红霉素、阿仑膦酸钠抑制磨损颗粒对小鼠巨噬细胞分泌白细胞介素1,6及肿瘤坏死因子α的影响

背景：阿仑膦酸钠的抗骨吸收作用是通过其对破骨细胞的抑制作用完成的,近年亦有报道证实红霉素有直接抑制破骨细胞的作用。目的：观察阿仑膦酸钠和红霉素抑制钛颗粒刺激巨噬细胞分泌肿瘤坏死因子α、白细胞介素1,6的作用。方法：分离、培养小鼠腹腔巨噬细胞,14h后分为红霉素组及阿仑膦酸钠组,每组分为6个亚组。红霉素组：A组：仅为巨噬细胞;B组：巨噬细胞＋钛颗粒;C组：巨噬细胞＋钛颗粒＋红霉素1μg/L;D组：巨噬细胞＋钛颗粒＋红霉素10μg/L;E组：巨噬细胞＋钛颗粒＋红霉素100μg/L;F组：巨噬细胞＋钛颗粒＋红霉素1000μg/L。阿仑膦酸钠组分组及剂量同红霉素组。培养24h后,用酶联免疫法检测细胞培养上清液中白细胞介素1,6及肿瘤坏死因子α的质量浓度。结果与结论：B组白细胞介素1,6及肿瘤坏死因子α的质量浓度明显高于其他组（P〈0.05）,F组白细胞介素1,6及肿瘤坏死因子α的质量浓度明显低于C组（P〈0.05）。同剂量阿仑膦酸钠和红霉素组间差异无显著性意义（P〉0.05）。提示钛颗粒可以刺激巨噬细胞分泌大量的白细胞介素1,6及肿瘤坏死因子α,红霉素、阿仑膦酸钠能够呈剂量依赖型地有效抑制钛颗粒诱导的巨噬细胞分泌白细胞介素1,6及肿瘤坏死因子α。     

阿仑膦酸钠对髋关节假体周围界膜组织分泌肿瘤坏死因子α的影响

背景:目前肿瘤坏死因子α的研究有望成为防治假体周围骨丢失的新的切入点,但阿仑膦酸钠对界膜分泌肿瘤坏死因子α的影响及作用机制尚不清楚.目的:观察阿仑膦酸钠对髋关节假体周围界膜组织分泌肿瘤坏死因子α的影响.设计、时间及地点:随机分组设计,对比观察,实验于2006-02/03在广州中医药大学附属骨科医院骨科实验室完成.材料和对象:界膜组织(15 g)取自广州中医药大学第一附属医院(患者知情并同意);雄性新西兰白兔6只用于制备阿仑膦酸钠含药血清,阿仑膦酸钠为石家庄制药厂产品.方法:无菌条件下从人工髋关节假体周围股骨分离界膜组织(15 g),并将其放入RPMI培养液中培养,将界膜组织剪成约250 mg的碎块,计30块,随机分为3组:空白对照组、低浓度阿仑膦酸钠组(100 g/L)、高浓度阿仑膦酸钠组(200 g/L),每组10个样本.低浓度阿仑膦酸钠组和高浓度阿仑膦酸钠组分别加入1.8 mL和1.6 mL,空白对照组加入1.8 mL.然后在低浓度阿仑膦酸钠组、高浓度阿仑膦酸钠组的培养孔中分别加入入0.2 mL和0.4 mL的阿仑膦酸钠含药血清,空白对照组加入0.2 mL空白血清.添加血清后在37℃、含体积分数为5%CO2的饱和湿度条件下培养72 h.主要观察指标:酶联法检测各组界膜组织中肿瘤坏死因子α的表达.结果:低浓度阿仑膦酸钠组和高浓度阿仑膦酸钠组界膜组织分泌肿瘤坏死因子α的量分别为(3.93±0.03),(3.92±0.04)pg/g,与空白对照组(4.04±0.13)pg/g相比,差异有显著性意义(P<0.01).结论:阿仑膦酸钠能显著抑制髋关节假体周围界膜组织分泌肿瘤坏死因子α.     

