者肾移植前后外周血白细胞介素18受体（IL-18R）表达的变化。方法：将2004-09/2005-09在解放军济南军区总医院进行的肾移植尿毒症32例患者分为肾功能稳定组24例,急性排斥组8例。另选择以往手术后移植肾功能正常,平均存活时间4年的肾移植患者12例为长期存活组。同期查体未发现异常健康自愿者7例为健康对照组。提取受试者的外周血淋巴细胞,加入双荧光标记的鼠抗人CD4/IL-18Rα及CD8/IL-18Rα单克隆抗体,利用流式细胞仪进行测定。结果与结论：流式细胞技术检测CD4/IL-18Rα及CD8/IL-18Rα在尿毒症组的阳性率明显高于健康对照组（P=0.02,P=0.04）。肾功能稳定组及长期存活组外周血CD4/IL-18Rα及CD8/IL-18Rα的表达明显低于急性排斥组（P〈0.05）。急性排斥反应组激素冲击治疗后其阳性率明显低于激素冲击治疗前（P〈0.05）。3例耐激素排斥反应者IL-18R表达值高于5例激素治疗有效者。结果显示外周血CD4/IL-18Rα及CD8/IL-18Rα的变化可以较早预测肾移植后急性排斥反应,预测肾移植后恢复情况,评估排斥反应对激素治疗的效果。     

转化生长因子β1基因克隆及重组真核表达质粒的构建

背景：通过基因转移方法研究某一外源性基因在细胞中的作用是目前分子生物学常用的技术手段，与病毒载体相比，应用DcDNA4．0-TGF-β1真核表达质粒的转染方法无遗传毒性和细胞毒性，有更高的安全性。目的：通过克隆转化生长因子β1基因，观察构建重组转化生长因子β1基因真核表达质粒的可行性。设计、时间及地点：以细胞为观察对象的体外实验，于200703／2008—02在河南省高等学校临床医学重点学科开放实验室完成。材料：2月龄清洁级健康SD大鼠，雌雄不拘，用于分离细胞中RNA。方法：从大鼠骨髓细胞中分离提取转化生长因子B1的mRNA，反转录聚合酶链反应扩增后，克隆入pGEM—T载体，PCR鉴定重组子；酶切下目的基因转化生长因子B1，再亚克隆入载体pcDNA4．0质粒，酶切鉴定亚克隆重组子并进行DNA序列分析。主要观察指标：TGF-β1 cDNA、pGEM—T—TGF-β1和重组子pcDNA4．0-TGF—β1的表达。结果：经过反转录一聚合酶链反应扩增得到TGF-β1 cDNA条带；PCR扩增鉴定得到pGEM—T—TGF-β1。酶切鉴定得到亚克隆重组子pcDNA4．0-TGF-β1，经测序鉴定证实插入DNA序列与设计完全一致。结论：成功克隆大鼠转化生长因子β1基因，并构建出重组转化生长因子β1真核表达载体。     