人工关节磨屑颗粒诱导巨噬细胞分泌细胞因子与依那西普干预的影响

目的:已证实肿瘤坏死因子α在人工关节无菌性松动过程中发挥重要作用,依那西普为肿瘤坏死因子α拮抗剂,拟验证采用依那西普预防人工关节钛颗粒刺激巨噬细胞分泌肿瘤坏死因子α等细胞因子导致无菌性松动的可能性.方法:实验于2007-03/07在宁夏医学院生殖与遗传实验室完成(宁夏省部级重点实验室).①主要试剂与药物:依那西普 (Enbrel, Amgen and Wyeth),钛颗粒(美国ZIM-MER公司提供,微粒直径为3～5 μm).②小鼠腹腔巨噬细胞的分离、培养:麻醉后处死12只6～8周的清洁级BALB/C小鼠,腹腔注入无血清RPMI1640 培养液5 mL,3～5 min后,无菌条件下打开小鼠腹腔,吸取腹腔液,离心洗涤后显微镜下计数,调整细胞至3×109/L,均匀加入24孔培养板中,置于37 ℃、体积分数为0.05的CO2培养箱中培养.12 h后更换培养液,去掉未贴壁的细胞,得到巨噬细胞.实验过程中对动物处置符合动物伦理学要求.③评估指标:24 h后将培养细胞分为5组,每组有8个平行孔:单纯细胞组,1×1012/L钛颗粒组,1×1012 /L 钛颗粒 10 μg/L依那西普组,1×1012/L钛颗粒 100 μg/L 依那西普组,1×1012/L钛颗粒 1 000 μg/L依那西普组.继续培养18 h后,用酶联免疫法检测上述各组细胞上清液中肿瘤坏死因子α、白细胞介素1、白细胞介素6的浓度.结果:1×1012/L钛颗粒组细胞培养上清液肿瘤坏死因子α、白细胞介素6、白细胞介素1浓度明显高于单纯细胞组、1×1012/L钛颗粒 100,1 000 μg/L依那西普组(P < 0.001).1 000 μg/L依那西普组低于10 μg/L依那西普组,差异有显著性(P < 0.001).结论:依那西普呈剂量依赖性的有效抑制磨屑颗粒诱导的巨噬细胞分泌细胞因子,有望成为预防人工关节无菌性松动的药物.     

破骨细胞活化过程中免疫内酯LY267108对核因子κB的抑制

背景：目前尚无理想药物可用于人工关节无菌性松动的防治。研究表明红霉素对假体周围骨溶解具有较强的抑制作用,然而其抗菌活性却限制了其在人工关节松动防治中的应用。免疫内酯 LY267108是一种新型红霉素衍生物,其消除了抗菌活性,同时保留了抗炎活性。 目的：评价LY267108在破骨细胞活化过程中对核因子kB的抑制作用。 方法：将RANKL、巨噬细胞-集落刺激因子加入小鼠RAW264.7细胞系建立破骨细胞诱导模型,同时分别加入不同浓度的阿仑膦酸钠、红霉素及LY267108共培养48 h,分别采用电泳迁移率分析法及蛋白免疫印迹法测定胞核内核因子κB活性和胞浆内κB抑制蛋白α含量。 结果与结论：LY267108对核因子κB具有较强的抑制活性,10 mg/L LY267108、25 mg/L红霉素与10 mg/L阿仑膦酸钠对核因子κB具有相似的抑制作用,且显著强于10 mg/L红霉素,而25 mg/L LY267108具有最强的抑制作用。10 mg/L LY267108、25 mg/L红霉素与10 mg/L阿仑膦酸钠组中胞浆内κB抑制蛋白α水平差异无显著性意义,但显著高于10 mg/L红霉素组,而25 mg/L LY267108组胞浆内κB抑制蛋白α水平最高。提示免疫内酯LY267108在破骨细胞活化过程中,对核因子kB具有较红霉素更强的抑制作用,且安全性高于阿仑膦酸钠。同时 LY267108无抗菌活性的特性,使其成为防治人工关节无菌性松动的潜在理想药物。而LY267108对κB抑制蛋白α降解的抑制作用,可能是其抑制核因子κB活化的机制之一。     

不同质量浓度阿仑膦酸钠干预牙槽骨吸收模型兔牙周组织破骨细胞分化因子和骨保护素的表达

背景:有研究表明阿仑膦酸钠能够防治骨质疏松症,但其应用到口腔牙周组织干预牙槽骨吸收较为罕见。目的:建立兔牙槽骨吸收模型,观察不同质量浓度阿仑膦酸钠干预下炎症牙周组织破骨细胞分化因子和骨保护素的表达。方法:将大耳白兔建立牙槽骨吸收模型,建模成功后随机分成5组,胶原+0.5g/L阿仑膦酸钠组、胶原+1g/L阿仑膦酸钠组、胶原+2g/L阿仑膦酸钠组、胶原组、对照组。各组分别于用药后2和4周用免疫组织化学方法检测破骨细胞分化因子和骨保护素表达情况,并进行药效评价。结果与结论:破骨细胞分化因子和骨保护素阳性细胞在5组成骨细胞、破骨细胞、成纤维细胞中均有表达,胞浆呈棕色或棕褐色颗粒状着色。对照组和胶原组牙槽嵴吸收区破骨细胞分化因子和骨保护素比率明显大于其他3组(P<0.05),胶原+1g/L阿仑膦酸钠组在用药后2和4周均高于胶原+0.5g/L阿仑膦酸钠组(P<0.05)。结果证实,阿仑膦酸钠能够降低牙槽骨破骨细胞分化因子和骨保护素的比率,应用含1g/L阿仑膦酸钠胶原海绵载体更适合抑制牙槽骨吸收。     

高迁移率族蛋白B1对磷酸钙诱导巨噬细胞释放炎症因子的协同作用

背景：研究表明,巨噬细胞及其炎症反应参与了肾结石的发生发展。前期实验发现结石晶体可刺激巨噬细胞释放高迁移率族蛋白B1。目的：观察高迁移率族蛋白B1对磷酸钙诱导巨噬细胞释放白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1的协同作用。方法：实验分两部分：①将成功诱导为巨噬细胞的U937细胞分为空白组、100 mg/L磷酸钙组、100μg/L高迁移率族蛋白B1组、100 mg/L磷酸钙＋100μg/L高迁移率族蛋白B1组,干预1,2,4 h后收集细胞上清液。②将已成功诱导为巨噬细胞的U937细胞分为100 mg/L磷酸钙组、磷酸钙＋10μg/L高迁移率族蛋白B1组、磷酸钙＋50μg/L高迁移率族蛋白B1组、磷酸钙＋100μg/L高迁移率族蛋白B1组,干预4 h后收集细胞上清液。Elisa法检测白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1水平。结果与结论：ELISA结果显示,磷酸钙组,100μg/L高迁移率族蛋白B1组上清液白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1质量浓度均高于空白组,磷酸钙＋100μg/L高迁移率族蛋白B1组上清液上述因子质量浓度均显著高于其他3组（P〈0.05）,且呈时间依赖性。不同质量浓度高迁移率族蛋白B1＋磷酸钙组细胞上清液白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1水平均显著高于磷酸钙组（P〈0.05）,且呈浓度依赖性。结果表明,磷酸钙及高迁移率族蛋白B1均可诱导巨噬细胞释放白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1;高迁移率族蛋白B1可协同磷酸钙诱导巨噬细胞释放白细胞介素1β、白细胞介素6、肿瘤坏死因子α、单核细胞趋化因子1。     