白细胞相关免疫球蛋白样受体2(CD306)真核表达载体构建及蛋白的纯化与鉴定

背景:目前国内外对白细胞相关免疫球蛋白样受体(leukocyte-associated immunoglobulin like-receptor,LAIR)家族中LAIR1 的生物学功能研究已较清晰,而关于LAIR-2分子在机体内的生物学功能尚缺乏相关报道.目的:为进一步研究LAlR-2的体内生物学功能,构建其真核表达载体,纯化并鉴定蛋白.设计、时间及地点:单一样本观察实验,于2007-06/2008-06在解放军第四军医大学完成.材料:载体pig/3c由英国牛津大学徐小宁博士惠赠.载体pcDNA3.1由荷兰Meyaard博士惠赠.中国仓鼠卵巢细胞系CHO由解放军第四军医大学免疫学教研室冻存.方法:构建plg/3c-LAIR-2及pcDNA3.1-LAIR-2两个真核表达载体,电转染法转染CHO细胞建立稳定表达LAIR-2-Fc融合蛋白及LAIR-2全长蛋白分子的细胞株,通过Western blot、细胞免疫化学、流式细胞术分析实验鉴定真核表达LAIR-2蛋白分子与抗原核表达LAIR-2 mAbs的结合活性.主要观察指标:稳定转染细胞系的建立及真核表达蛋白的纯化和鉴定.LAIR-2蛋白与相应单克隆抗体的结合活性.结果:构建真核表达载体并成功转染CHO细胞,建立稳定分泌LAIR-2-Fc融合蛋白的转染细胞系及稳定分泌LAIR-2蛋白的转染细胞系,分别命名为CHO/LAIR-2-Fc以及CHO/LAIR-2.Western blot实验表明真核表达的LAIR-2全长蛋白分子均可与三株抗LAIR-2 mAb 1A7、3H12及4A9发生特异性结合反应.免疫细胞化学、流式细胞术分析实验结果表明抗原核细胞表达蛋白的三株LAIR-2单克隆抗体中,1A7不能结合pcDNA3.1-LAIR-2转染的CHO细胞内的LAIR-2,面3H12和4A9均可以很好结合细胞内的LAIR-2.结论:实验成功构建了plg/3c-LAIR-2及pcDNA3.1-LAIR-2两个真核表达载体,成功转染CHO细胞并建立稳定表达LAIR-2-Fc融合蛋白及LAIR-2全长蛋白分子的细胞株.真核表达的LAIR-2蛋白分子与抗原核表达的LAIR-2 mAbs有良好的结合活性.     

HLA-E真核表达载体的构建及其在K562细胞中的表达与鉴定

本研究旨在亚克隆HLA-E真核表达载体，并使其在HLA-I类阴性的靶细胞K562细胞上获得稳定表达。首先采用PcR方法从多顺反子表达载体(pG/A2E)扩增出目的片段A2／ E cDNA，与真核表达载体pcDNA3．1( )重组，构建成HLA—E真核表达载体pcDNA3．1( )／A2E，然后采用脂质体转染的方法将重组质粒转入K562细胞，最后经G418筛选及有限稀释，利用抗HLA-E特异的单克隆抗体K0126-3进行FACS检测，以观察HLA—E分子在靶细胞表面的表达情况。结果显示，HLA-E分子在经pcDNA3．1( )／A2E转染的靶细胞表面获得表达(27．76％)，而经空载体pcDNA3．1( )转染的靶细胞则未获得表达。结论：成功构建了pcDNA3．1( )／A2E真核表达载体，并使HLA-E分子在HLA-I类阴性的K562细胞表面表达，为进一步研究HLA-E作用的分子机制以及探索HLA-E与NK受体之间的相互作用和HLA-E体外表达对NK细胞功能的影响奠定了基础。     

重组人MBL在CHO细胞中的表达及检测

[目的]在CHO-K1细胞中表达重组MBL并进行检测。[方法]构建真核表达载体pcDNA3．1（＋）-mbl，用阳离子转染试剂Transfast转染CHO-K1细胞，G418筛选稳定转染的细胞。应用RT-PCR、ELISA及免疫荧光检测重组MBL的表达。[结果]RT-PCR从稳定转染pcDNA3.1（＋）-mbl的CHO-K1细胞中检测到mbl基因的表达，夹心ELISA并未从转染细胞上清中检测到重组MBL，免疫荧光表明稳定转染pcDNA3．1（＋）-mbl的CHO-K1细胞能够在胞浆中表达重组人MBL。[结论]成功构建真核表达载体pcDNA3．1（＋）-mbl并转染CHO-K1细胞，转染的细胞能够表达重组人MBL。     

肌腱愈合及粘连形成机制的研究：胰岛素样生长因子1基因真核表达载体的构建及表达

目的：探讨真核表达载体介导外源性胰岛素样生长因子-1(IGF-1)基因转染肌腱细胞的可能性。方法：组织块法培养原代肌腱细胞，体外重组真核表达载体pcDNA3．1(+)-IGF-1，并以脂多胺DOGS为介导转染培养的肌腱细胞，反转录多聚酶链反应(RT-PCR)检测IGF-1mRNA的表达，ELISA法检测IGF-1活性蛋白表达。结果：成功构建真核表达载体pcDNA3．1(+)-IGF-1；RT-PCR检测显示转染后的肌腱细胞成功表达IGF-1mRNA；ELISA结果显示转染组IGF-1蛋白表达较对照组明显增高(t=-2．873，P&;lt;0．05)。结论：重组的真核表达载体pcDNA3．1(+)-IGF-1能够将IGF-1cDNA成功导入肌腱细胞并表达。     