构建创伤性股骨头塌陷坏死大鼠模型及阿仑膦酸钠预防的机制

背景：临床随访研究表明阿仑膦酸钠对于预防股骨头坏死塌陷有效,但尚缺乏其预防塌陷作用的机制研究。目的：分析阿仑膦酸钠预防股骨头坏死塌陷的效果及其作用机制。方法：将45只SD大鼠随机分成3组,每组15只。安慰剂组在建立股骨头坏死模型后给予生理盐水治疗;阿仑膦酸钠组建立股骨头坏死模型后给予药物阿仑膦酸钠治疗;假手术组给予同样剂量的生理盐水治疗。造模后5周处死大鼠,取造模侧股骨标本分别行大体标本观察,X射线、Micro-CT及组织学检测。结果与结论：大体标本观察安慰剂组股骨头明显塌陷畸形,阿仑膦酸钠组股骨头轻度变形。股骨头高度与宽度的比值假手术组〉阿仑膦酸钠组〉安慰剂组,差异均有显著性意义。Micro-CT扫描结果显示阿仑膦酸钠组骨小梁平均数量多于安慰剂组,少于假手术组,差异均有显著性意义。阿仑膦酸钠组骨小梁平均厚度小于安慰剂组,但和假手术组比差异无显著性意义。阿仑膦酸钠组骨小梁平均间距小于安慰剂组,但大于假手术组,差异均有显著性意义。阿仑膦酸钠组股骨头骨组织体积、骨表面积、骨矿盐密度均大于安慰剂组,小于假手术组,差异均有显著性意义。组织学检测结果显示,阿仑膦酸钠组存在明显的死骨,破骨细胞明显受到抑制,破骨细胞数量较安慰剂组明显减少,成骨细胞和新生血管也受到了一定程度的抑制。结果表明阿仑膦酸钠可通过全面抑制破骨细胞、成骨细胞及血管新生而抑制骨坏死的修复反应,减慢坏死骨的吸收,保存骨量及股骨头形态,对大鼠创伤性股骨头坏死早期塌陷具有一定的预防作用。     

不同质量浓度阿仑膦酸钠干预牙槽骨吸收模型兔牙周组织破骨细胞分化因子和骨保护素的表达

背景：有研究表明阿仑膦酸钠能够防治骨质疏松症,但其应用到口腔牙周组织干预牙槽骨吸收较为罕见。目的：建立兔牙槽骨吸收模型,观察不同质量浓度阿仑膦酸钠干预下炎症牙周组织破骨细胞分化因子和骨保护素的表达。方法：将大耳白兔建立牙槽骨吸收模型,建模成功后随机分成5组,胶原＋0.5g/L阿仑膦酸钠组、胶原＋1g/L阿仑膦酸钠组、胶原＋2g/L阿仑膦酸钠组、胶原组、对照组。各组分别于用药后2和4周用免疫组织化学方法检测破骨细胞分化因子和骨保护素表达情况,并进行药效评价。结果与结论：破骨细胞分化因子和骨保护素阳性细胞在5组成骨细胞、破骨细胞、成纤维细胞中均有表达,胞浆呈棕色或棕褐色颗粒状着色。对照组和胶原组牙槽嵴吸收区破骨细胞分化因子和骨保护素比率明显大于其他3组（P〈0.05）,胶原＋1g/L阿仑膦酸钠组在用药后2和4周均高于胶原＋0.5g/L阿仑膦酸钠组（P〈0.05）。结果证实,阿仑膦酸钠能够降低牙槽骨破骨细胞分化因子和骨保护素的比率,应用含1g/L阿仑膦酸钠胶原海绵载体更适合抑制牙槽骨吸收。     

川芎嗪可影响肿瘤坏死因子α刺激肝星状细胞结缔组织生长因子、核因子κB及相关基因产物的表达

背景：川芎嗪延缓肝纤维化形成的机制尚不清楚。目的：观察川芎嗪对肿瘤坏死因子α刺激的肝星状细胞增殖及结缔组织生长因子、核因子κB及其相关基因产物白细胞介素6表达的影响。方法：体外培养HSC-T6细胞株,取对数生长期的细胞用于实验。实验分为4组：对照组仅加入细胞;TNF-α组加入10μg/L TNF-α;川芎嗪干预组先加入终浓度为10μg/L的TNF-α作用30min后,分别加入川芎嗪50,100,200,400,600,1000mg/L;PDTC组先加入终浓度为10μg/L的TNF-α作用30min后,再加入终浓度18μmol/L的核因子κB阻断剂PDTC。结果与结论：MTT结果显示100,200,400,600,1000mg/L的川芎嗪均能抑制HSC-T6增殖,且呈剂量依赖性。免疫细胞化学染色及Western blot检测发现10μg/L的肿瘤坏死因子α刺激后,HSC-T6细胞结缔组织生长因子、核因子κB及白细胞介素6的表达明显增多（P〈0.01或P〈0.05）,200,400,600mg/L的川芎嗪及18μmol/L的PDTC均可明显降低肿瘤坏死因子α刺激后HSC-T6细胞结缔组织生长因子、核因子κB及白细胞介素6的表达（P〈0.01）,且随着川芎嗪质量浓度的增加,抑制作用增强,PDTC的抑制作用最明显。相关性分析结果显示HSC-T6细胞结缔组织生长因子和核因子κB的表达呈正相关（r=0.980,P〈0.01）。说明川芎嗪可以抑制肝星状细胞结缔组织生长因子、核因子κB及白细胞介素6的表达,抑制肝星状细胞的增殖。     

瘦素对RAW264.7细胞肿瘤坏死因子α表达的影响

背景:瘦素是脂肪组织分泌的一种多肽激素,研究显示瘦素在动脉粥样硬化形成中发挥了一定重要的作用。目的:观察瘦素对鼠源性巨噬细胞系RAW264.7细胞肿瘤坏死因子α表达的影响,并从核转录因子κB活性变化角度探讨其可能机制。设计:对照观察实验。单位:华中科技大学同济医学院生物化学及分子生物学系。材料:实验于2005-04/2006-02在华中科技大学同济医学院生物化学及分子生物学系及附属协和医院普外科实验室完成。将培养的RAW264.7细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)、IkappaB激酶抑制剂组及空白对照组。每组3瓶,重复实验3次。方法:将鼠源性巨噬细胞株RAW264.7细胞以1×109L-1密度接种于6孔板中,用含体积分数为0.1的小牛血清的RPMI-1640培养基培养。待RAW264.7细胞生长至80%时,换用无血清培养基Opti-MEM继续培养24h后,将细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)及空白对照组,瘦素孵育4h后采用反转录-聚合酶链反应检测肿瘤坏死因子α在mRNA水平的表达。上述分组细胞经瘦素分别孵育1,3,6和9h后采用双抗夹心酶联免疫吸附实验检测肿瘤坏死因子α在蛋白水平的表达。上述分组细胞经瘦素孵育不同时间后采用凝胶迁移率实验检测细胞核内核转录因子κB活性。将RAW264.7细胞分为以下4组:空白对照组、IkappaB激酶特异性抑制剂PS1145(10μmol/L)处理组、瘦素(50μg/L)处理组、瘦素(50μg/L) PS1145(10μmol/L)组,各组孵育时间均为6h,分别检测细胞核内核转录因子κB活性及肿瘤坏死因子α在mRNA水平的表达。主要观察指标:①不同浓度瘦素对RAW264.7细胞肿瘤坏死因子α:mRNA表达水平的影响;蛋白分泌的影响。②不同浓度瘦素对RAW264.7细胞核内核转录因子κB活性的影响。③抑制IkappaB激酶活性对瘦素诱导RAW264.7细胞肿瘤坏死因子α的影响。结果:①RAW264.7细胞经不同浓度的瘦素处理后,肿瘤坏死因子α在mRNA水平呈瘦素剂量依赖性增加,50μg/L瘦素处理组达峰值。②蛋白水平的表达呈瘦素剂量时间依赖性增加,50μg/L瘦素处理6h即可达峰值。③核转录因子κB的活性亦与瘦素浓度正相关,50μg/L瘦素处理6h后核转录因子κB活性最高(P<0.05)。④抑制IkappaB激酶活性可部分抑制肿瘤坏死因子α的表达。结论:瘦素可直接促进RAW264.7细胞肿瘤坏死因子α的表达和分泌,并呈剂量时间依赖性,其机制可能与瘦素激活核转录因子κB有关。这可能是瘦素致动脉粥样硬化的机制之一。     