PCI-hTGF-β1转染体外培养成人退变椎间盘细胞及其表达产物的测定

背景：人转化生长因子β1基因可用于椎间盘退变的基因治疗，但有效载体的构建是实验的关键。目的：测定真核表达载体PCI-hTGF-β1转染体外培养成人退变椎间盘细胞可否表达产物人转化生长因子β1，为椎间盘退变的基因治疗提供实验基础。设计：单一样本实验。单位：山东省创伤骨科研究所，一所市立医院骨科。材料：实验于1999—10／2001—01在山东省创伤骨科研究所实验室进行。椎间盘标本均为椎间盘突出症患者手术中取材（获患者知情同意）。标本1为30岁女性的L4/5椎间盘，标本2为38岁女性的L5／S1椎间盘。方法：①培养成人退变椎间盘细胞：标本离体30min内取回实验室，收集原代培养的纤维环细胞和髓核细胞。②转染：将细胞置于24孔培养板中，每孔细胞数目在5．5&;#215;10^5个，应用构建的PCI-hTGF-β1真核表达载体对其进行转染，并设立转染PCI组和未转染组。③用免疫组织化学方法对转染48h后的细胞进行表达产物测定。主要观察指标：两个标本中，各组原代纤维环细胞和髓核细胞中真核表达载体PCI-hTGF-β1的阳性细胞产物的吸光度值比较。结果：①标本1：原代纤维环细胞和原代髓核细胞中真核表达载体PCI—hTGF—β1的阳性细胞产物的吸光度值为PCI组的3．49，3.69倍，为未转染组的3.55倍。②标本2：原代纤维环细胞和原代髓核细胞中真核表达载体PCI—hTGF-β1的阳性细胞产物的吸光度值为PCI组的3．56，3.46倍，为未转染组的3．43，3.33倍。结论：PCI-hTGF—β1是人转化生长因子β1基因在体外培养退变椎间盘细胞转染中有效的真核表达载体，可以成功转染体外培养的成人退变椎间盘细胞，并可获得人转化生长因子β1基因产物的高表达。     

Effect of beta 2-adrenergic receptor stimulation on interleukin-18-induced intercellular adhesion molecule-1 expression and cytokine production

beta-Adrenergic receptor (AR) agonists have been demonstrated to modulate the production of inflammatory mediators. Recent studies implied that beta 2-AR agonists might be useful for chronic inflammatory diseases caused by interleukin (IL)-18. In the present study, we found that norepinephrine, epinephrine, or isoproterenol down-regulated IL-18 (100 ng/ml)-induced intercellular adhesion molecule (ICAM)-1 expression on monocytes in a dose-dependent manner (10(-8)-10(-4) M), but did not effect B7.1 and B7.2 expression after 24-h incubation. The modulatory effect of these catecholamines on ICAM-1 expression was antagonized by beta 2-AR antagonist, but not by alpha 1-, alpha 2-, or beta 1-AR antagonist. beta 2-AR-selective agonists salbutanol and terbutaline down-regulated IL-18-induced ICAM-1 expression on monocytes, but alpha 1-, alpha 2-, or beta1-AR agonist had no effect. In the same manner, salbutanol and terbutaline as well as norepinephrine, epinephrine, and isoproterenol regulated the IL-18-induced cytokine production, including IL-12, tumor necrosis factor-alpha or interferon-gamma through the stimulation of beta 2-AR. Together with the previous finding that ICAM-1/lymphocyte function-associated antigen-1 interaction plays a crucial role in the IL-18-initiated cytokine network, the present study strongly suggested that the stimulation of beta 2-AR inhibited the IL-18-activated cytokine cascade through the inhibitory effect on ICAM-1 expression, contributing to finding a new method for clinical treatment.     

IL-18及其受体在特发性血小板减少性紫癜患者Th1类细胞优势应答中的作用

Objective To evaluate the role of interleukin(IL)-18 and IL-18 receptor(IL-18R)in the predominant Thl type cytokine response in patients with immune thrombocytopenia(ITP).Methods Fifteen patients with active phase ITP,eighteen in remission and thirteen healthy controls were enrolled in this study.T-bet and GATA-3 mRNA levels in peripheral blood mononueleated cells(PBMNC)were measured by reverse transcfiptase polymerase chain reaction(RT-PCR);the plasma IL-18 level by enzyme linked immunosorbent assay(ELISA),the expression of IL-18R on CD3+ lymphocytes and total lymphocytes by flow cytometry(FCM).Results The T-bet mRNA levels in patients with active phase ITP was 3.572 fold as much as that in the controls(P＜0.05),while the GATA-3 mRNA levels were 0.378 foId of that in controls(P＜O.05).The levels of plasma IL-18 and IL-18R on CD3+ lymphocytes were significantly increased in active ph8se ITP than in remission phase and controls.There was no differenee in ratio of T-bet/GATA-3 between remitted ITP and controls and so was for T-bet mRNA,GATA-3 mRNA,plasma IL-18 and IL-18R on CD3+lymphocytes.Conclusion ITP as a disease of Thl-dominant response there is an unbalance between T-bet and GATA-3 in its active phase:IL-18 and IL-18R being upregulated.     

IL-18及其受体在特发性血小板减少性紫癜患者Th1类细胞优势应答中的作用

目的 探讨白细胞介素18(IL-18)及其受体(IL-18R)在特发性血小板减少性紫癜(ITP)患者Th1类细胞因子优势应答中的作用.方法 应用逆转录聚合酶链反应(RT-PCR)检测15例活动期和18例缓解期ITP患者外周血单个核细胞(PBMNC)中转录因子T-bet和GATA-3 mRNA的表达;应用ELISA法检测血浆中IL-18的变化;应用流式细胞术检测CD3+细胞和淋巴细胞表面IL-18R的表达.13名健康志愿者为正常对照.结果 ITP活动期患者PBMNC中T-bet mRNA表达水平(0.069±0.013)明显高于对照组(0.019±0.010)(P＜0.05),而GATA-3 mRNA的表达水平(0.002±0.001)明显低于对照组(0.005±0.002)(P＜0.05);血浆IL-18和CD3+细胞表面IL-18R表达水平较ITP缓解期患者和对照组显著增高.ITP缓解期患者T-bet与GATA-3 mRNA表达水平与对照组比较差异均无统计学意义(P值均＞0.05),T-bet/GATA-3比例基本恢复正常,血浆中IL-18和CD3+细胞表面IL-18R的表达水平也基本恢复正常.结论 ITP活动期患者表现为Th1优势应答,T-bet/GATA-3比例明显失衡,T-bet/GATA-3比例可作为ITP患者Th1类细胞极化的敏感指标;ITP活动期患者IL-18和IL-18R表达上调,可能为ITP患者的治疗提供一个新的治疗靶点.     

细胞因子中白细胞介素1β、白细胞介素1Ra及γ-氨基丁酸B受体与癫痫的发病

目的:综述细胞因子中的白细胞介素1β、白细胞介素1Ra及γ-氨基丁酸B受体在癫痫发病中的作用及其与癫痫的关系。资料来源:应用计算机检索PubMed数据库1990-01/2006-10相关文章,检索词“epilepsy,IL-1β,IL-1Ra,GABABR”,并限定文章语言种类为English;同时计算机检索CNKI数据库1990-01/2006-10相关文章,检索词“癫痫,白细胞介素1β,白细胞介素1受体拮抗剂,γ-氨基丁酸B受体”,限定文章语言种类为中文。资料选择:对资料进行初审,纳入标准:①随机对照研究。②基础或临床研究。排除标准:重复研究、重复临床报道的文献、个案报告。资料提炼:共收集到51篇关于癫痫,白细胞介素1β,白细胞介素1受体拮抗剂,γ-氨基丁酸B受体的随机和未随机试验,30个试验符合纳入标准。资料综合:①癫痫的发病有免疫学机制参与。白细胞介素1作为一种重要的细胞因子,在中枢神经系统中的作用尤为突出,在一些神经系统疾病有不同程度的升高,内源性增高的白细胞介素1可明显促进这些疾病所导致的脑损伤。内源性细胞介素1β增多及外源性给予白细胞介素1β与癫痫的形成有密切关系。白细胞介素1受体拮抗剂是白细胞介素1受体的天然拮抗剂。白细胞介素1β与其受体结合后,通过细胞内信息传递途径,多环节调节中枢神经系统的兴奋性,最终影响癫痫的发病过程。②γ-氨基丁酸是中枢神经系统中最主要的抑制性神经递质,各类癫痫的发生几乎都与脑内γ-氨基丁酸的功能变化有关。结论:细胞因子中的白细胞介素1β、神经递质中的γ-氨基丁酸B受体在癫痫的发病过程中均起着重要的作用。但二者在癫痫发病机制中的关系有待于进一步研究。     

Atorvastatin affects interleukin-2 signaling by altering the lipid raft enrichment of the interleukin-2 receptor beta chain.

Although the immunomodulatory properties of statins are in part independent of their lipid-lowering effects, cholesterol is a major component of lipid rafts. We therefore studied the effects of atorvastatin (AS) on the raft enrichment of the interleukin-2 receptor (IL-2R) beta chain previously described by us and on early IL-2R signaling events in activated human T cells. We found that concomitant AS exposure during a 3-day stimulation with phytohemagglutinin (PHA) attenuates activation-associated events, such as the enhanced surface expression of the raft marker GM-1 and the induced expression of the activation marker CD25 (the IL-2R alpha chain). In contrast, brief AS treatment after PHA stimulation increased GM-1 surface expression and virtually abolished the selective raft enrichment of the IL-2R beta chain. Although this AS-associated increase in GM-1 expression resembled that seen in the presence of the raft-disrupting cholesterol chelator methyl-beta-cyclodextrin (MBCD), the two agents had contrasting effects on the tyrosine phosphorylation of the IL-2R beta chain by exogenous IL-2: MBCD essentially abolished this event, whereas AS tended to enhance it and shifted its occurrence out of rafts. We conclude that AS affects IL-2R signaling by altering the raft enrichment of the IL-2R beta chain and propose that this effect is one mechanism underlying the immunomodulatory properties of statins.     

Stimulation of Toll-like receptor 3 and 4 induces interleukin-1beta maturation by caspase-8

The cytokine interleukin (IL)-1β is a key mediator of the inflammatory response and has been implicated in the pathophysiology of acute and chronic inflammation. IL-1β is synthesized in response to many stimuli as an inactive pro–IL-1β precursor protein that is further processed by caspase-1 into mature IL-1β, which is the secreted biologically active form of the cytokine. Although stimulation of membrane-bound Toll-like receptors (TLRs) up-regulates pro–IL-1β expression, activation of caspase-1 is believed to be mainly initiated by cytosolic Nod-like receptors. In this study, we show that polyinosinic:polycytidylic acid (poly[I:C]) and lipopolysaccharide stimulation of macrophages induces pro–IL-1β processing via a Toll/IL-1R domain–containing adaptor-inducing interferon-β–dependent signaling pathway that is initiated by TLR3 and TLR4, respectively. Ribonucleic acid interference (RNAi)–mediated knockdown of the intracellular receptors NALP3 or MDA5 did not affect poly(I:C)-induced pro–IL-1β processing. Surprisingly, poly(I:C)- and LPS-induced pro–IL-1β processing still occurred in caspase-1–deficient cells. In contrast, pro–IL-1β processing was inhibited by caspase-8 peptide inhibitors, CrmA or vFLIP expression, and caspase-8 knockdown via RNAi, indicating an essential role for caspase-8. Moreover, recombinant caspase-8 was able to cleave pro–IL-1β in vitro at exactly the same site as caspase-1. These results implicate a novel role for caspase-8 in the production of biologically active IL-1β in response to TLR3 and TLR4 stimulation.     

Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist.

Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis.     

P2X7 receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1beta into mature IL-1beta, which is then secreted. Because both LPS (in vivo) and IL-1beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco's modified Eagle's medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-l-arginine methyl ester or N-([3-(aminomethyl)phenyl]methyl)ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1beta induced iNOS protein expression. Thus, activation of P2X(7) receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1beta-mediated release of nitric oxide by iNOS.