Inhibitory effect of erythromycin on interleukin 8 production by 1 alpha,25-dihydroxyvitamin D3-stimulated THP-1 cells.

We have recently reported that long-term administration of erythromycin at a low dose reduced the number of neutrophils and concentrations of interleukin 8 (IL-8) in bronchoalveolar lavage fluid in patients with chronic lower respiratory tract disease. To investigate the mechanism of action of erythromycin, we evaluated its effect on IL-8 production in the 1 alpha,25-dihydroxyvitamin D3-stimulated human monocytic cell line THP-1. Erythromycin at a concentration of 10 micrograms/ml significantly reduced IL-8 production by THP-1 cells stimulated with lipopolysaccharide (10 ng/ml) and 1% normal human serum compared with the amount produced by untreated cells (untreated cells, 2,448 pg/ml; erythromycin-treated cells, 872 pg/ml). Our results suggest that erythromycin may impair IL-8 production by alveolar macrophages, ultimately reducing neutrophil accumulation in the airspace.     

Osteoblasts mediate interleukin 1 stimulation of bone resorption by rat osteoclasts

A monocyte-derived factor with IL-1-like properties has recently been shown to cause resorption of bone in organ culture. We have investigated the action of IL-1 on disaggregated populations of osteoclasts, incubated alone or in the presence of osteoblastic cells, in an attempt to identify the target cell for IL-1 in bone, and to elucidate the mechanism by which IL-1 induces osteoclastic resorption. Osteoclasts were disaggregated from neonatal rat long bones and incubated on slices of human femoral cortical bone. Under these conditions, the majority of osteoclasts form distinctive excavations in the bone surface within 24 h, the volume of which can be quantified by computer-assisted morphometric and stereophotogrammetic techniques. IL-1 had no effect on bone resorption by osteoclasts alone, but when incubated in the presence of calvarial cells or cloned osteosarcoma cells, it induced a 3.8 (+/- 0.38)-fold increase in osteoclastic bone resorption, with significant enhancement at concentrations of greater than or equal to 30 pg/ml. The osteoblastic populations themselves did not resorb bone. The mechanism by which osteoblastic cells stimulate osteoclasts did not appear to depend upon PG synthesis; nor could we detect a diffusible substance in the medium of stimulated cocultures. These results indicate that IL-1 stimulates bone resorption through a primary action on osteoblasts, which are induced by IL-1 to transmit a short-range signal that stimulates osteoclastic bone resorption.     

MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.     

Regulation and enzymatic basis of bone resorption by human osteoclasts

Although much has been learned recently of the mechanisms that regulate osteoclastic differentiation, much less is known of the means through which their resorptive activity is controlled. This is especially so for human osteoclasts. We have recently developed an assay that allows us to measure resorptive activity while minimizing confounding effects on differentiation by optimizing osteoclastogenesis, so that measurable resorption occurs over a short period, and by relating resorption in each culture during the test period to the resorption that had occurred in the same culture in a prior control period. In the present study, we found that RANKL (receptor activator of nuclear factor kappaB ligand) strongly stimulated the release of CTX-I (C-terminal telopeptide degradation product of type I collagen) by osteoclasts over a similar range to that over which it induces osteoclastic differentiation, consistent with a distinct action on osteoclastic function. CT (calcitonin) dose-dependently inhibited bone resorption, whereas PTH (parathyroid hormone), IL (interleukin)-1, TNF-alpha (tumour necrosis factor-alpha), IL-6, IL-8, VEGF (vascular endothelial growth factor), MCP-1 (monocyte chemoattractant protein-1), MIP-1gamma (macrophage inflammatory protein-1gamma), IFN (interferon)-gamma and dibutyryl cGMP had no significant effect. Ca(2+), cyclosporin A, IFN-beta and dibutyryl cAMP all strongly suppressed resorption. Bone resorption was also strongly suppressed by alendronate, the cysteine protease inhibitor E64 and the cathepsin K inhibitor MV061194. Inhibitors of MMPs (matrix metalloproteinases) had no effect on CTX-I release. Moreover, the release of the MMP-derived collagen fragment ICTP (C-terminal cross-linked telopeptide of type I collagen) represented less that 0.01% of the quantity of CTX-I released in our cultures. This suggests that MMPs make, at most, a very small contribution to the bone-resorptive activity of osteoclasts.     

Role of interleukin 2, interleukin 4, and alpha, beta, and gamma interferon in stimulating macrophage antibody-dependent tumoricidal activity

Pretreatment of murine peritoneal exudate macrophages with 1-5 U/ml rIFN-gamma or rIL-2, or higher concentrations of IFN-alpha or IFN-beta greatly stimulated ADCC to Rl lymphoma targets. The assay was direct counting of viable target cells after 9 and 24 h using an E/T ratio of 5:1. 2d of pretreatment was optimal for enhancing ADCC. rIL-4 was inactive and IL-4-depleted Con A-induced spleen lymphokine retained its ADCC-stimulating activity. Antibody to IFN-gamma blocked the ADCC-promoting effect of the lymphokine, suggesting a major role for this factor.     

Listeria meningitis: identification of a cerebrospinal fluid inhibitor of macrophage listericidal function as interleukin 10

The killing of bacteria gaining access to the central nervous system is insufficient and requires bactericidal antibiotics for treatment. The inefficient host response in cerebrospinal fluid (CSF) is thought to be due to impaired phagocytosis in CSF, and low local concentration of antibody and complement. In addition, the CSF may contain inhibitors, disabling phagocytes to eliminate bacteria. We have assessed the bactericidal activity of macrophages in the presence of CSF from mice infected intracerebrally with Listeria monocytogenes (LM). Pretreatment of J774A.1 macrophages with interferon gamma (IFN-gamma) resulted in high levels of nitric oxide-dependent intracellular killing of LM. CSF taken from mice 24 h after infection (CSF-LM 24) contained IFN-gamma and induced killing of LM by macrophages. However, pulsing J774A.1 cells with IFN-gamma in the presence of CSF obtained from mice at later time points (48 h) rendered macrophages partly permissive for intracellular Listeria growth. The inhibitor detected in CSF-LM 48 was identified as IL-10 since: (a) IL-10 dose dependently impaired the listericidal activity of IFN-gamma-activated macrophages; (b) anti-IL- 10 antibodies abrogated the bacterial growth permissive effect of CSF- LM 48; and (c) IL-10 was detected in CSF-LM 48 but not in CSF-LM 24 or CSF of mock-injected animals (CSF-Co). Likewise, IL-10 was found in the CSF of 95% of patients with bacterial meningitis.     

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3H]alendronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 microns away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone. In vitro alendronate bound to bone particles with a Kd of approximately 1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 x 10(-3) fmol/microns 2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts.