补体抗动脉粥样硬化和调节Toll样受体致炎作用的实验研究
|
摘要
中文摘要Ⅰ补体膜调节蛋白抗动脉粥样硬化作用的实验研究
背景
补体系统的可溶性成分多以前体的形式存在于外周血,当病原入侵或者组织损伤时,可通过经典途径、凝集素激活途径或替代途径被激活。激活的补体系统通过释放白细胞趋化因子C3a和C5a促进炎症反应,并可通过合成膜攻击复合体介导细胞损伤。为了防止自体细胞受到补体系统的攻击,生物体内的多种细胞,尤其是那些接触血液循环的血细胞和内皮细胞,表达了多种结合于细胞膜的补体调节蛋白,其中包括通过GPI结构与细胞膜锚链的CD55和CD59。CD55可以通过促进C3和C5转化酶的降解而调控补体激活,而CD59主要通过抑制膜攻击复合体的形成而发挥作用。
近年的研究表明,动脉粥样硬化(atherosclerosis,AS)是一种发生于动脉血管壁的慢性炎症,天然性与获得性免疫系统的多个成分参与了其发生发展过程。作为天然性免疫系统的一个重要组成部分,补体系统致AS的作用已为许多研究所揭示。各种补体系统的激活产物,调节蛋白和补体受体均可在AS病灶,尤其是易损斑块和已破裂斑块中被探及,并且对人体病变的研究发现,膜攻击复合体的沉积量与病灶进展程度呈现正相关。对补体敲基因动物的研究亦初步揭示了补体致AS的机理:补体激活的中心分子—C3的基因缺陷可以通过上调血甘油三酯的浓度促进AS病灶的发展;C6基因缺陷可以明显减少高胆固醇饮食所诱发的兔主AS,而C5基因敲除却并没有明显地影响ApoE~(-/-)小鼠的斑块大小,意味着补体终末效应物-膜攻击复合体在AS发展的过程中发挥着较为复杂的作用;在补体激活通路的研究方面,替代激活途径的关键因子-Factor B缺陷并没有对AS产生明显的影响,提示另外两条通路可能发挥着更为重要的作用;而经典激活途径的关键因子-Clq基因缺陷却通过影响坏死细胞的清除促进了病变的发展。总之,近来的研究表明,补体系统参与了AS的发病过程,但是其具体的作用机制十分复杂,仍然需要进一步的研究来加以阐明。
作为补体系统的重要组成部分,膜调节蛋白亦被发现存在于AS病灶中,并且研究发现,阿托伐他汀和C反应蛋白均可诱导内皮细胞表达补体膜调节蛋白,从而使之幸免于补体系统的裂解作用。补体膜调节蛋白是否参与了AS病灶的发生和发展过程,具体的作用机制是什么,目前均无研究加以阐明。由于该组蛋白具有明显的补体激活抑制作用,我们提出如下科学假说:补体膜调节蛋白通过抑制补体激活或补体效应物的产生而发挥了抗AS作用。
目的
1.通过分别把CD55~(-/-)和CD59~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得易形成AS斑块的双基因敲除小鼠;
2.通过对双敲小鼠的病变范围、斑块大小和病灶内容物进行分析,研究补体膜调节蛋白-CD55和CD59在AS发生发展中的作用;
3.通过对血脂、血尿、补体激活和T细胞免疫的分析,探讨CD55和CD59抗AS的机制。
方法
1.获取双基因敲除小鼠及高脂喂养
把CD55~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得CD55~(+/+)ApoE~(-/-)杂合子,以杂合子小鼠交配繁殖获得CD55~(-/-)ApoE~(-/-)小鼠和同龄野生型对照-CD55~(+/+)ApoE~(-/-)小鼠。将6周龄的小鼠根据高脂喂养时间和性别分为4组:短期雌性组包括雌性CD55~(-/-)ApoE~(-/-)小鼠和雌性CD55~(+/+)ApoE~(-/-)对照,高脂喂养8周;短期雄性组包括雄性CD55~(-/-)ApoE~(-/-)小鼠和雄性CD55~(+/+)ApoE~(-/-)对照,高脂喂养8周;长期雌性组包括雌性CD55~(-/-)ApoE~(-/-)小鼠和雌性CD55~(+/+)ApoE~(-/-)对照,高脂喂养16周;长期雄性组包括雄性CD55~(-/-)ApoE~(-/-)小鼠和雄性CD55~(+/+)ApoE~(-/-)对照,高脂喂养16周。以与CD55同样的方式,把CD59~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得获得CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠。将6周龄的CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠按照高脂喂养时间和性别分为4组。
各组高脂喂养结束后,小鼠空腹过夜后被处死。自下腔静脉收集血液标本,并冻存。PBS冲洗左侧心腔及动脉腔内的残血后,将心脏分离并包埋于OCT。钝性分离自主动脉弓到腹主动脉分叉处的主动脉节段,进行染色分析。所有的动物实验及相关处理均通过了美国宾夕法尼亚大学动物管理及应用委员会的批准。
2.血液及尿液分析
应用酶法测定血清总胆固醇水平。应用代谢笼连续24小时收集小鼠的尿液,并应用HPLC/MS/MS法分析尿液中脂质代谢产物8,12-iso-iPF2_α-V和肌酐的水平。
3.斑块大小分析
将主动脉纵剖后钉于蜡版,并进行苏丹Ⅳ染色分析。病变范围表示为AS斑块占主动脉表面积的百分比。将小鼠心脏进行6μm连续冰冻切片,收集自出现三个主动脉瓣叶起至瓣尖的切片。对冰冻切片进行油红0染色,照相并应用软件分析。斑块大小表示为斑块占主动脉根面积的百分比。
4.病理学检测
对主动脉窦部冰冻切片行免疫组化染色和天狼星红染色,测量斑块内平滑肌细胞、巨噬细胞、C3、C9和胶原的阳性染色面积,其含量表示为阳性染色面积占斑块总面积的百分比。对切片行CD4和CD8免疫组化染色,计数单位面积内的阳性细胞个数。
5.统计学分析
定量数据均以均数±标准误表示,应用Graphpad公司的Prism3.0进行统计分析。在做分析之前,对所有的数据进行正态分布检验。对于正态分布的数据应用双侧t检验,对非正态分布的数据应用Mann—Whiteney分析。设P＜0.05为有统计学意义。
结果
1.CD59的抗AS作用
短期雌性组内,虽然主动脉表面病灶范围并无组间差异(2.07±0.27%vs.1.34±0.21%,P=0.06),但是CD59~(-/-)ApoE~(-/-)小鼠主动脉窦部的斑块明显大于CD59~(+/+)ApoE~(-/-)对照(20.74±1.33%vs.13.12±1.46%;P＜0.005)。然而,短期雄性组内,雄性CD59~(-/-)ApoE~(-/-)小鼠与CD59~(+/+)+ApoE~(-/-)对照间未发现显著差异(病变范围:1.35±0.16%vs.1.57±0.19%,P=0.3936;斑块大小:11.23±1.23%vs.12.75±1.41%,P=0.536)。长期雌性组内,CD59基因缺陷明显地增加了雌性小鼠的病变覆盖范围(17.13±1.14%vs.9.72±1.14%;P＜0.001),但是却未对主动脉窦部的斑块大小产生明显的影响(32.67±1.58%vs.34.44±2.52%;P=0.315)。与短期雄性组的结果相似,在长期雄性组内未发现显著差异(病变范围:12.88±1.49%vs.10.98±0.95%,P=0.779;斑块大小:26.84±1.97%vs.29.46±2.3%,P=0.408)。CD59基因缺陷导致了病变覆盖范围的扩大或局部斑块的增大,提示CD59具有明显的抗AS作用。然而,无论是在短期还是长期雄性组内,均未发现显著的差异,提示CD59的抗AS作用存在明显的性别差异。
2 CD55缺陷与AS
与CD59基因缺陷的结果不同,无论在雄性还是雌性组,CD55基因敲除均未引起明显的病变范围或斑块大小的改变。在短期雌性组,雌性CD55~(-/-)ApoE~(-/-)和CD55~(+/+)ApoE~(-/-)小鼠的病变范围(0.80±0.17%vs 1.37±0.22%;P=0.07)和斑块大小(21.45±1.33%vs 23.58±2.1096;P=0.337)无显著差异:同样的结果也见于短期雄性组(病变范围:1.14±0.17%vs 0.80±0.14%,P=0.207:斑块大小:15.68±1.58%vs 16.89±1.65%,P=0.921)。在长期雌性组,CD55~(-/-)ApoE~(-/-)与CD55~(+/+)ApoE~(-/-)小鼠的病变范围(6.39±0.59%vs 5.81±0.51%:P=0.303)和斑块大小(29.81±2.78%vs 29.11±3.25%;P=0.918)无显著差异;长期雄性组内,也未发现明显的差异(病变范围:6.09±0.83%vs 6.16±0.69%,P=0.95;斑块大小:25.25±3.02%vs 30.94±3.22%;P=0.281)。
3 CD59缺陷对斑块构成的影响
通过对短期雌性组的病理学分析,我们研究了CD59基因缺陷对斑块内各种成份的影响。CD68染色显示,CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠巨噬细胞的分布和相对含量较为一致(18.74±2.79%vs.18.66±2.41%;P=0.983),提示CD59并没有参与巨噬细胞的粘附、浸润或增殖过程,仅仅通过其他途径促进了斑块的发展,而巨噬细胞呈现等比例的增加;α-SMC actin染色显示,CD59~(-/-)ApoE~(-/-)小鼠的平滑肌细胞显著少于对照组(3.69±0.51%vs.6.42±0.86%;P＜0.05);苦味酸-天狼星红染色发现,CD59基因缺陷小鼠的胶原含量较对照小鼠明显增加(8.14±0.33%vs.6.07±0.82%:P＜0.05)。
2.4 CD59抗AS作用的机制
血清学分析显示,短期雌性组内,CD59~(-/-)ApoE~(-/-)小鼠的血清总胆固醇显著高于CD59~(+/+)ApoE~(-/-)小鼠(1404±43.74vs.1208±57.78mg/dL;P＜0.05),提示CD59可能通过降低血清总胆固醇发挥抗AS作用。通过对尿液中氧化应激标志物的质谱分析,发现在雌性CD59~(-/-)ApoE~(-/-)小鼠和CD59~(+/+)ApoE~(-/-)对照间无显著差异(短期雌性组:2.98±1.01 vs.3.12±0.96 ng/mg肌酐,P=0.92;长期雌性组:1.65±0.56 vs.1.59±0.38 ng/mg肌酐,P=0.92),因而CD59基因缺陷并未对该系统产生显著的影响。短期雌性组的C3免疫组化染色发现,CD59~(-/-)ApoE~(-/-)小鼠与对照小鼠间斑块内的C3沉积无显著差异(28.65±1.69%vs.25.33±3.07%,P=0.37),提示CD59基因缺陷并没有影响局部的补体激活;而C9染色发现,CD59~(-/-)ApoE~(-/-)小鼠斑块内的C9沉积明显多于对照组(17.32±1.52%vs.8.74±3.63%,p＜0.05),提示CD59基因缺陷时,局部的补体膜攻击复合体形成明显增加。为了明确CD59是否通过影响T细胞而发挥抗AS作用,对短期雌性组的小鼠切片做了CD4和CD8染色分析。结果发现,CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠斑块内的T细胞含量无差异(CD4~+T细胞:35.89±8.505 vs.40.99±7.091/mm~2,P=0.66:CD8~+T细胞:1.622±1.622 vs.5.384±3.946/mm~2,P=0.40)。
结论
1 CD59可能通过降低血清总胆固醇和减少补体效应产物-膜攻击复合体的生成而发挥抗AS的作用。该作用与氧化应激和GPI介导的T细胞激活无明显的相关性。CD59的抗AS作用仅见于雌性,具有明显的性别差异。
2 CD59并没有参与斑块内巨噬细胞的粘附、迁移和增殖过程,该因子缺陷时通过影响其他方面导致了等幅度的巨噬细胞浸润;CD59通过抑制膜攻击复合体的形成保护血管壁平滑肌细胞免受补体系统的攻击。
3 CD55基因缺陷并没有引起明显的斑块大小改变,提示在动脉壁保护的过程中,其他的C3转化酶抑制剂如Factor H和Crry可能发挥着更重要的作用。在CD55基因缺陷的情况下,C3转化酶的功能仍然得到了有效的抑制,补体系统并未被有效地激活。
4主动脉表面的病变范围和窦部的斑块大小存在失偶联的现象,很可能是由于根部的空间有限,在ApoE缺陷较强的致AS背景下,其他基因很难引起明显的斑块大小改变。主动脉表面具有广阔的空间,可以作为更恒定的评价指标。
中文摘要Ⅱ补体过敏毒素调节Toll样受体致炎作用的体外研究
背景
天然免疫系统是机体抵御病原微生物感染的第一道防线。Toll样受体(tolllike receptors,TLRs)和补体是天然免疫系统的两个重要组成部分,通过迅速诱发炎症反应和激活继发的获得性免疫而在应对致病原的过程中发挥着重要的作用。
哺乳动物的TLRs,是果蝇Toll样蛋白的同源体,属于病原模式识别受体家族,可以通过识别病原或坏死细胞的相关分子活化天然免疫系统,同时也可以通过向抗原递呈细胞提供信号而启动获得性免疫系统,在细胞吞噬的调节、炎症反应的激活等方面发挥着重要的作用。补体是由30余种可溶性蛋白和膜结合性蛋白组成的多分子系统。其可溶性成分多以无活性的前体形式存在于血液循环中,需要时,在激活物如抗原-抗体复合物等的作用下,被依次激活,形成膜攻击复合体,发挥溶解、破坏细菌、病毒等致病微生物的作用。除膜攻击复合体外,补体系统激活时尚可以通过产生过敏毒素-C3a和C5a,参与机体的抗感染免疫,扩大体液免疫效应,调节免疫应答。
研究发现,许多TLRs的天然激活物如革兰阴性菌来源的脂多糖和来源于酵母细胞壁的酵母多糖也可以激活补体系统,提示在这两系统间可能存在一定的相互联系。探讨补体和TLRs之间是否存在相互作用及其具体的作用机制,对于深入理解炎症和机体的免疫反应具有极其重要的意义。日前,我们的一项研究发现,LPS等TLRs激活剂在CD55~(-/-)小鼠诱发的炎症反应明显强于对照小鼠,提示补体上调了TLRs的致炎作用。后继的机制研究发现,该作用是由补体过敏毒素-C3a和C5a所介导的。体内实验中,补体与TLRs的协同作用发生迅速,TLRs激活剂注入体内后3小时即达到顶峰,提示该作用很可能是由与循环系统密切接触的器官或者组织所介导的。内皮细胞构成了机体内最大的“内分泌器官”,并且与循环系统直接接触,故而我们提出如下科学假说:内皮细胞介导了补体对TLRs致炎作用的调节。在本研究中,我们应用人脐静脉内皮细胞(HUVEC)和小鼠心脏内皮细胞(H5V)对内皮系统在这一过程中的作用进行了探讨。
目的
研究内皮细胞是否介导了补体C3a和C5a对TLRs致炎作用的调控,并探讨其具体的作用机制。
方法
1细胞及其培养
人脐静脉内皮细胞,培养于含10%胎牛血清、0.05 mg/ml内皮细胞生长因子、2mML—谷氨酰胺、0.1mg/ml肝素和100 U混合抗生素的以M199为主体的培养基中。H5V为来源于小鼠心脏的内皮细胞系,培养于含10%胎牛血清的DMEM培养基内。内皮细胞贴壁生长于以0.1%明胶预先包被的培养皿中,37℃、5%CO_2的条件下,2-3天传代一次。其中第5-7代的HUVEC用于实验。
2细胞毒性实验
以单剂细胞增殖分析法研究C3a、C5a、Pam3CKS4、Poly I:C、LPS和CPG对内皮细胞的毒性作用,以选出可用的浓度范围。
3内皮细胞C5a受体的测定
以流式细胞仪分析HUVEC和H5V表面的C5a受体的表达情况,以确认内皮细胞可表达C5aR。
4干预实验
以1×10~5个/孔的浓度将细胞接种于24孔板,培养24小时后细胞大约融合至80%。在分析浓度-效应时,将各TLRs激活剂以培养基梯度稀释成5个浓度,刺激24小时;在分析时间-效应时,以选定的浓度稀释TLRs激活剂,分别刺激内皮细胞3、6、9、12、15和24个小时。刺激实验结束后,收集培养液用于IL-6或IL-8的ELISA分析,以选出合适的作用浓度和时间。过敏毒素和TLRs激活剂联合作用时,用不同浓度的C3a和C5a合并选定浓度的TLRs激活剂刺激80%融合的单层内皮细胞24小时。取培养液用于IL-6、IL-8或chemokines KC的ELISA分析,研究过敏毒素是否上调了TLRs的致炎作用。
5酶联免疫吸附实验(enzyme linked immunoad sorbent assay,ELISA)按照生产厂家提供的实验步骤,对稀释过的培养液进行IL-6、IL-8或chemokine KC的ELISA分析。
结果
1细胞毒性实验
应用单剂细胞增殖分析试剂盒,检测了补体过敏毒素和TLRs对H5V的细胞毒性作用。结果显示,C3a在0-100nM,C5a在0-200nM,Pam3CKS4在0-10000 ng/ml,PolyI:C在0-50μg/ml,LPS在0-1000 ng/ml,CPG在0-160ng/ml为安全的浓度区域,并未引起明显的细胞坏死。
2 C5a受体表达
应用流式分析,发现在HUVEC和H5V表面有相当数量的C5aR表达,而同时染色的阴性对照-HEK细胞上几乎没有荧光信号。
3 C5a和C3a对TLR4致炎作用的影响
LPS浓度-效应的分析发现,10ng/ml LPS即可以明显地增加H5V细胞的IL-6表达,随后IL-6的生成随着LPS浓度的增高而增加,当LPS浓度为500ng/ml时达到顶峰;时间-效应的分析发现,100ng/ml LPS作用3小时即可以诱导H5V细胞IL-6的释放增加,其效应随着作用时间的延长而增加,在刺激24小时时达到顶峰。
根据上述实验结果,我们选用10 ng/ml和100 ng/ml作为LPS的作用浓度,与C3a和C5a联合作用于内皮细胞。刺激24小时后,100 ng/ml的LPS明显地增加了IL-6的生成,其作用约为10 ng/ml组的2.7倍。但无论是合并10 ng/ml还是100 ng/ml的LPS,50nM C3a或C5a均未显著地增加LPS的诱导IL-6或chemokine KC的生成。应用不同浓度的C5a与100 ng/ml LPS同时刺激H5V,结果在其他浓度时,并未发现明显的上调作用。应用HUVEC重复了上述实验,也只获得了类似的结果。
4 C3a和C5a对其他补体激活物致炎作用的影响
100ng/ml Pam3CSK 4即可引起明显的IL-6表达增加,其刺激作用在1000ng/ml时达到最高;IL-6的生成亦随着Pam3CSK 4作用时间的延长而增加,刺激24小时达到顶峰。联合刺激时,100ng/ml Pam3CSK 4明显增加了内皮细胞的IL-6生成,但50hM C3a或50nM C5a均未对Pam3CSK4的致炎作用产生明显影响。
1μg/ml PolyI:C即可引起明显IL-6表达增加,其刺激作用在50μg/ml时达到最高:IL-6的生成随着作用时间的延长而增加,刺激24小时达到顶峰。联合刺激时,1μg/mlPolyI:C明显增加了内皮细胞的IL-6表达,但50hM C3a或50nM C5a均未对PolyI:C的致炎作用产生明显影响。
分析CPG的浓度-效应时,先后分析了5个浓度:200ng/ml、2μg/ml、20μg/ml、100μg/ml和200μg/ml,均没有引起明显的IL-6表达增加;时间-效应分析发现,20μg/mlCPG刺激24小时后,未引起明显的IL-6表达增加。合并50nM C3a或C5a时,20μg/mlCPG对内皮细胞刺激24小时,未发现明显的IL-6和chemokine KC表达增加。
应用HUVEC替代H5V后,发现了Pam3CSK4和Poly I:C可以明显地诱导IL-8表达,但并未发现C3a和C5a可以上调TLRs激活物的致炎作用。
结论
1 Pam3CSK4、Poly I:C和LPS可以激活相应的TLRs,引起明显的炎性细胞因子释放。心脏内皮细胞也具有与人脐静脉内皮细胞相似的反应性,可以在Pam3CSK4、Poly I:C或LPS的刺激下,产生大量的炎性因子。
2无论是HUVEC还是H5V,过敏毒素-C3a或C5a的联合刺激均未显著地增加TLRs的致炎作用,提示内皮细胞并未参与补体上调TLRs致炎作用这一过程。
Background
The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury via three different pathways,the classical pathway,the lectin pathway and the alternative pathway. Activated complement system promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).To prevent complement-mediated autologous tissue injury,many kinds of host cells,particularly those in the vascular space such as blood cells and endothelial cells,express several membrane-bound complement regulatory proteins.DAF and CD59 are two of them that link to the cell membrane via a glycosylphosphatidylinositol(GPI) anchor.DAF regulates complement activation by promoting the decay of C3 and C5 convertases,whereas CD59 inhibits the formation of the MAC.
Recent studies have indicated that atherosclerosis is a chronic inflammatory condition in which many components of innate and adaptive immune systems are involved.As an important part of innate immunity,complement system has been implicated in the pathogenesis of atherosclerosis by several studies.For example,various complement activation products,regulatory proteins,and complement receptors have been detected in human atherosclerotic lesions,particularly in vulnerable and ruptured plaques,and deposition of C5b-9 has been shown to correlate with the disease state. Experimental studies with complement deficient animals have also been performed to elucidate the role of the complement system in atherogenesis.A cholesterol-rich diet was shown to induce less atherosclerosis in rabbits deficient in C6 than in controls with a fully functional complement system.Contrasting results were obtained, however,in C5 deficient ApoE~(-/-) mice,which showed a similar extent of atherosclerotic lesions compared to complement-competent control mice.In other studies,C3 deficiency was found to increase lipid-positive lesions in the mouse aorta, as well as altering the plasma lipid profile.On the other hand,deficiency of factor B affected neither lipid levels nor lesion size.More recently,Clq deficiency was found to cause larger atherosclerotic lesions in low-density lipoprotein receptor knockout mice,possibly reflecting a role of Clq in the disposal of dying cells.Together,these results suggested a variable and complex role of complement components in atherosclerosis.
In contrast to the above studies on individual complement components in atherogenesis,there have been few studies testing the role of complement regulatory proteins in the development of this disease.Previous studies have detected the presence of complement regulators in atherosclerotic lesions and recently,both statins and C-reactive protein,the latter a marker of chronic inflammation and strong predictor of vascular diseases,have been shown to induce the expression of endothelial complement regulators.However,it is still not clear whether complement membrane-bound regulatory proteins are involved in the atherosclerosis.In this study, our hypothesis is that complement membrane-bound regulatory proteins protect agaist atherosclerosis through inhibiting the activation of complement system or the production of complement effectors.
Aims
1.To produce CD55~(-/-) ApoE~(-/-) and CD59~(-/-) ApoE~(-/-) mice by crossing CD55~(-/-)and CD59 ~(-/-) mice with ApoE~(-/-) mice respectively.
2.To illustrate the anti-atherosclerotic role of membrane-bound regulatory proteins, by analyzing the lesion size and lesion contents of double knockout mice after a high-fat diet.
3.To reveal the mechanism of the anti-atherosclerotic role of membrane-bound regulatory proteins.
Methods
1.Mice and diets
CD55 knockout mice and CD59 knockout mice were crossed with ApoE~(-/-) mice respectively to produce CD55~(-/-) ApoE~(-/-) and CD59~(-/-) ApoE~(-/-) mice.CD55~(+/+) ApoE~(-/-) mice and CD59~(+/+) ApoE~(-/-) mice were used as controls.Starting from 6 weeks of age,they were fed a high fat diet.At 14 or 22 weeks of age,they were fasted overnight and killed by CO2 asphyxiation.Blood was collected for plasma lipid analysis by vena cava nicking and then stored in -80℃refrigerators.The aorta was perfused with cold PBS by inserting a cannula into the left ventricle.The hearts were dissected and embedded in the OCT.Perfused aortas were dissected from aortic arch to iliac bifurcation.All animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania.
2.Serum and urine sample analysis
Plasma samples were collected after 12 hours of fasting and stored at -80℃.Total cholesterol was measured.Twenty-four hour urine samples were collected in metabolic cages.The nonenzymatic lipid peroxidation product,8,12-iso-iPF2α-Ⅵ, was measured by HPLC/MS/MS.Metabolite levels were corrected for urinary creatinine.
3.Processing and analysis of the aorta
Aortas were pinned on wax plates and stained with SudanⅣ.The extent of atherosclerosis was expressed as the percentage of the aorta surface covered by positive staining.Serial 6-μm-thick cryostat sections were prepared from the origin of the aortic valve cusps and cross-sectional analysis for atherosclerotic lesion was performed every 60μm over 360μm by staining with oil-red O.The extent of atherosclerosis was expressed as the percentage of aortic root covered by lesions. Images were captured digitally with a video camera and analyzed by computerized image analysis.
4.Histological analysis of aortic lesions
The aortic root sections were stained for smooth muscle cells,macrophages,collagen, C3,C9,CD4 and CD8.The ratio of positive area or cells to aortic root area was recorded.
5.Statistical Analysis
Statistical analyses were performed with GraphPad software(Prism 3.0).Data were analyzed by two-tailed Student t-test(for data with normal distribution) or Mann-Whitney test(for data with non-parametric distribution).Results are considered to be significant at values of P＜0.05 and are presented as mean±SEM.
Results
1.CD59 protects against atherosclerosis
After 8 weeks of HFD,we found that female CD59~(-/-) ApoE~(-/-) mice developed significantly larger atherosclerotic lesions in their aortas,as shown by both en face (2.07±0.27%vs.1.34±0.21%,P=0.06) and aortic root section analysis(20.74±1.33% vs.13.12±1.46%,P＜0.005).Interestingly,the increased sensitivity to atherosclerosis development in CD59~(-/-) ApoE~(-/-) mice appeared to be gender-biased as we observed no significant difference between male CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice by either en face(1.35±0.16%vs.1.57±0.19%,P=0.393) or aortic root section analysis (11.23±1.23%vs.12.75±1.41%,P=0.536).
After 16 weeks of HFD,a more pronounced difference between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice was observed by en face staining (17.13±1.14%vs.9.72±1.14%,P＜0.001).However,a difference could not be detected between the two groups mice by aortic root section analysis at this advanced stage of lesion development(32.67±1.58%vs.34.44±2.52%,P=0.315).Similar to the finding at 8 weeks of HFD feeding,we detected no significant difference at 16 weeks between male CD59~(+/+) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice by either the en face (12.88±1.49%vs.10.98±0.95%,P=0.779) or aortic root section analysis (26.845±1.97%vs.29.46±2.3%,P=0.408).
2.CD55 has no influence on the development of atherosclerosis
In contrast to the effect of CD59 deficiency on atherosclerosis in female ApoE~(-/-) mice, we observed no significant difference in either gender between CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice,regardless of the length of HFD feeding.En face analysis showed that the average lesion areas in female CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice at 8 and 16 weeks of HFD feeding were 0.80±0.17%vs 1.37±0.22%(P=0.07), and 6.39±0.59%vs 5.81±0.51%,(P=0.303),and that for male mice were 1.14±0.17% vs 0.805±0.14%(P=0.207),and 6.09±0.83%vs 6.16±0.69%(P=0.693),respectively. Aortic root section analysis showed the average lesion areas in female CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice at 8 and 16 weeks of HFD to be 21.45±1.33%vs 23.58±2.10%(P=0.337),and 29.81±2.78%vs 29.11±3.25%(P=0.918),and that for male mice were 15.68±1.58%vs 16.89±1.65%(P=0.921),and 25.25±3.02%vs 30.94±3.22%(P=0.281),respectively.
3.The influence of CD59 knockout on the components of atherosclerotic lesions We next performed histological studies in female CD59-deficient and-sufficient mice to evaluate the complexity of the atherosclerotic lesions.Using CD68 as a marker,we measured the lesion areas that were positive for macrophage infiltration.No significant difference was observed between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice in macrophage distribution and content(18.74±2.79%vs. 18.66±2.41%,P=0.983).On the other hand,after 8 weeks of HFD feeding,we detected more collagen(8.14±0.33%vs.6.07±0.82%,P＜0.05) and less smooth muscle cell staining(3.69±0.51%vs.6.42±0.86%,P＜0.05) in the lesions of CD59-deficient mice compared with their littermate controls.However,the difference in smooth muscle cell content was not observed at 16 weeks of HFD feeding (2.25±0.24%vs.2.28±0.54%,P=0.97).
4.The anti-atheroslerotic mechanisms of CD59 Plasma lipid analysis showed a moderate increase in total plasma cholesterol levels in female CD59~(-/-) ApoE~(-/-) mice compared with their CD59~(+/+) ApoE~(-/-) littermates,but the difference reached statistical significance only at 8 weeks of HFD feeding (1404±43.74 vs.1208±57.78 mg/dL,P＜0.05 ).We observed no significant difference in 8,12-iso-iPF2α-Ⅵbetween female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice either at 8 weeks or 16 weeks of HFD feeding(8 weeks:2.98±1.01 vs.3.12±0.96 ng/mg creatinine,P=0.92;16 weeks:1.59±0.38 vs.1.65±0.56 ng/mg creatinine,P=0.92). Staining of aortic root sections with anti-C3 and anti-C9 antibodies showed a similar C3 deposition(28.65±1.69%vs.25.33±3.07%,P=0.371) in female CD59~(-/-) ApoE~(-/-) mice and controls,but significantly increased C9 deposition was found in the sections of female CD59~(-/-) ApoE~(-/-) mice than those of the controls(17.32±1.52%vs.8.74±3.63%,P＜0.05) after 8 weeks of HFD feeding.This result suggested that CD59 deficiency led to more deposition of the membrane attack complex within the atherosclerotic lesions.We also examined CD4+ and CDS+ T cell infiltration into the lesions but observed no significant difference between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice(for CD4+ T cells:35.89±8.505 vs.40.99±7.091/mm~2,P=0.66; for CDS+ T cells:1.622±1.622 vs.5.384±3.946/mm~2,P=0.40).
Conclusions
1.CD59 offers protection against atherosclerosis in the context of ApoE deficiency, most likely through lowering total cholesterol and inhibiting the formation of membrane attack complex.Its anti-atherosclerotic role is independent of oxidative stress and GPI-mediated T cell activation,and gender-biased,which maybe related to the different level of sex hormones.
2.CD59 is not involved in the adhesion,migration and proliferation of macrophages; CD59 protect smooth muscle cells against complement attack by inhibiting the formation of MAC
3.CD55 shows no obvious influence on atherosclerosis.This maybe because that other complement regulators such as factor H or Crry have compensated for the lack of CD55.Even under the deficiency of CD55,C3 convertase can be inhibited effectively.
4.There is a loss of correlation between en face and aortic root analysis.The aortic roots may be particularly sensitive to lesion development caused by ApoE-deficiency, and any additive effect brought upon by other genetic alterations could be difficult to be detected,especially after prolonged HFD feeding.Thus,en face analysis appears to be a more stable method than aortic root analysis.
Background
Innate immunity is the first line of host defense against pathogens.The Toll-like receptors(TLRs) and complement are 2 critical components of the innate immune system.They play an essential role in host defense by eliciting rapid inflammatory reactions and orchestrating adaptive immune responses to microbial infection. The mammalian TLRs,one kind of the pattern recognition receptors,are horologes of Toll proteins from drosophila.Through recognizing the invading microbes and dead cells,they can activate the innate immunity,and initiate the adaptive immunity by interacting with the antigen-presenting cells.They play critical roles in the regulation of phagocytosis,signal transduetion and cell apoptosis.The complement system consists of soluble proteins and membrane-bound proteins.The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury.Activated complement promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).
Many common PAMPs,such as lipopolysaccharide(LPS) from gram-negative bacteria and zymosan,an insoluble carbohydrate from the yeast cell wall,act both as TLR ligands and activators of complement.Whether and how the TLR and the complement systems,when coactivated in vivo,interact with each other and how potential cross talks between the 2 systems might impact the inflammatory and adaptive immune responses of the host has not been well studied.Recently,one of our studies showed that TLRs agonist induced significant inflammatory response in CD55 deficient mice,indicating that complement system upregulates the TLR-mediated inflammatory response.Subsequent analysis showed that this function was mediated by the anaphylatoxins.In vivo study showed that complement upregulated the cytokines production 3 hours after LPS challenge.This means that the function is most likely mediated by the organs or tissues which have direct contaction with circulation system.Endothelial system is the largest endocrinal organ and has direct contaction with blood.This led to our hypothesis that endothelial cells mediate the upregulation of TLRs-mediated inflammatory response by complement.
Aim
To elucidate whether and how endothelial cedis are involved in the upregulation of the TLR-mediated inflammatory response by complement.
Methods
1 Cells culture
HUVEC were cultured in Medium 199 containing 10%fetal bovine serum,0.05 mg/ml endothelial cell growth supplement,2mML-glutamine,0.1mg/ml heparin,and 100 U penieillinstreptomycin.H5V,an endothelial cell line derived from mouse heart, was cultured in DMEM supplemented by 10%fetal bovine serum.Endothelial cells were cultured at 37℃in a humidified atmosphere containing.5%CO2 and passaged every two or three days at a split ratio of 1:3.Passages 5-7 of HUVEC were used for experiments.
2 Cell toxicity testing
The cell toxicity of C3a,C5a,LPS,Poly I:C,Pam3CKS4 and CPG to endothelial cells was conducted with promega's one solution cell proliferation assay.This test was performed to find the dose range for the stimulation of endothelial cells.
3 C5aR expressions in endothelial cells
C5aR expression in HUVEC or H5V was measured by flow cytometry
4 Treatment of HUVEC
Endothelial cells were seed in 24-well plate at 1×105 per well.After 24 hours,the cells were 80%confluent and were stimulated by various concentration of TLRs agonists or for different time to find the way for the joint stimulation. For the joint stimulation,80%confluent endothelial cells were stimulated by anaphylatoxins and different TLRs agonists for 24 hours.
5 ELISA analyses
ELISA for IL-6,IL-8 and chemokine KC was conducted according to the instructions from companies.
Results
1 The following dose ranges were found with no obvious cell toxicity to endothelial cells:0-100nM for C3a,0-200nM for C5a,0-10000 ng/ml for Pam3CKS4,0-50μg/m for Poly I:C,0-1000 ng/ml for LPS and 0-160ng/ml for CPG.
2 A number of C5aR were found in HUVEC and H5V,but not in the negative controls.
3 10 ng/ml LPS significantly induced IL-6 expression.More IL-6 was produced along with the increase of LPS concentration until 500ng/ml.Time course analysis showed that significant IL-6 production was detected after 3 hours'LPS stimulation and peaked after 24 hours.According to the concentration and time course analysis,we chose 10 ng/ml and 100 ng/ml LPS for the joint stimulation.Either 50nM C3a or C5a didn't show any influence on the IL-6 or Chemokine KC production induced by LPS. Then this conclusion was confirmed by using different level of C5a.
4 In the same way,we analyzed the influence of C3a and C5a on other TLR-mediated inflammatory response.50nM C3a or C5a showed no influence on IL-6,IL-8 or chemokine KC production induced by Pam3CSK4,Poly I:C and CPG.
Conclusions
1 Pam3CSK4,Poly I:C and LPS but not CPG can activate toll like receptors on the membrane of endothelial cells.Similar to HUVEC,heart endothelial cells can produce a lot of cytokines under the stimulation of Pam3CSK4,Poly I:C and LPS.
2 Endothelial cells are not involved in the regulation of TLR-mediated inflammatory response by complement
背景
补体系统的可溶性成分多以前体的形式存在于外周血,当病原入侵或者组织损伤时,可通过经典途径、凝集素激活途径或替代途径被激活。激活的补体系统通过释放白细胞趋化因子C3a和C5a促进炎症反应,并可通过合成膜攻击复合体介导细胞损伤。为了防止自体细胞受到补体系统的攻击,生物体内的多种细胞,尤其是那些接触血液循环的血细胞和内皮细胞,表达了多种结合于细胞膜的补体调节蛋白,其中包括通过GPI结构与细胞膜锚链的CD55和CD59。CD55可以通过促进C3和C5转化酶的降解而调控补体激活,而CD59主要通过抑制膜攻击复合体的形成而发挥作用。
近年的研究表明,动脉粥样硬化(atherosclerosis,AS)是一种发生于动脉血管壁的慢性炎症,天然性与获得性免疫系统的多个成分参与了其发生发展过程。作为天然性免疫系统的一个重要组成部分,补体系统致AS的作用已为许多研究所揭示。各种补体系统的激活产物,调节蛋白和补体受体均可在AS病灶,尤其是易损斑块和已破裂斑块中被探及,并且对人体病变的研究发现,膜攻击复合体的沉积量与病灶进展程度呈现正相关。对补体敲基因动物的研究亦初步揭示了补体致AS的机理:补体激活的中心分子—C3的基因缺陷可以通过上调血甘油三酯的浓度促进AS病灶的发展;C6基因缺陷可以明显减少高胆固醇饮食所诱发的兔主AS,而C5基因敲除却并没有明显地影响ApoE~(-/-)小鼠的斑块大小,意味着补体终末效应物-膜攻击复合体在AS发展的过程中发挥着较为复杂的作用;在补体激活通路的研究方面,替代激活途径的关键因子-Factor B缺陷并没有对AS产生明显的影响,提示另外两条通路可能发挥着更为重要的作用;而经典激活途径的关键因子-Clq基因缺陷却通过影响坏死细胞的清除促进了病变的发展。总之,近来的研究表明,补体系统参与了AS的发病过程,但是其具体的作用机制十分复杂,仍然需要进一步的研究来加以阐明。
作为补体系统的重要组成部分,膜调节蛋白亦被发现存在于AS病灶中,并且研究发现,阿托伐他汀和C反应蛋白均可诱导内皮细胞表达补体膜调节蛋白,从而使之幸免于补体系统的裂解作用。补体膜调节蛋白是否参与了AS病灶的发生和发展过程,具体的作用机制是什么,目前均无研究加以阐明。由于该组蛋白具有明显的补体激活抑制作用,我们提出如下科学假说:补体膜调节蛋白通过抑制补体激活或补体效应物的产生而发挥了抗AS作用。
目的
1.通过分别把CD55~(-/-)和CD59~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得易形成AS斑块的双基因敲除小鼠;
2.通过对双敲小鼠的病变范围、斑块大小和病灶内容物进行分析,研究补体膜调节蛋白-CD55和CD59在AS发生发展中的作用;
3.通过对血脂、血尿、补体激活和T细胞免疫的分析,探讨CD55和CD59抗AS的机制。
方法
1.获取双基因敲除小鼠及高脂喂养
把CD55~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得CD55~(+/+)ApoE~(-/-)杂合子,以杂合子小鼠交配繁殖获得CD55~(-/-)ApoE~(-/-)小鼠和同龄野生型对照-CD55~(+/+)ApoE~(-/-)小鼠。将6周龄的小鼠根据高脂喂养时间和性别分为4组:短期雌性组包括雌性CD55~(-/-)ApoE~(-/-)小鼠和雌性CD55~(+/+)ApoE~(-/-)对照,高脂喂养8周;短期雄性组包括雄性CD55~(-/-)ApoE~(-/-)小鼠和雄性CD55~(+/+)ApoE~(-/-)对照,高脂喂养8周;长期雌性组包括雌性CD55~(-/-)ApoE~(-/-)小鼠和雌性CD55~(+/+)ApoE~(-/-)对照,高脂喂养16周;长期雄性组包括雄性CD55~(-/-)ApoE~(-/-)小鼠和雄性CD55~(+/+)ApoE~(-/-)对照,高脂喂养16周。以与CD55同样的方式,把CD59~(-/-)小鼠与ApoE~(-/-)小鼠杂交,获得获得CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠。将6周龄的CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠按照高脂喂养时间和性别分为4组。
各组高脂喂养结束后,小鼠空腹过夜后被处死。自下腔静脉收集血液标本,并冻存。PBS冲洗左侧心腔及动脉腔内的残血后,将心脏分离并包埋于OCT。钝性分离自主动脉弓到腹主动脉分叉处的主动脉节段,进行染色分析。所有的动物实验及相关处理均通过了美国宾夕法尼亚大学动物管理及应用委员会的批准。
2.血液及尿液分析
应用酶法测定血清总胆固醇水平。应用代谢笼连续24小时收集小鼠的尿液,并应用HPLC/MS/MS法分析尿液中脂质代谢产物8,12-iso-iPF2_α-V和肌酐的水平。
3.斑块大小分析
将主动脉纵剖后钉于蜡版,并进行苏丹Ⅳ染色分析。病变范围表示为AS斑块占主动脉表面积的百分比。将小鼠心脏进行6μm连续冰冻切片,收集自出现三个主动脉瓣叶起至瓣尖的切片。对冰冻切片进行油红0染色,照相并应用软件分析。斑块大小表示为斑块占主动脉根面积的百分比。
4.病理学检测
对主动脉窦部冰冻切片行免疫组化染色和天狼星红染色,测量斑块内平滑肌细胞、巨噬细胞、C3、C9和胶原的阳性染色面积,其含量表示为阳性染色面积占斑块总面积的百分比。对切片行CD4和CD8免疫组化染色,计数单位面积内的阳性细胞个数。
5.统计学分析
定量数据均以均数±标准误表示,应用Graphpad公司的Prism3.0进行统计分析。在做分析之前,对所有的数据进行正态分布检验。对于正态分布的数据应用双侧t检验,对非正态分布的数据应用Mann—Whiteney分析。设P＜0.05为有统计学意义。
结果
1.CD59的抗AS作用
短期雌性组内,虽然主动脉表面病灶范围并无组间差异(2.07±0.27%vs.1.34±0.21%,P=0.06),但是CD59~(-/-)ApoE~(-/-)小鼠主动脉窦部的斑块明显大于CD59~(+/+)ApoE~(-/-)对照(20.74±1.33%vs.13.12±1.46%;P＜0.005)。然而,短期雄性组内,雄性CD59~(-/-)ApoE~(-/-)小鼠与CD59~(+/+)+ApoE~(-/-)对照间未发现显著差异(病变范围:1.35±0.16%vs.1.57±0.19%,P=0.3936;斑块大小:11.23±1.23%vs.12.75±1.41%,P=0.536)。长期雌性组内,CD59基因缺陷明显地增加了雌性小鼠的病变覆盖范围(17.13±1.14%vs.9.72±1.14%;P＜0.001),但是却未对主动脉窦部的斑块大小产生明显的影响(32.67±1.58%vs.34.44±2.52%;P=0.315)。与短期雄性组的结果相似,在长期雄性组内未发现显著差异(病变范围:12.88±1.49%vs.10.98±0.95%,P=0.779;斑块大小:26.84±1.97%vs.29.46±2.3%,P=0.408)。CD59基因缺陷导致了病变覆盖范围的扩大或局部斑块的增大,提示CD59具有明显的抗AS作用。然而,无论是在短期还是长期雄性组内,均未发现显著的差异,提示CD59的抗AS作用存在明显的性别差异。
2 CD55缺陷与AS
与CD59基因缺陷的结果不同,无论在雄性还是雌性组,CD55基因敲除均未引起明显的病变范围或斑块大小的改变。在短期雌性组,雌性CD55~(-/-)ApoE~(-/-)和CD55~(+/+)ApoE~(-/-)小鼠的病变范围(0.80±0.17%vs 1.37±0.22%;P=0.07)和斑块大小(21.45±1.33%vs 23.58±2.1096;P=0.337)无显著差异:同样的结果也见于短期雄性组(病变范围:1.14±0.17%vs 0.80±0.14%,P=0.207:斑块大小:15.68±1.58%vs 16.89±1.65%,P=0.921)。在长期雌性组,CD55~(-/-)ApoE~(-/-)与CD55~(+/+)ApoE~(-/-)小鼠的病变范围(6.39±0.59%vs 5.81±0.51%:P=0.303)和斑块大小(29.81±2.78%vs 29.11±3.25%;P=0.918)无显著差异;长期雄性组内,也未发现明显的差异(病变范围:6.09±0.83%vs 6.16±0.69%,P=0.95;斑块大小:25.25±3.02%vs 30.94±3.22%;P=0.281)。
3 CD59缺陷对斑块构成的影响
通过对短期雌性组的病理学分析,我们研究了CD59基因缺陷对斑块内各种成份的影响。CD68染色显示,CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠巨噬细胞的分布和相对含量较为一致(18.74±2.79%vs.18.66±2.41%;P=0.983),提示CD59并没有参与巨噬细胞的粘附、浸润或增殖过程,仅仅通过其他途径促进了斑块的发展,而巨噬细胞呈现等比例的增加;α-SMC actin染色显示,CD59~(-/-)ApoE~(-/-)小鼠的平滑肌细胞显著少于对照组(3.69±0.51%vs.6.42±0.86%;P＜0.05);苦味酸-天狼星红染色发现,CD59基因缺陷小鼠的胶原含量较对照小鼠明显增加(8.14±0.33%vs.6.07±0.82%:P＜0.05)。
2.4 CD59抗AS作用的机制
血清学分析显示,短期雌性组内,CD59~(-/-)ApoE~(-/-)小鼠的血清总胆固醇显著高于CD59~(+/+)ApoE~(-/-)小鼠(1404±43.74vs.1208±57.78mg/dL;P＜0.05),提示CD59可能通过降低血清总胆固醇发挥抗AS作用。通过对尿液中氧化应激标志物的质谱分析,发现在雌性CD59~(-/-)ApoE~(-/-)小鼠和CD59~(+/+)ApoE~(-/-)对照间无显著差异(短期雌性组:2.98±1.01 vs.3.12±0.96 ng/mg肌酐,P=0.92;长期雌性组:1.65±0.56 vs.1.59±0.38 ng/mg肌酐,P=0.92),因而CD59基因缺陷并未对该系统产生显著的影响。短期雌性组的C3免疫组化染色发现,CD59~(-/-)ApoE~(-/-)小鼠与对照小鼠间斑块内的C3沉积无显著差异(28.65±1.69%vs.25.33±3.07%,P=0.37),提示CD59基因缺陷并没有影响局部的补体激活;而C9染色发现,CD59~(-/-)ApoE~(-/-)小鼠斑块内的C9沉积明显多于对照组(17.32±1.52%vs.8.74±3.63%,p＜0.05),提示CD59基因缺陷时,局部的补体膜攻击复合体形成明显增加。为了明确CD59是否通过影响T细胞而发挥抗AS作用,对短期雌性组的小鼠切片做了CD4和CD8染色分析。结果发现,CD59~(-/-)ApoE~(-/-)和CD59~(+/+)ApoE~(-/-)小鼠斑块内的T细胞含量无差异(CD4~+T细胞:35.89±8.505 vs.40.99±7.091/mm~2,P=0.66:CD8~+T细胞:1.622±1.622 vs.5.384±3.946/mm~2,P=0.40)。
结论
1 CD59可能通过降低血清总胆固醇和减少补体效应产物-膜攻击复合体的生成而发挥抗AS的作用。该作用与氧化应激和GPI介导的T细胞激活无明显的相关性。CD59的抗AS作用仅见于雌性,具有明显的性别差异。
2 CD59并没有参与斑块内巨噬细胞的粘附、迁移和增殖过程,该因子缺陷时通过影响其他方面导致了等幅度的巨噬细胞浸润;CD59通过抑制膜攻击复合体的形成保护血管壁平滑肌细胞免受补体系统的攻击。
3 CD55基因缺陷并没有引起明显的斑块大小改变,提示在动脉壁保护的过程中,其他的C3转化酶抑制剂如Factor H和Crry可能发挥着更重要的作用。在CD55基因缺陷的情况下,C3转化酶的功能仍然得到了有效的抑制,补体系统并未被有效地激活。
4主动脉表面的病变范围和窦部的斑块大小存在失偶联的现象,很可能是由于根部的空间有限,在ApoE缺陷较强的致AS背景下,其他基因很难引起明显的斑块大小改变。主动脉表面具有广阔的空间,可以作为更恒定的评价指标。
中文摘要Ⅱ补体过敏毒素调节Toll样受体致炎作用的体外研究
背景
天然免疫系统是机体抵御病原微生物感染的第一道防线。Toll样受体(tolllike receptors,TLRs)和补体是天然免疫系统的两个重要组成部分,通过迅速诱发炎症反应和激活继发的获得性免疫而在应对致病原的过程中发挥着重要的作用。
哺乳动物的TLRs,是果蝇Toll样蛋白的同源体,属于病原模式识别受体家族,可以通过识别病原或坏死细胞的相关分子活化天然免疫系统,同时也可以通过向抗原递呈细胞提供信号而启动获得性免疫系统,在细胞吞噬的调节、炎症反应的激活等方面发挥着重要的作用。补体是由30余种可溶性蛋白和膜结合性蛋白组成的多分子系统。其可溶性成分多以无活性的前体形式存在于血液循环中,需要时,在激活物如抗原-抗体复合物等的作用下,被依次激活,形成膜攻击复合体,发挥溶解、破坏细菌、病毒等致病微生物的作用。除膜攻击复合体外,补体系统激活时尚可以通过产生过敏毒素-C3a和C5a,参与机体的抗感染免疫,扩大体液免疫效应,调节免疫应答。
研究发现,许多TLRs的天然激活物如革兰阴性菌来源的脂多糖和来源于酵母细胞壁的酵母多糖也可以激活补体系统,提示在这两系统间可能存在一定的相互联系。探讨补体和TLRs之间是否存在相互作用及其具体的作用机制,对于深入理解炎症和机体的免疫反应具有极其重要的意义。日前,我们的一项研究发现,LPS等TLRs激活剂在CD55~(-/-)小鼠诱发的炎症反应明显强于对照小鼠,提示补体上调了TLRs的致炎作用。后继的机制研究发现,该作用是由补体过敏毒素-C3a和C5a所介导的。体内实验中,补体与TLRs的协同作用发生迅速,TLRs激活剂注入体内后3小时即达到顶峰,提示该作用很可能是由与循环系统密切接触的器官或者组织所介导的。内皮细胞构成了机体内最大的“内分泌器官”,并且与循环系统直接接触,故而我们提出如下科学假说:内皮细胞介导了补体对TLRs致炎作用的调节。在本研究中,我们应用人脐静脉内皮细胞(HUVEC)和小鼠心脏内皮细胞(H5V)对内皮系统在这一过程中的作用进行了探讨。
目的
研究内皮细胞是否介导了补体C3a和C5a对TLRs致炎作用的调控,并探讨其具体的作用机制。
方法
1细胞及其培养
人脐静脉内皮细胞,培养于含10%胎牛血清、0.05 mg/ml内皮细胞生长因子、2mML—谷氨酰胺、0.1mg/ml肝素和100 U混合抗生素的以M199为主体的培养基中。H5V为来源于小鼠心脏的内皮细胞系,培养于含10%胎牛血清的DMEM培养基内。内皮细胞贴壁生长于以0.1%明胶预先包被的培养皿中,37℃、5%CO_2的条件下,2-3天传代一次。其中第5-7代的HUVEC用于实验。
2细胞毒性实验
以单剂细胞增殖分析法研究C3a、C5a、Pam3CKS4、Poly I:C、LPS和CPG对内皮细胞的毒性作用,以选出可用的浓度范围。
3内皮细胞C5a受体的测定
以流式细胞仪分析HUVEC和H5V表面的C5a受体的表达情况,以确认内皮细胞可表达C5aR。
4干预实验
以1×10~5个/孔的浓度将细胞接种于24孔板,培养24小时后细胞大约融合至80%。在分析浓度-效应时,将各TLRs激活剂以培养基梯度稀释成5个浓度,刺激24小时;在分析时间-效应时,以选定的浓度稀释TLRs激活剂,分别刺激内皮细胞3、6、9、12、15和24个小时。刺激实验结束后,收集培养液用于IL-6或IL-8的ELISA分析,以选出合适的作用浓度和时间。过敏毒素和TLRs激活剂联合作用时,用不同浓度的C3a和C5a合并选定浓度的TLRs激活剂刺激80%融合的单层内皮细胞24小时。取培养液用于IL-6、IL-8或chemokines KC的ELISA分析,研究过敏毒素是否上调了TLRs的致炎作用。
5酶联免疫吸附实验(enzyme linked immunoad sorbent assay,ELISA)按照生产厂家提供的实验步骤,对稀释过的培养液进行IL-6、IL-8或chemokine KC的ELISA分析。
结果
1细胞毒性实验
应用单剂细胞增殖分析试剂盒,检测了补体过敏毒素和TLRs对H5V的细胞毒性作用。结果显示,C3a在0-100nM,C5a在0-200nM,Pam3CKS4在0-10000 ng/ml,PolyI:C在0-50μg/ml,LPS在0-1000 ng/ml,CPG在0-160ng/ml为安全的浓度区域,并未引起明显的细胞坏死。
2 C5a受体表达
应用流式分析,发现在HUVEC和H5V表面有相当数量的C5aR表达,而同时染色的阴性对照-HEK细胞上几乎没有荧光信号。
3 C5a和C3a对TLR4致炎作用的影响
LPS浓度-效应的分析发现,10ng/ml LPS即可以明显地增加H5V细胞的IL-6表达,随后IL-6的生成随着LPS浓度的增高而增加,当LPS浓度为500ng/ml时达到顶峰;时间-效应的分析发现,100ng/ml LPS作用3小时即可以诱导H5V细胞IL-6的释放增加,其效应随着作用时间的延长而增加,在刺激24小时时达到顶峰。
根据上述实验结果,我们选用10 ng/ml和100 ng/ml作为LPS的作用浓度,与C3a和C5a联合作用于内皮细胞。刺激24小时后,100 ng/ml的LPS明显地增加了IL-6的生成,其作用约为10 ng/ml组的2.7倍。但无论是合并10 ng/ml还是100 ng/ml的LPS,50nM C3a或C5a均未显著地增加LPS的诱导IL-6或chemokine KC的生成。应用不同浓度的C5a与100 ng/ml LPS同时刺激H5V,结果在其他浓度时,并未发现明显的上调作用。应用HUVEC重复了上述实验,也只获得了类似的结果。
4 C3a和C5a对其他补体激活物致炎作用的影响
100ng/ml Pam3CSK 4即可引起明显的IL-6表达增加,其刺激作用在1000ng/ml时达到最高;IL-6的生成亦随着Pam3CSK 4作用时间的延长而增加,刺激24小时达到顶峰。联合刺激时,100ng/ml Pam3CSK 4明显增加了内皮细胞的IL-6生成,但50hM C3a或50nM C5a均未对Pam3CSK4的致炎作用产生明显影响。
1μg/ml PolyI:C即可引起明显IL-6表达增加,其刺激作用在50μg/ml时达到最高:IL-6的生成随着作用时间的延长而增加,刺激24小时达到顶峰。联合刺激时,1μg/mlPolyI:C明显增加了内皮细胞的IL-6表达,但50hM C3a或50nM C5a均未对PolyI:C的致炎作用产生明显影响。
分析CPG的浓度-效应时,先后分析了5个浓度:200ng/ml、2μg/ml、20μg/ml、100μg/ml和200μg/ml,均没有引起明显的IL-6表达增加;时间-效应分析发现,20μg/mlCPG刺激24小时后,未引起明显的IL-6表达增加。合并50nM C3a或C5a时,20μg/mlCPG对内皮细胞刺激24小时,未发现明显的IL-6和chemokine KC表达增加。
应用HUVEC替代H5V后,发现了Pam3CSK4和Poly I:C可以明显地诱导IL-8表达,但并未发现C3a和C5a可以上调TLRs激活物的致炎作用。
结论
1 Pam3CSK4、Poly I:C和LPS可以激活相应的TLRs,引起明显的炎性细胞因子释放。心脏内皮细胞也具有与人脐静脉内皮细胞相似的反应性,可以在Pam3CSK4、Poly I:C或LPS的刺激下,产生大量的炎性因子。
2无论是HUVEC还是H5V,过敏毒素-C3a或C5a的联合刺激均未显著地增加TLRs的致炎作用,提示内皮细胞并未参与补体上调TLRs致炎作用这一过程。
Background
The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury via three different pathways,the classical pathway,the lectin pathway and the alternative pathway. Activated complement system promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).To prevent complement-mediated autologous tissue injury,many kinds of host cells,particularly those in the vascular space such as blood cells and endothelial cells,express several membrane-bound complement regulatory proteins.DAF and CD59 are two of them that link to the cell membrane via a glycosylphosphatidylinositol(GPI) anchor.DAF regulates complement activation by promoting the decay of C3 and C5 convertases,whereas CD59 inhibits the formation of the MAC.
Recent studies have indicated that atherosclerosis is a chronic inflammatory condition in which many components of innate and adaptive immune systems are involved.As an important part of innate immunity,complement system has been implicated in the pathogenesis of atherosclerosis by several studies.For example,various complement activation products,regulatory proteins,and complement receptors have been detected in human atherosclerotic lesions,particularly in vulnerable and ruptured plaques,and deposition of C5b-9 has been shown to correlate with the disease state. Experimental studies with complement deficient animals have also been performed to elucidate the role of the complement system in atherogenesis.A cholesterol-rich diet was shown to induce less atherosclerosis in rabbits deficient in C6 than in controls with a fully functional complement system.Contrasting results were obtained, however,in C5 deficient ApoE~(-/-) mice,which showed a similar extent of atherosclerotic lesions compared to complement-competent control mice.In other studies,C3 deficiency was found to increase lipid-positive lesions in the mouse aorta, as well as altering the plasma lipid profile.On the other hand,deficiency of factor B affected neither lipid levels nor lesion size.More recently,Clq deficiency was found to cause larger atherosclerotic lesions in low-density lipoprotein receptor knockout mice,possibly reflecting a role of Clq in the disposal of dying cells.Together,these results suggested a variable and complex role of complement components in atherosclerosis.
In contrast to the above studies on individual complement components in atherogenesis,there have been few studies testing the role of complement regulatory proteins in the development of this disease.Previous studies have detected the presence of complement regulators in atherosclerotic lesions and recently,both statins and C-reactive protein,the latter a marker of chronic inflammation and strong predictor of vascular diseases,have been shown to induce the expression of endothelial complement regulators.However,it is still not clear whether complement membrane-bound regulatory proteins are involved in the atherosclerosis.In this study, our hypothesis is that complement membrane-bound regulatory proteins protect agaist atherosclerosis through inhibiting the activation of complement system or the production of complement effectors.
Aims
1.To produce CD55~(-/-) ApoE~(-/-) and CD59~(-/-) ApoE~(-/-) mice by crossing CD55~(-/-)and CD59 ~(-/-) mice with ApoE~(-/-) mice respectively.
2.To illustrate the anti-atherosclerotic role of membrane-bound regulatory proteins, by analyzing the lesion size and lesion contents of double knockout mice after a high-fat diet.
3.To reveal the mechanism of the anti-atherosclerotic role of membrane-bound regulatory proteins.
Methods
1.Mice and diets
CD55 knockout mice and CD59 knockout mice were crossed with ApoE~(-/-) mice respectively to produce CD55~(-/-) ApoE~(-/-) and CD59~(-/-) ApoE~(-/-) mice.CD55~(+/+) ApoE~(-/-) mice and CD59~(+/+) ApoE~(-/-) mice were used as controls.Starting from 6 weeks of age,they were fed a high fat diet.At 14 or 22 weeks of age,they were fasted overnight and killed by CO2 asphyxiation.Blood was collected for plasma lipid analysis by vena cava nicking and then stored in -80℃refrigerators.The aorta was perfused with cold PBS by inserting a cannula into the left ventricle.The hearts were dissected and embedded in the OCT.Perfused aortas were dissected from aortic arch to iliac bifurcation.All animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania.
2.Serum and urine sample analysis
Plasma samples were collected after 12 hours of fasting and stored at -80℃.Total cholesterol was measured.Twenty-four hour urine samples were collected in metabolic cages.The nonenzymatic lipid peroxidation product,8,12-iso-iPF2α-Ⅵ, was measured by HPLC/MS/MS.Metabolite levels were corrected for urinary creatinine.
3.Processing and analysis of the aorta
Aortas were pinned on wax plates and stained with SudanⅣ.The extent of atherosclerosis was expressed as the percentage of the aorta surface covered by positive staining.Serial 6-μm-thick cryostat sections were prepared from the origin of the aortic valve cusps and cross-sectional analysis for atherosclerotic lesion was performed every 60μm over 360μm by staining with oil-red O.The extent of atherosclerosis was expressed as the percentage of aortic root covered by lesions. Images were captured digitally with a video camera and analyzed by computerized image analysis.
4.Histological analysis of aortic lesions
The aortic root sections were stained for smooth muscle cells,macrophages,collagen, C3,C9,CD4 and CD8.The ratio of positive area or cells to aortic root area was recorded.
5.Statistical Analysis
Statistical analyses were performed with GraphPad software(Prism 3.0).Data were analyzed by two-tailed Student t-test(for data with normal distribution) or Mann-Whitney test(for data with non-parametric distribution).Results are considered to be significant at values of P＜0.05 and are presented as mean±SEM.
Results
1.CD59 protects against atherosclerosis
After 8 weeks of HFD,we found that female CD59~(-/-) ApoE~(-/-) mice developed significantly larger atherosclerotic lesions in their aortas,as shown by both en face (2.07±0.27%vs.1.34±0.21%,P=0.06) and aortic root section analysis(20.74±1.33% vs.13.12±1.46%,P＜0.005).Interestingly,the increased sensitivity to atherosclerosis development in CD59~(-/-) ApoE~(-/-) mice appeared to be gender-biased as we observed no significant difference between male CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice by either en face(1.35±0.16%vs.1.57±0.19%,P=0.393) or aortic root section analysis (11.23±1.23%vs.12.75±1.41%,P=0.536).
After 16 weeks of HFD,a more pronounced difference between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice was observed by en face staining (17.13±1.14%vs.9.72±1.14%,P＜0.001).However,a difference could not be detected between the two groups mice by aortic root section analysis at this advanced stage of lesion development(32.67±1.58%vs.34.44±2.52%,P=0.315).Similar to the finding at 8 weeks of HFD feeding,we detected no significant difference at 16 weeks between male CD59~(+/+) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice by either the en face (12.88±1.49%vs.10.98±0.95%,P=0.779) or aortic root section analysis (26.845±1.97%vs.29.46±2.3%,P=0.408).
2.CD55 has no influence on the development of atherosclerosis
In contrast to the effect of CD59 deficiency on atherosclerosis in female ApoE~(-/-) mice, we observed no significant difference in either gender between CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice,regardless of the length of HFD feeding.En face analysis showed that the average lesion areas in female CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice at 8 and 16 weeks of HFD feeding were 0.80±0.17%vs 1.37±0.22%(P=0.07), and 6.39±0.59%vs 5.81±0.51%,(P=0.303),and that for male mice were 1.14±0.17% vs 0.805±0.14%(P=0.207),and 6.09±0.83%vs 6.16±0.69%(P=0.693),respectively. Aortic root section analysis showed the average lesion areas in female CD55~(-/-) ApoE~(-/-) and CD55~(+/+) ApoE~(-/-) mice at 8 and 16 weeks of HFD to be 21.45±1.33%vs 23.58±2.10%(P=0.337),and 29.81±2.78%vs 29.11±3.25%(P=0.918),and that for male mice were 15.68±1.58%vs 16.89±1.65%(P=0.921),and 25.25±3.02%vs 30.94±3.22%(P=0.281),respectively.
3.The influence of CD59 knockout on the components of atherosclerotic lesions We next performed histological studies in female CD59-deficient and-sufficient mice to evaluate the complexity of the atherosclerotic lesions.Using CD68 as a marker,we measured the lesion areas that were positive for macrophage infiltration.No significant difference was observed between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice in macrophage distribution and content(18.74±2.79%vs. 18.66±2.41%,P=0.983).On the other hand,after 8 weeks of HFD feeding,we detected more collagen(8.14±0.33%vs.6.07±0.82%,P＜0.05) and less smooth muscle cell staining(3.69±0.51%vs.6.42±0.86%,P＜0.05) in the lesions of CD59-deficient mice compared with their littermate controls.However,the difference in smooth muscle cell content was not observed at 16 weeks of HFD feeding (2.25±0.24%vs.2.28±0.54%,P=0.97).
4.The anti-atheroslerotic mechanisms of CD59 Plasma lipid analysis showed a moderate increase in total plasma cholesterol levels in female CD59~(-/-) ApoE~(-/-) mice compared with their CD59~(+/+) ApoE~(-/-) littermates,but the difference reached statistical significance only at 8 weeks of HFD feeding (1404±43.74 vs.1208±57.78 mg/dL,P＜0.05 ).We observed no significant difference in 8,12-iso-iPF2α-Ⅵbetween female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice either at 8 weeks or 16 weeks of HFD feeding(8 weeks:2.98±1.01 vs.3.12±0.96 ng/mg creatinine,P=0.92;16 weeks:1.59±0.38 vs.1.65±0.56 ng/mg creatinine,P=0.92). Staining of aortic root sections with anti-C3 and anti-C9 antibodies showed a similar C3 deposition(28.65±1.69%vs.25.33±3.07%,P=0.371) in female CD59~(-/-) ApoE~(-/-) mice and controls,but significantly increased C9 deposition was found in the sections of female CD59~(-/-) ApoE~(-/-) mice than those of the controls(17.32±1.52%vs.8.74±3.63%,P＜0.05) after 8 weeks of HFD feeding.This result suggested that CD59 deficiency led to more deposition of the membrane attack complex within the atherosclerotic lesions.We also examined CD4+ and CDS+ T cell infiltration into the lesions but observed no significant difference between female CD59~(-/-) ApoE~(-/-) and CD59~(+/+) ApoE~(-/-) mice(for CD4+ T cells:35.89±8.505 vs.40.99±7.091/mm~2,P=0.66; for CDS+ T cells:1.622±1.622 vs.5.384±3.946/mm~2,P=0.40).
Conclusions
1.CD59 offers protection against atherosclerosis in the context of ApoE deficiency, most likely through lowering total cholesterol and inhibiting the formation of membrane attack complex.Its anti-atherosclerotic role is independent of oxidative stress and GPI-mediated T cell activation,and gender-biased,which maybe related to the different level of sex hormones.
2.CD59 is not involved in the adhesion,migration and proliferation of macrophages; CD59 protect smooth muscle cells against complement attack by inhibiting the formation of MAC
3.CD55 shows no obvious influence on atherosclerosis.This maybe because that other complement regulators such as factor H or Crry have compensated for the lack of CD55.Even under the deficiency of CD55,C3 convertase can be inhibited effectively.
4.There is a loss of correlation between en face and aortic root analysis.The aortic roots may be particularly sensitive to lesion development caused by ApoE-deficiency, and any additive effect brought upon by other genetic alterations could be difficult to be detected,especially after prolonged HFD feeding.Thus,en face analysis appears to be a more stable method than aortic root analysis.
Background
Innate immunity is the first line of host defense against pathogens.The Toll-like receptors(TLRs) and complement are 2 critical components of the innate immune system.They play an essential role in host defense by eliciting rapid inflammatory reactions and orchestrating adaptive immune responses to microbial infection. The mammalian TLRs,one kind of the pattern recognition receptors,are horologes of Toll proteins from drosophila.Through recognizing the invading microbes and dead cells,they can activate the innate immunity,and initiate the adaptive immunity by interacting with the antigen-presenting cells.They play critical roles in the regulation of phagocytosis,signal transduetion and cell apoptosis.The complement system consists of soluble proteins and membrane-bound proteins.The soluble components of complement are present in the blood in precursor forms and can be activated during pathogen invasion or tissue injury.Activated complement promotes inflammation through the generation of leukocyte chemoattractants C3a and C5a,and causes cellular injury by the formation of the membrane attack complex(MAC).
Many common PAMPs,such as lipopolysaccharide(LPS) from gram-negative bacteria and zymosan,an insoluble carbohydrate from the yeast cell wall,act both as TLR ligands and activators of complement.Whether and how the TLR and the complement systems,when coactivated in vivo,interact with each other and how potential cross talks between the 2 systems might impact the inflammatory and adaptive immune responses of the host has not been well studied.Recently,one of our studies showed that TLRs agonist induced significant inflammatory response in CD55 deficient mice,indicating that complement system upregulates the TLR-mediated inflammatory response.Subsequent analysis showed that this function was mediated by the anaphylatoxins.In vivo study showed that complement upregulated the cytokines production 3 hours after LPS challenge.This means that the function is most likely mediated by the organs or tissues which have direct contaction with circulation system.Endothelial system is the largest endocrinal organ and has direct contaction with blood.This led to our hypothesis that endothelial cells mediate the upregulation of TLRs-mediated inflammatory response by complement.
Aim
To elucidate whether and how endothelial cedis are involved in the upregulation of the TLR-mediated inflammatory response by complement.
Methods
1 Cells culture
HUVEC were cultured in Medium 199 containing 10%fetal bovine serum,0.05 mg/ml endothelial cell growth supplement,2mML-glutamine,0.1mg/ml heparin,and 100 U penieillinstreptomycin.H5V,an endothelial cell line derived from mouse heart, was cultured in DMEM supplemented by 10%fetal bovine serum.Endothelial cells were cultured at 37℃in a humidified atmosphere containing.5%CO2 and passaged every two or three days at a split ratio of 1:3.Passages 5-7 of HUVEC were used for experiments.
2 Cell toxicity testing
The cell toxicity of C3a,C5a,LPS,Poly I:C,Pam3CKS4 and CPG to endothelial cells was conducted with promega's one solution cell proliferation assay.This test was performed to find the dose range for the stimulation of endothelial cells.
3 C5aR expressions in endothelial cells
C5aR expression in HUVEC or H5V was measured by flow cytometry
4 Treatment of HUVEC
Endothelial cells were seed in 24-well plate at 1×105 per well.After 24 hours,the cells were 80%confluent and were stimulated by various concentration of TLRs agonists or for different time to find the way for the joint stimulation. For the joint stimulation,80%confluent endothelial cells were stimulated by anaphylatoxins and different TLRs agonists for 24 hours.
5 ELISA analyses
ELISA for IL-6,IL-8 and chemokine KC was conducted according to the instructions from companies.
Results
1 The following dose ranges were found with no obvious cell toxicity to endothelial cells:0-100nM for C3a,0-200nM for C5a,0-10000 ng/ml for Pam3CKS4,0-50μg/m for Poly I:C,0-1000 ng/ml for LPS and 0-160ng/ml for CPG.
2 A number of C5aR were found in HUVEC and H5V,but not in the negative controls.
3 10 ng/ml LPS significantly induced IL-6 expression.More IL-6 was produced along with the increase of LPS concentration until 500ng/ml.Time course analysis showed that significant IL-6 production was detected after 3 hours'LPS stimulation and peaked after 24 hours.According to the concentration and time course analysis,we chose 10 ng/ml and 100 ng/ml LPS for the joint stimulation.Either 50nM C3a or C5a didn't show any influence on the IL-6 or Chemokine KC production induced by LPS. Then this conclusion was confirmed by using different level of C5a.
4 In the same way,we analyzed the influence of C3a and C5a on other TLR-mediated inflammatory response.50nM C3a or C5a showed no influence on IL-6,IL-8 or chemokine KC production induced by Pam3CSK4,Poly I:C and CPG.
Conclusions
1 Pam3CSK4,Poly I:C and LPS but not CPG can activate toll like receptors on the membrane of endothelial cells.Similar to HUVEC,heart endothelial cells can produce a lot of cytokines under the stimulation of Pam3CSK4,Poly I:C and LPS.
2 Endothelial cells are not involved in the regulation of TLR-mediated inflammatory response by complement
引文
1.Braunwald E.Shattuck lecture—cardiovascular medicine at the turn of the millennium:triumphs,concerns,and opportunities.N Engl J Med.1997;337:1360-9.
2.Hansson GK.Inflammation,atherosclerosis,and coronary artery disease.N Engl J Med.2005;352:1685-95.
3.Libby P.Inflammation in atherosclerosis.Nature.2002;420:868-74.
4.Curtiss LK.Reversing atherosclerosis? NEngl J Med.2009;360:1144-6.
5.Ait-Oufella H,Salomon BL,Potteaux S,Robertson AK,Gourdy P,Zoll J,Merval R,Esposito B,Cohen JL,Fisson S,Flavell RA,Hansson GK,Klatzmann D,Tedgui A,Mallat Z.Natural regulatory T cells control the development of atherosclerosis in mice.Nat Med.2006;12:178-80.
6.Bjorkbacka H,Kunjathoor VV,Moore K J,Koehn S,Ordija CM,Lee MA,Means T,Halmen K,Luster AD,Golenbock DT,Freeman MW.Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways.Nat Med.2004;10:416-21.
7.Yasojima K,Schwab C,McGeer EG,McGeer PL.Complement components,but not complement inhibitors,are upregulated in atherosclerotic plaques.Arterioscler Thromb Vasc Biol.2001;21:1214-9.
8.Oksjoki R,Kovanen PT,Pentikainen MO.Role of complement activation in atherosclerosis.Curr Opin Lipidol.2003;14:477-82.
9.Acosta J,Qin X,Halperin J.Complement and complement regulatory proteins as potential molecular targets for vascular diseases.Curr Pharm Des.2004;10:203-11.
10.McFarland HF,Martin R.Multiple sclerosis:a complicated picture of autoimmunity.Nat Immunol.2007;8:913-9.
11.Seifert PS,Hansson GK.Complement receptors and regulatory proteins in human atherosclerotic lesions.Arteriosclerosis.1989;9:802-11.
12.Bhakdi S,Torzewski M,Klouche M,Hemmes M.Complement and atherogenesis:binding of CRP to degraded,nonoxidized LDL enhances complement activation.Arterioscler Thromb Vasc Biol.1999;19:2348-54.
13.Li SH,Szrnitko PE,Weisel RD,Wang CH,Fedak PW,Li RK,Mickle DA,Verma S.C-reactive protein upregulates complement-inhibitory factors in endothelial cells.Circulation.2004;109:833-6.
14.Mason JC,Ahmed Z,Mankoff R,Lidington EA,Ahmad S,Bhatia V,Kinderlerer A,Randi AM,Haskard DO.Statin-induced expression of decay-accelerating factor protects vascular endothelium against complement-mediated injury.Circ Res.2002;91:696-703.
15.Kinderlerer AR,Ali F,Johns M,Lidington EA,Leung V,Boyle J J,Hamdulay SS,Evans PC,Haskard DO,Mason JC.KLF2-dependent,shear stress-induced expression of CD59:a novel cytoprotective mechanism against complement-mediated injury in the vasculature.J Biol Chem.2008;283:14636-44.
16.Sun X,Funk CD,Deng C;Sahu A,Lambris JD,Song WC.Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.Proc Natl Acad Sci U S A.1999;96:628-33.
17.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
18.Song WL,Lawson JA,Wang M,Zou H,FitzGerald GA.Noninvasive assessment of the role of cyclooxygenases in cardiovascular health:a detailed HPLC/MS/MS method.Methods Enzymol.2007;433:51-72.
19.Custodis F,Baumhakel M,Schlimmer N,List F,Gensch C,Bohm M,Laufs U.Heart rate reduction by ivabradine reduces oxidative stress,improves endothelial function,and prevents atherosclerosis in apolipoprotein E-deficient mice.Circulation.2008;117:2377-87.
20.Mehta JL,Sanada N,Hu CP,Chen J,Dandapat A,Sugawara F,Satoh H,Inoue K,Kawase Y,Jishage K,Suzuki H,Takeya M,Sehnaekenberg L,Beger R,Hermonat PL,Thomas M,Sawamura T.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res.2007;100:1634-42.
21.Wick G,Knoflach M,Xu Q.Autoimmune and inflammatory mechanisms in atherosclerosis.Annu Rev Immunol.2004;22:361-403.
22.Shah PK.Plaque disruption and thrombosis.Potential role of inflammation and infection.Cardiol Clin.1999;17:271-81.
23.Ross R.Atherosclerosis—an inflammatory disease.N Engl J Med.1999;340:115-26.
24.Nilsson J,Hansson GK.Autoimmunity in atherosclerosis:a protective response losing control? J Intern Med.2008;263:464-78.
25.Ridker PM,Silvertown JD.Inflammation,C-reactive protein,and atherothrombosis.J Periodontol.2008;79:1544-51.
26.Robertson AK,Hansson GK.T cells in atherogenesis:for better or for worse?Arterioscler Thromb Vasc Biol.2006;26:2421-32.
27.Taleb S,Tedgui A,Mallat Z.Regulatory T-cell immunity and its relevance to atherosclerosis.J Intern Med.2008;263:489-99.
28.Udi N,Yehuda S.Intravenous immunoglobulin-indications and mechanisms in cardiovascular diseases.Autoimmun Rev.2008;7:445-52.
29.Medzhitov R.,Janeway C,Jr.Innate immunity.N Engl J Med.2000;343:338-44.
30.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
31.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
32.Yasojima K,Schwab C,McGeer EG,McGeer PL.Generation of C-reactive protein and complement components in atherosclerotic plaques.Am J Pathol.2001;158:1039-51.
33.Vlaicu R,Niculescu F,Rus HG,Cristea A.Immunohistochemical localization of the terminal C5b-9 cmplement complex in human aortic fibrous plaque.Atherosclerosis.1985;57:163-77.
34.Torzewski M,Torzewski J,Bowyer DE,Waltenberger J,Fitzsimmons C,Hombach V,Gabbert HE.Immunohistochemical colocalization of the terminal complex of human complement and smooth muscle cell alpha-actin in early atherosclerotic lesions.Arterioscler Thromb Vase Biol.1997;17:2448-52.
35.Torzewsld M,Klouche M,Hock J,Messner M,Dorweiler B,Torzewski J,Gabbert HE,Bhakdi S.Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion.Arterioscler Thromb Vase Biol.1998;18:369-78.
36.Seifert PS,Hugo F,Hansson GK,Bhakdi S.Prelesional complement activation in experimental atherosclerosis.Terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits.Lab Invest.1989;60:747-54.
37.Laine P,Pentikainen MO,Wurzner R,Penttila A,Paavonen T,Meri S,Kovanen PT.Evidence for complement activation in mptured coronary plaques in acute myocardial infarction.Am J Cardiol.2002;90:404-8.
38.Hammerschmidt DE,Greenberg CS,Yamada O,Craddock PR,Jacob HS.Cholesterol and atheroma lipids activate complement and stimulate granulocytes.A possible mechanism for amplification of ischemic injury in atherosclerotic states.J Lab Clin Med.1981;98:68-77.
39.Bhakdi S,Dorweiler B,Kirchmann R,Torzewsld J,Weise E,Tranum-Jensen J,Walev Ⅰ,Wieland E.On the pathogenesis of atherosclerosis:enzymatic transformation of human low density lipoprotein to an atherogenic moiety.J Exp Med.1995;182:1959-71.
40.Bhakdi S,Torzewski M,Paprotka K,Schmitt S,Barsoom H,Suriyaphol P,Han SR,Lackner K J,Husmann M.Possible protective role for C-reactive protein in atherogenesis:complement activation by modified lipoproteins halts before detrimental terminal sequence.Circulation.2004;109:1870-6.
41.Biro A,Thielens NM,Cervenak L,Prohaszka Z,Fnst G,Arlaud GJ.Modified low density lipoproteins differentially bind and activate the C1 complex of complement.Mol Immunol.2007;44:1169-77.
42.Ji H,Ohmura K,Mahmood U,Lee DM,Hofhuis FM,Boaekle SA,Takahashi K,Holers VM,Walport M,Gerard C,Ezekowitz A,Carroll MC,Brenner M,Weissleder R,Verbeek JS,Duehatelle V,Degott C,Benoist C,Mathis D.Arthritis critically dependent on innate immune system players.Immunity.2002;16:157-68.
43.Vlaicu R,Rus HG,Niculescu F,Cristea A.Quantitative determinations of immunoglobulins and complement components in human aortic atherosclerotic wall.Med Interne.1985;23:29-35.
44.Torzewski J,Torzewski M,Bowyer DE,Frohlich M,Koenig W,Waltenberger J,Fitzsimmons C,Hombach V.C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries.Arterioscler Thromb Vasc Biol.1998;18:1386-92.
45.Reynolds GD,Vance RP.C-reactive protein immunohistochemieal localization in normal and atherosclerotic human aortas.Arch Pathol Lab Med.1987;111:265-9.
46.Ji SR,Wu Y,Potempa LA,Liang YH,Zhao J.Effect of modified C-reactive protein on complement activation:a possible complement regulatory role of modified or monomeric C-reactive protein in atherosclerotic lesions.Arterioscler Thromb Vasc Biol.2006;26:935-41.
47.Rus HG,Niculescu F,Constantinescu E,Cristea A,Vlaicu R.Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque.Atherosclerosis.1986;61:35-42.
48.Seifert PS,Kazatchkine MD.Generation of complement anaphylatoxins and C5b-9 by crystalline cholesterol oxidation derivatives depends on hydroxyl group number and position.Mol Immunol.1987;24:1303-8.
49.Saito E,Fujioka T,Kanno H,Hata E,Ueno T,Matsumoto T,Takahashi Y, Tochihara T,Yasugi T.Complement receptors in atherosclerotic Iesions.Artery.1992;19:47-62.
50.Mevorach D,Mascarenhas JO,Gershov D,Elkon KB.Complement-dependent clearance of apoptotic cells by human macrophages.J Exp Med.1998;188:2313-20.
51.Aderem A,Underhill DM.Mechanisms of phagocytosis in macrophages.Annu Rev Immunol.1999;17:593-623.
52.Husemann J,Obstfeld A,Febbraio M,Kodama T,Silverstein SC.CD11b/CD18 mediates production of reactive oxygen species by mouse and human maerophages adherent to matrixes containing oxidized LDL.Arterioscler Thromb Vase Biol.2001;21:1301-5.
53.Helmy KY,Katschke KJ,Jr.,Gorgani NN,Kljavin NM,Elliott JM,Diehl L,Scales S J,Ghilardi N,van Lookeren Campagne M.CRIg:a maerophage complement receptor required for phagocytosis of circulating pathogens.Cell.2006;124:915-27.
54.Lee MY,Kim WJ,Kang YJ,Jung YM,Kang YM,Suk K,Park JE,Choi EM,Choi BK,Kwon BS,Lee WH.Z39Ig is expressed on maerophages and may mediate inflammatory reactions in arthritis and atherosclerosis.J Leukoc Biol.2006;80:922-8.
55.Niculescu F,Rus H.Mechanisms of signal transduction activated by sublytic assembly of terminal complement complexes on nucleated cells,Immunol Res.2001;24:191-9.
56.Seeger W,Suttorp N,Hellwig A,Bhakdi S.Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes.J Immunol.1986;137:1286-93.
57.Torzewski J,Oldroyd R,Lachmarm P,Fitzsimmons C,Proudfoot D,Bowyer D.Complement-induced release of monocyte chemotaetic protein-1 from human smooth muscle cells.A possible initiating event in atherosclerotic lesion formation.Arterioscler Thromb Vasc Biol.1996;16:673-7.
58.Niculescu F,Badea T,Rus H.Sublytic C5b-9 induces proliferation of human aortic smooth muscle cells:role of mitogen activated protein kinase and phosphatidylinositol 3-kinase.Atherosclerosis.1999;142:47-56.
59.Benzaquen LR,Nicholson-Weller A,Halperin JA.Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells.J Exp Med.1994;179:985-92.
60.Kilgore KS,Schmid E,Shanley TP,Flory CM,Maheswari V,Tramontini NL,Cohen H,Ward PA,Friedl HP,Warren JS.Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.Am J Pathol.1997;150:2019-31.
61.Hattori R,Hamilton KK,McEver RP,Sims PJ.Complement proteins C5b-9induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.J IBiol Chem.1989;264:9053-60.
62.Tedesco F,Pause M,Nardon E,Introna M,Mantovani A,Dobrina A.The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.J Exp Med.1997;185:1619-27.
63.Kilgore KS,Shen JP,Miller BF,Ward PA,Warren JS.Enhancement by the complement membrane attack complex of tumor necrosis factor-alpha-induced endothelial cell expression of E-selectin and ICAM-1.J Immunol.1995;155:1434-41.
64.Oksjoki R,Laine P,Helske S,Vehmaan-Kreula P,Mayranpaa MI,Gasque P,Kovanen PT,Pentikainen MO.Receptors for the anaphylatoxins C3a and CSa are expressed in human atherosclerotic coronary plaques.Atheroselerosis.2007;195:90-9.
65.Arend WP,Massoni RJ,Niemann MA,Giclas PC.Absence of induction of IL-1 production in human monocytes by complement fragments.J Immunol.1989;142:173-8.
66.Murakami Y,Imarnichi T,Nagasawa S.Characterization of C3a anaphylatoxin receptor on guinea-pig macrophages.Immunology.1993;79:633-8.
67.Takabayashi T,Vannier E,Clark BD,Margolis NH,Dinarello CA,Burke JF,Gelfand JA.A new biologic role for C3a and C3a desArg:regulation of TNF-alpha and IL-1 beta synthesis.J Immunol.1996;156:3455-60.
68.Takabayashi T,Vannier E,Burke JF,Tompkins RG,Gelfand JA,Clark BD.Both C3a and C3a(desArg) regulate interleukin-6 synthesis in human peripheral blood mononuelear cells.J Infect Dis.1998;177:1622-8.
69.Verdeguer F,Castro C,Kubicek M,Pla D,Vila-Caballer M,Vinue A,Civeira F,Pocovi M,Calvete JJ,Andres V.Complement regulation in murine and human hypercholesterolemia and role in the control of macrophage and smooth muscle cell proliferation.Cardiovasc Res.2007;76:340-50.
70.Nataf S,Davoust N,Ames RS,Barnum SR.Human T cells express the C5a receptor and are chemoattracted to C5a.J Immunol.1999;162:4018-23.
71.Schulman ES,Post TJ,Henson PM,Giclas PC.Differential effects of the complement peptides,C5a and C5a des Arg on human basophil and lung mast cell histamine release.J Clin Invest.1988;81:918-23.
72.Foreman KE,Vaporciyan AA,Bonish BK,Jones ML,Johnson KJ,Glovsky MM,Eddy SM,Ward PA.C5a-induced expression of P-selectin in endothelial cells.J Clin Invest.1994;94:1147-55.
73.Okusawa S,Yancey KB,van der Meer JW,Endres S,Lonnemann G,Hefter K,Frank MM,Burke JF,Dinarello CA,Gelfand JA.C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro.Comparison with secretion of interleukin 1 beta and interleukin 1 alpha.J Exp Med.1988;168:443-8.
74.McCarthy K,Henson PM.Induction of lysosomal enzyme secretion by alveolar maerophages in response to the purified complement fragments C5a and C5a des-arg.J Immunol.1979;123:2511-7.
75.Kastl SP,Speidl WS,Kaun C,Rega G,Assadian A,Weiss TW,Valent P,Hagrnueller GW,Maurer G,Huber K,Wojta J.The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophagcs via NF-kappaB activation.J Thromb Haemost.2006;4:1790-7.
76.Kastl SP,Speidl WS,Kaun C,Katsaros KM,Rega G,Afonyushkin T,Bochkov VN,Valcnt P,Assadian A,Hagmueller GW,Hoeth M,de Martin R,Ma Y,Maurer G,Huber K,Wojta J.In human macrophages the complement component CSa induces the expression of oncostatin M via AP-1 activation.Arterioscler Thromb Vasc Biol.2008;28:498-503.
77.Schmiedt W,Kinseherf R,Deigner HP,Kamencic H,Nauen O,Kilo J,Oelert H,Metz J,Bhakdi S.Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits.Arterioscler Thromb Vasc Biol.1998;18:1790-5.
78.Patel S,Thelander EM,Hernandez M,Montenegro J,Hassing H,Burton C,Mundt S,Hermanowski-Vosatka A,Wright SD,Chao YS,Detmers PA.ApoE(-/-) mice develop atheroselerosis in the absence of complement component C5.Biochem Biophys Res Commun.2001;286:164-70.
79.Buono C,Come CE,Witztum JL,Maguire GF,Colmelly PW,Carroll M,Lichtman AH.Influence of C3 deficiency on atheroselerosis.Circulation.2002;105:3025-31.
80.Persson L,Boren J,Robertson AK,Wallenius V,Hansson GK,Pekna M.Lack of complement factor C3,but not factor B,increases hyperlipidemia and atheroselerosis in apolipoprotein E-/-low-density lipoprotein receptor-/-mice.Arterioscler Thromb Vasc Biol.2004;24:1062-7.
81.Bhatia Vk,Yun S,Leung V,Grimsditch DC,Benson GM,Botto MB,Boyle JJ,Haskard DO.Complement Clq reduces early atherosclerosis in low-density lipoprotein receptor-deficient mice.Am J Pathol.2007;170:416-26.
82.Molines TE,Song WC,Lambris JD.Complement in inflammatory tissue damage and disease.Trends Immunol.2002;23:61-4.
83.Miwa T,Song WC.Membrane complement regulatory proteins:insight from animal studies and relevance to human diseases,Int Immunopharmacol.2001;1:445-59.
84.Liszewski MK,Post TW,Atkinson JP.Membrane cofactor protein(MCP or CD46):newest member of the regulators of complement activation gene cluster. Annu Rev Immunol. 1991 ;9:431 -55.
85. Lublin DM, Atkinson JP. Decay-accelerating factor: biochemistry, molecular biology, and function. Annu Rev Immunol. 1989;7:35-58.
86. Ahearn JM, Fearon DT. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv Immunol. 1989;46:183-219.
87. Spicer AP, Seldin MF, Gendler SJ. Molecular cloning and chromosomal localization of the mouse decay-accelerating factor genes. Duplicated genes encode glycosylphosphatidylinositol-anchored and transmembrane forms. J Immunol 1995;155:3079-91.
88. Song WC, Deng C, Raszmann K, Moore R, Newbold R, McLachlan JA, Negishi M. Mouse decay-accelerating factor: selective and tissue-specific induction by estrogen of the gene encoding the glycosylphosphatidylinositol-anchored form. J Immunol. 1996; 157:4166-72.
89. Powell MB, Marchbank KJ, Rushmere NK, van den Berg CW, Morgan BP. Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59. J Immunol. 1997;158:1692-702.
90. Qian YM, Qin X, Miwa T, Sun X, Halperin JA, Song WC. Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59. J Immunol. 2000; 165:2528-34.
91. Nickells MW, Atkinson JP. Characterization of CR1- and membrane cofactor protein-like proteins of two primates. J Immunol. 1990; 144:4262-8.
92. Johnstone RW, Loveland BE, McKenzie IF. Identification and quantification of complement regulator CD46 on normal human tissues. Immunology. 1993;79:341-7.
93. Miwa T, Nonaka M, Okada N, Wakana S, Shiroishi T, Okada H. Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1. Immunogenetics. 1998;48:363-71.
94. Holers VM, Kinoshita T, Molina H. The evolution of mouse and human complement C3-binding proteins:divergence of form but conservation of function.Immunol Today.1992;13:231-6.
95.Li B,Sallee C,Dehoff M,Foley S,Molina H,Holers VM.Mouse Crry/p65.Characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF.J Immunol.1993;151:4295-305.
96.Song WC.Membrane complement regulatory proteins in autoimmune and inflammatory tissue injury.Curr Dir Autoimmun.2004;7:181-99.
97.Goodfellow RM,Williams AS,Levin JL,Williams BD,Morgan BP.Soluble complement receptor one(sCR1) inhibits the development and progression of rat collagen-induced arthritis.Clin Exp Immunol.2000;119:210-6.
98.Mizuno M,Nishikawa K,Morgan BP,Matsuo S.Comparison of the suppressive effects of soluble CR1 and C5a receptor antagonist in acute arthritis induced in rats by blocking of CD59.Clin Exp Immunol.2000;119:368-75.
99.Hamaeher J,Sadallah S,Schifferli JA,Villard J,Nicod LP.Soluble complement receptor type 1(CD35) in bronchoalveolar lavage of inflammatory lung diseases.Eur Respir J.1998;11:112-9.
100.Mulligan MS,Warner RL,Rittershaus CW,Thomas LJ,Ryan US,Foreman KE,Crouch LD,Till GO,Ward PA.Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewisx moieties.J Immunol.1999;162:4952-9.
101.Molina H,Kinoshita T,Inoue K,Carel JC,Holers VM.A molecular and immunochemical characterization of mouse CR2.Evidence for a single gene model of mouse complement receptors 1 and 2.J Immunol.1990;145:2974-83.
102.Kinoshita T,Takeda J,Hong K,Kozono H,Sakai H,Inoue K.Monoclonal antibodies to mouse complement receptor type 1(CR1).Their use in a distribution study showing that mouse erythrocytes and platelets are CR1-negative.J Immunol.1988;140:3066-72.
103.Holers VM,Boackle SA.Complement receptor 2 and autoimmunity.Curr Dir Autoimmun.2004;7:33-48.
104.Burge J,Nicholson-Weller A,Austen KF.Isolation of C4-binding protein from guinea pig plasma and demonstration of its function as a control protein of the classical complement pathway C3 convertase.J Immunol.1981;126:232-5.
105.Davitz MA,Low MG,Nussenzweig V.Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C(PIPLC).Selective modification of a complement regulatory protein.J Exp Med.1986;163:1150-61.
106.Kinoshita T,Rosenfeld SI,Nussenzweig V.A high m.w.form of decay-accelerating factor(DAF-2) exhibits size abnormalities in paroxysmal nocturnal hemoglobinuria erythrocytes.J Immunol.1987;138:2994-8.
107.Lublin DM,Lemons RS,Le Beau MM,Holers VM,Tykocinski ML,Medof ME,Atkinson JP.The gene encoding decay-accelerating factor(DAF) is located in the complement-regulatory locus on the long arm of chromosome 1.J Exp Med.1987;165:1731-6.
108.Medof ME,Lublin DM,Holers VM,Ayers DJ,Getty RR,Leykam JF,Atkinson JP,Tykocinski ML.Cloning and characterization of cDNAs encoding the complete sequence of decay-accelerating factor of human complement.Proc Natl Acad Sci USA.1987;84:2007-11.
109.Mizuno M,Donev RM,Harris CL,Morgan BP.CD55 in rat male reproductive tissue:differential expression in testis and expression of a unique truncated isoform on spermatozoa.Mol Immunol.2007;44:1613-22.
110.Yan J,Allendorf DJ,Li B,Yan R,Hansen R,Donev R.The role of membrane complement regulatory proteins in cancer immunotherapy.Adv Exp Med Biol.2008;632:159-74.
111.Pettigrew DM,Williams DT,Kerrigan D,Evans DJ,Lea SM,Bhella D.Structural and functional insights into the interaction of echoviruses and decay-accelerating factor.J Biol Chem.2006;281:5169-77.
112.Weeks C,Moratz C,Zacharia A,Stracener C,Egan R,Peckham R,Moore FD, Jr.,Tsokos GC.Decay-accelerating factor attenuates remote ischemia-reperfusion-initiated organ damage.Clin Immunol.2007;124:311-27.
113.Yamada K,Miwa T,Liu J,Nangaku M,Song WC.Critical protection from renal ischemia reperfusion injury by CD55 and CD59.J Immunol.2004;172:3869-75.
114.Pavlov V,Raedler H,Yuan S,Leisman S,Kwan WH,Lalli PN,Medof ME,Heeger PS.Donor deficiency of decay-accelerating factor accelerates murine T cell-mediated cardiac allograft rejection.J Immunol.2008;181:4580-9.
115.Barros MM,Yamamoto M,Figueiredo MS,Cancado R,Kimura EY,Langhi DM,Jr.,Chiattone CS,Bordin JO.Expression levels of CD47,CD35,CD55,and CD59 on red blood cells and signal-regulatory protein-alpha,beta on monocytes from patients with warm autoimmune hemolytic anemia.Transfusion.2009;49:154-60.
116.Parker C.Eculizumab for paroxysmal nocturnal haemoglobinuria.Lancet.2009;373:759-67.
117.Shichishirna T,Noji H.A new aspect of the molecular pathogenesis of paroxysmal nocturnal hemoglobinuria.Hematology.2002;7:211-27.
118.Sloand EM,Mainwaring L,Keyvanfar K,Chen J,Maciejewski J,Klein HG;Young NS.Transfer of glycosylphosphatidylinositol-anchored proteins to deficient cells after erythrocyte transfusion in paroxysmal nocturnal hemoglobinuria.Blood.2004;104:3782-8.
119.Yin W,Ghebrehiwet B,Weksler B,Peerschke EI.Regulated complement deposition on the surface of human endothelial cells:effect of tobacco smoke and shear stress.Thromb Res.2008;122:221-8.
120.Liu J,Miwa T,Hilliard B,Chen Y,Lambris JD,Wells AD,Song WC.The complement inhibitory protein DAF(CD55) suppresses T cell immunity in vivo.J Exp Med.2005;201:567-77.Epub 2005 Feb 14.
121.Sogabe H,Nangaku M,Ishibashi Y,Wada T,Fujita T,Sun X,Miwa T,Madaio MP,Song WC.Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis,J Immunol. 2001;167:2791-7.
122.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
123.Molina H,Miwa T,Zhou L,Hilliard B,Mastellos D,Maldonado MA,Lambris JD,Song WC.Complement-mediated clearance of erythrocytes:mechanism and delineation of the regulatory roles of Crry and DAF.Decay-accelerating factor.Blood.2002;100:4544-9.
124.Kim DD,Miwa T,Kimttra Y,Schwendener RA,van Lookeren Campagne M,Song WC.Deficiency of DAF and Crry on murine platelets leads to complement-dependent clearance via the macrophage phagocytic receptor CRIg.Blood.2008.
125.Ratnoff WD,Knez JJ,Prince GM,Okada H,Lachmarm PJ,Medof ME.Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59.Clin Exp Immunol.1992;87:415-21.
126.Qin X,Miwa T,Aktas H,Gao M,Lee C,Qian YM,Morton CC,Shahsafaei A,Song WC,Halperin JA.Genomic structure,functional comparison,and tissue distribution of mouse Cd59a and Cd59b.Mamm Genome.2001;12:582-9.
127.Sawada R,Ohashi K,Anaguchi H,Okazaki H,Hattori M,Minato N,Naruto M.Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.DNA Cell Biol.1990;9:213-20.
128.Baalasubramanian S,Harris CL,Donev RM,Mizuno M,Omidvar N,Song WC,Morgan BP.CD59a is the primary regulator of membrane attack complex assembly in the mouse.J Immunol.2004;173:3684-92.
129.Longhi MP,Sivasankar B,Omidvar N,Morgan BP,Gallimore A.Cutting edge:routine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner.J Immunol.2005;175:7098-102.
130.Morgan BP,van den Berg CW,Davies EV,Hallett MB,Horejsi V. Cross-linking of CD59 and of other glycosyl phosphatidylinositol-anchored molecules on nentrophils triggers cell activation via tyrosine kinase.Eur J Immunol.1993;23:2841-50.
131.Murray EW,Robbins SM.Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement.J Biol Chem.1998;273:25279-84.
132.Seifert PS,Roth I,Schmiedt W,Oelert H,Okada N,Okada H,Bhakdi S.CD59(homologous restriction factor 20),a plasma membrane protein that protects against complement C5b-9 attack,in human atherosclerotic lesions.Atherosclerosis.1992;96:135-45.
133.Li W,Tada T,Miwa T,Okada N,Ito J,Okada H,Tateyama H,Eimoto T.mRNA expression of complement components and regulators in rat arterial smooth muscle cells.Microbiol Immunol.1999;43:585-93.
134.Kinderlerer AR,Steinberg R,Johns M,Harten SK,Lidington EA,Haskard DO,Maxwell PH,Mason JC.Statin-induced expression of CD59 on vascular endothelium in hypoxia:a potential mechanism for the anti-inflammatory actions of statins in rheumatoid arthritis.Arthritis Res Ther.2006;8:R130.
135.Thomas CM,Smart EJ.Gender as a regulator of atherosclerosis in routine models.Curr Drug Targets.2007;8:1172-80.
136.Patrono C,FitzGerald GA.Isoprostanes:potential markers of oxidant stress in atherothrombotic disease.Arterioscler Thromb Vasc Biol.1997;17:2309-15.
137.Pratico D,Tangirala RK,Rader DJ,Rokach J,FitzGerald GA.Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice.Nat Med.1998;4:1189-92.
138.Babaev VR,Patel MB,Semenkovich CF,Fazio S,Linton MF.Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice.J Biol Chem.2000;275:26293-9.
1.Anggard EE.The endothelium—the body's largest endocrine gland? J Endocrinol.1990;127:371-5.
2.Schmid D,Munz C.Innate and adaptive immunity through autophagy.Immunity.2007;27:11-21.
3.Medzhitov R.Recognition of microorganisms and activation of the immune response.Nature.2007;449:819-26.
4.Kirk P,Bazan JF.Pathogen recognition:TLRs throw us a curve:Immunity.2005;23:347-50.
5.Means TK,Wang S,Lien E,Yosbimura A,Golenboek DT,Fenton MJ.Human toll-like receptors mediate cellular activation by Mycobaetedum tuberculosis.J Immunol.1999;163:3920-7.
6.Sansonetti PJ.The innate signaling of dangers and the dangers of innate signaling.Nat Immunol.2006;7:1237-42.
7.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
8.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
9.Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol.2004;4:499-511.
10.Kawai T,Akira S.TLR signaling.CellDeath Differ.2006;13:816-25.
11.Zhang X,Kimura Y,Fang C,Zhou L,Sfyroera G,Larnbris JD,Wetsel RA,Miwa T,Song WC.Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo.Blood.2007;110:228-36.
12.Meneghin A,Hogaboam CM.Infectious disease,the innate immune response,and fibrosis.J Clin Invest.2007;117:530-8.
13.Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol.2004;5:987-95.
14.Takeda K,Kaisho T,Akira S.Toll-like receptors.Annu Rev Immunol. 2003;21:335-76.
15.Medzhitov R,Preston-Hurlburt P,Janeway CA,Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature.1997;388:394-7.
16.O'Neill LA.Signal transduction pathways activated by the IL-1receptor/toll-like receptor superfamily.Curr Top Microbiol Immunol.2002;270:47-61.
17.Homung V,Rothenfusser S,Britsch S,Krug A,Jahrsdorfer B,Giese T,Endres S,Hartmann G.Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides.J Immunol.2002;168:4531-7.
18.Kokkinopoulos I,Jordan WJ,Ritter MA.Toll-like receptor mRNA expression patterns in human dendritic cells and monocytes.Mol Immunol.2005;42:957-68.
19.Swantek JL,Tsen MF,Cobb MH,Thomas JA.IL-1 receptor-associated kinase modulates host responsiveness to endotoxin,J Immunol.2000;164:4301-6.
20.Poltorak A,Smirnova I,He X,Liu MY,Van Huffel C,McNally O,Birdwell D,Alejos E,Silva M,Du X,Thompson P,Chan EK,Ledesma J,Roe B,Clifton S,Vogel SN,Beutler B.Genetic and physical mapping of the Lps locus:identification of the toll-4 receptor as a candidate gene in the critical region.Blood Cells Mol Dis.1998;24:340-55.
21.Michelsen KS,Wong MH,Shah PK,Zhang W,Yano J,Doherty TM,Akira S,Rajavashisth TB,Arditi M.Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E.Proc Natl Acad Sci U S A.2004;101:10679-84.
22.Xu XH,Shah PK,Faure E,Equils O,Thomas L,Fishbein MC,Luthringer D,Xu XP,Rajavashisth TB,Yano J,Kaul S,Arditi M.Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL.Circulation.2001;104:3103-8.
23.Ozinsky A,Underhill DM,Fontenot JD,Hajjar AM,Smith KD,Wilson CB,Schroeder L,Aderem A.The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.Proc Natl Acad Sci USA.2000;97:13766-71.
24.Jin MS,Lee JO.Structures of the toll-like receptor family and its ligand complexes.Immunity.2008;29:182-91.
25.Massari P,Hermeke P,Ho Y,Latz E,Golenbock DT,Wetzler LM.Cutting edge:Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent.J Immunol.2002;168:1533-7.
26.Lee HK,Iwasald A.Innate control of adaptive immunity:dendritic cells and beyond.Semin Immunol.2007;19:48-55.
27.Alexopoulou L,Thomas V,Schnare M,Lobet Y,Anguita J,Schoen RT,Medzhitov R,Fikrig E,Flavell RA.Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1-and TLR2-deficient mice.Nat Med.2002;8:878-84.
28.Wooten RM,Ma Y,Yoder RA,Brown JP,Weis JH,Zachary JF,Kirschning CJ,Weis JJ.Toll-like receptor 2 is required for innate,but not acquired,host defense to Borrelia burgdorferi.J Immunol.2002;168:348-55.
29.Barton GM,Medzhitov R.Toll signaling:RIPping off the TNF pathway.Nat Immunol.2004;5:472-4.
30.Matsumoto M,Kikkawa S,Kohase M,Miyake K,Seya T.Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling.Biochem Biophys Res Commun.2002;293:1364-9.
31.Celia M,Salio M,Sakakibara Y,Langen H,Julkunen I,Lanzavecchia A.Maturation,activation,and protection of dendritic cells induced by double-stranded RNA.J Exp Med.1999;189:821-9.
32.Khvalevsky E,Rivkin L,Rachmilewitz J,Galun E,Giladi H.TLR3 signaling in a hepatoma cell line is skewed towards apoptosis,J Cell Biochem.2007;100:1301-12.
33.Schnare M,Holt AC,Takeda K,Akira S,Medzhitov R.Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88.Curr Biol.2000;10:1139-42.
34.Latz E,Schoenemeyer A,Visintin A,Fitzgerald KA,Monks BG,Knetter CF,Lien E,Nilsen NJ,Espevik T,Golenbock DT.TLR9 signals after translocating from the ER to CpG DNA in the lysosome.Nat Immunol.2004;5:190-8.
35.Stacey KJ,Sweet MJ,Hume DA.Macrophages ingest and are activated by bacterial DNA.J Immunol.1996;157:2116-22.
36.Sparwasser T,Koch ES,Vabulas RM,Heeg K,Lipford GB,Ellwart JW,Wagner H.Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells.Eur J Immunol.1998;28:2045-54.
37.Zuiderweg ER,Nettesheim DG,Mollison KW,Carter GW.Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data.Biochemistry.1989;28:172-85.
38.Kohl J,Lubbers B,Klos A,Bautsch W,Casaretto M.Evaluation of the C-terminal C5a effeetor site with short synthetic C5a analog peptides.Eur J Immunol.1993;23:646-52.
39.Foreman KE,Vaporciyan AA,Bonish BK,Jones ML,Johnson KJ,Glovsky MM,Eddy SM,Ward PA.C5a-induced expression of P-selectin in endothelial cells.J Clin Invest.1994;94:1147-55.
40.Mollnes TE,Brekke OL,Fung M,Fure H,Christiansen D,Bergseth G;Videm V,Lappegard KT,Kohl J,Lambris JD.Essential role of the C5a receptor in E coli-indueed oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation.Blood.2002;100:1869-77.
41.Gutzmer R,Kother B,Zwirner J,Dijkstra D,Purwar R,Wittmann M,Werfel T.Human plasmacytoid dendritic cells express receptors for anaphylatoxins C3a and C5a and are chemoattracted to C3a and C5a.J Invest Dermatol.2006;126:2422-9.
42.Melendez AJ,Ibrahim FB.Antisense knockdown of sphingosine kinase 1 in human macrophages inhibits C5a receptor-dependent signal transduction,Ca2+ signals,enzyme release,cytokine production,and chemotaxis.J Immunol.2004;173:1596-603.
43.Kastl SP,Speidl WS,Kaun C,Rega G,Assadian A,Weiss TW,Valent P,Hagmueller GW,Maurer G,Huber K,Wojta J.The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-kappaB activation.J Thromb Haemost.2006;4:1790-7.
44.Malmsten M,Schmidtchen A.Antimicrobial C3a—biology,biophysics,and evolution.Adv Exp Med Biol.2007;598:141-58.
45.DiScipio RG,Schraufstatter IU.The role of the complement anaphylatoxins in the recruitment of eosinophils.Int Immunopharmacol.2007;7:1909-23.
46.Haas PJ,van Stdjp J.Anaphylatoxins:their role in bacterial infection and inflammation,Immunol Res.2007;37:161-75.
47.Fujita T,Matsushita M,Endo Y.The lectin-complement pathway—its role in innate immunity and evolution,Immunol Rev.2004;198:185-202.
48.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
49.Yamada K,Miwa T,Liu J,Nangaku M,Song WC.Critical protection from renal isehemia reperfusion injury by CD55 and CD59.J Immunol.2004;172:3869-75.
50.Triantafilou M,Triantafilou K.Lipopolysaccharide recognition:CD14,TLRs and the LPS-activation cluster.Trends Immunol.2002;23:301-4.
51.Coyne CB,Bergelson JM.Virus-induced Ab1 and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions.Cell.2006;124:119-31.
52.O'Brien DP,Israel DA,Krishna U,Romero-Gallo J,Nedrud J,Medof ME,Lin F,Redline R,Lublin DM,Nowicki BJ,Franco AT,Ogden S,Williams AD,Polk DB,Peek RM,Jr.The role of decay-accelerating factor as a receptor for Helicobacter pylori and a mediator of gastric inflammation.J Biol Chem.2006;281:13317-23.
1.Hansson GK.Inflammation,atherosclerosis,and coronary artery disease.N Engl J Med.2005;352:1685-95.
2.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
3.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
4.Song WC,Sarrias MR,Lambris JD.Complement and innate immunity.Immunopharmacology.2000;49:187-98.
5.Mollnes TE,Song WC,Lambris JD.Complement in inflammatory tissue damage and disease.Trends Immunol.2002;23:61-4.
6.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
7.Lublin DM,Atkinson JP.Decay-accelerating factor:biochemistry,molecular biology,and function.Annu Rev Immunol.1989;7:35-58.
8.Nicholson-Weller A,Burge J,Fearon DT,Weller PF,Austen KF.Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system.J Immunol.1982;129:184-9.
9.Okada N,Harada R,Fujita T,Okada H.A novel membrane glycoprotein capable of inhibiting membrane attack by homologous complement.Int Immunol.1989;1:205-8.
10.Rollins SA,Sims PJ.The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9.J Immunol.1990;144:3478-83.
11.Oksjoki R,Kovanen PT,Pentikainen MO.Role of complement activation in atherosclerosis.Curr Opin Lipidol.2003;14:477-82.
12.Vlaicu R,Rus HG,Niculescu F,Cristea A.Immunoglobulins and complement components in human aortic atherosclerotic intima.Atherosclerosis.1985;55:35-50.
13.Seifert PS,Hansson GK.Complement receptors and regulatory proteins in human atherosclerotic lesions.Arteriosclerosis.1989;9:802-11.
14.Vlaicu R,Rus HG,Niculescu F,Cristea A.Quantitative determinations of immunoglobulins and complement components in human aortic atherosclerotic wall.Med Interne.1985;23:29-35.
15.Niculescu F,Rus H.The role of complement activation in atherosclerosis.Immunol Res.2004;30:73-80.
16.Bhatia VK,Yun S,Leung V,Grimsditch DC,Benson GM,Botto MB,Boyle JJ,Haskard DO.Complement C1q reduces early atherosclerosis in low-density lipoprotein receptor-deficient mice.Am J Pathol.2007;170:416-26.
17.Schmiedt W,Kinscherf R,Deigner HP,Kamencic H,Nauen O,Kilo J,Oelert H,Metz J,Bhakdi S.Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits.Arterioscler Thromb Vasc Biol.1998;18:1790-5.
18.Patel S,Thelander EM,Hernandez M,Montenegro J,Hassing H,Burton C,Mundt S,Hermanowski-Vosatka A,Wright SD,Chao YS,Detmers PA.ApoE(-/-) mice develop atherosclerosis in the absence of complement component C5.Biochem Biophys Res Commun.2001;286:164-70.
19.Buono C,Coffee CE,Witztum JL,Maguire GF,Connelly PW,Carroll M,Lichtman AH.Influence of C3 deficiency on atherosclerosis.Circulation.2002;105:3025-31.
20.Persson L,Boren J,Robertson AK,Wallenius V,Hansson GK,Pekna M.Lack of complement factor C3,but not factor B,increases hyperlipidemia and atherosclerosis in apolipoprotein E-/- low-density lipoprotein receptor-/- mice.Arterioscler Thromb Vasc Biol.2004;24:1062-7.
21.Seifert PS,Hansson GK.Decay-accelerating factor is expressed on vascular smooth muscle cells in human atherosclerotic lesions.J Clin Invest.1989;84:597-604.
22.Niculescu F,Pus HG,Vlaicu R.Decay-accelerating factor regulates complement-mediated damage in the human atherosclerotic wall.Immunol Lett.1990;26:17-23.
23.Niculescu F,Rus HG,Vlaicu R.Immunohistochemical localization of C5b-9, S-protein,C3d and apolipoprotein B in human arterial tissues with atherosclerosis.Atherosclerosis.1987;65:1-11.
24.Mackness B,Hunt R,Durrington PN,Mackness MI.Increased immunolocalization of paraoxonase,clusterin,and apolipoprotein A-I in the human artery wall with the progression of atherosclerosis.Arterioscler Thromb Vasc Biol.1997;17:1233-8.
25.Li SH,Szmitko PE,Weisel RD,Wang CH,Fedak PW,Li RK,Mickle DA,Verma S.C-reactive protein upregulates complement-inhibitory factors in endothelial cells.Circulation.2004;109:833-6.
26.Bhakdi S,Torzewski M,Klouche M,Hemmes M.Complement and atherogenesis:binding of CRP to degraded,nonoxidized LDL enhances complement activation.Arterioscler Thromb Vase Biol.1999;19:2348-54.
27.Mason JC,Ahmed Z,Mankoff R,Lidington EA,Ahmad S,Bhatia V,Kinderlerer A,Randi AM,Haskard DO.Statin-induced expression of decay-accelerating factor protects vascular endothelium against complement-mediated injury.Circ Res.2002;91:696-703.
28.Sun X,Funk CD,Deng C,Sahu A,Lambris JD,Song WC.Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.Proc Natl Acad Sci U S A.1999;96:628-33.
29.Miwa T,Zhou L,Hilliard B,Molina H,Song WC.Crry,but not CD59 and DAF,is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack.Blood.2002;99:3707-16.
30.Tangirala RK,Rubin EM,Palinski W.Quantitation of atherosclerosis in murine models:correlation between lesions in the aortic origin and in the entire aorta,and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.J Lipid Res.1995;36:2320-8.
31.Nicoletti A,Kaveri S,Caligiuri G,Bariety J,Hansson GK.Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice.J Clin lnvest. 1998;102:910-8.
32.Wang M,Lee E,Song W,Ricciotti E,Rader DJ,Lawson JA,Pure E,FitzGerald GA.Microsomal prostaglandin E synthase-1 deletion suppresses oxidative stress and angiotensin Ⅱ-induced abdominal aortic aneurysm formation.Circulation.2008;117:1302-9.
33.Song WL,Lawson JA,Wang M,Zou H,FitzGerald GA.Noninvasive assessment of the role of cyclooxygenases in cardiovascular health:a detailed HPLC/MS/MS method.Methods Enzymol.2007;433:51-72.
34.Niculescu F,Rus H.Complement activation and atherosclerosis.Mol Immunol.1999;36:949-55.
35.Yun S,Leung VW,Botto M,Boyle JJ,Haskard DO.Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice Lacking the Membrane-Bound Complement Regulator CD59.Arterioscler Thromb Vasc Biol.2008.
36.Liu J,Miwa T,Hilliard B,Chen Y,Lambris JD,Wells AD,Song WC.The complement inhibitory protein DAF(CD55) suppresses T cell immunity in vivo.J Exp Med.2005;201:567-77.Epub 2005 Feb 14.
37.Sogabe H,Nangaku M,Ishibashi Y,Wada T,Fujita T,Sun X,Miwa T,Madaio MP,Song WC.Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.J Immunol.2001;167:2791-7.
38.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
39.Molina H,Miwa T,Zhou L,Hilliard B,Mastellos D,Maldonado MA,Lambris JD,Song WC.Complement-mediated clearance of erythrocytes:mechanism and delineation of the regulatory roles of Crry and DAF.Decay-accelerating factor.Blood.2002;100:4544-9.
40.Kim DD,Miwa T,Kimura Y,Schwendener RA,van Lookeren Campagne M, Song WC.Deficiency of DAF and Crry on murine platelets leads to complement-dependent clearance via the macrophage phagocytic receptor CRIg.Blood.2008.
41.Babaev VR,Patel MB,Semenkovich CF,Fazio S,Linton MF.Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice.J Biol Chem.2000;275:26293-9.
42.Patrono C,FitzGerald GA.Isoprostanes:potential markers of oxidant stress in atherothrombotic disease.Arterioscler Thromb Vasc Biol.1997;17:2309-15.
43.Pratico D,Iuliano L,Mauriello A,Spagnoli L,Lawson JA,Rokach J,Maclouf J,Violi F,FitzGerald GA.Localization of distinct F2-isoprostanes in human atherosclerotic lesions.J Clin Invest.1997;100:2028-34.
44.Rokach J,Khanapure SP,Hwang SW,Adiyaman M,Lawson JA,FitzGerald GA.Nomenclature of isoprostanes:a proposal.Prostaglandins.1997;54:853-73.
45.Rudic RD,Brinster D,Cheng Y,Fries S,Song WL,Austin S,Coffman TM,FitzGerald GA.COX-2-derived prostacyclin modulates vascular remodeling.Circ Res.2005;96:1240-7.
46.Benzaquen LR,Nicholson-Weller A,Halperin JA.Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells.J Exp Med.1994;179:985-92.
47.Halperin JA,Taratuska A,Nicholson-Weller A.Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells.J Clin Invest.1993;91:1974-8.
48.Morgan BP,van den Berg CW,Davies EV,Hallett MB,Horejsi V.Cross-linking of CD59 and of other glycosyl phosphatidylinositol-anchored molecules on neutrophils triggers cell activation via tyrosine kinase.Eur J Immunol.1993;23:2841-50.
49.Murray EW,Robbins SM.Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement.J Biol Chem.1998;273:25279-84.
50.Longhi MP,Sivasankar B,Omidvar N,Morgan BP,GaUimore A.Cutting edge:routine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner.J Immunol.2005;175:7098-102.
51.Longhi MP,Harris CL,Morgan BP,Gallimore A.Holding T cells in check—a new role for complement regulators? Trends Immunol.2006;27:102-8.
1.Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity.Cell.2006;124:783-801.
2.Carroll MC.The complement system in regulation of adaptive immunity.Nat Immunol.2004;5:981-6.
3.Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol.2004;5:987-95.
4.Medzhitov R,Preston-Hurlburt P,Janeway CA,Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature.1997;388:394-7.
5.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
6.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
7.Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol.2004;4:499-511.
8.Fujita T,Matsushita M,Endo Y.The lectin-complement pathway—its role in innate immunity and evolution.Immunol Rev.2004;198:185-202.
9.Zhang X,Kimura Y,Fang C,Zhou L,Sfyroera G,Lambris JD,Wetsel RA,Miwa T,Song WC.Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo.Blood.2007;110:228-36.
10.Lee H,Zahra D,Vogelzang A,Newton R,Thatcher J,Quan A,So T,Zwirner J,Koentgen F,Padkjaer SB,Mackay F,Whitfeld PL,Mackay CR.Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies.Nat Biotechnol.2006;24:1279-84.
11.Takeda K,Kaisho T,Akira S.Toll-like receptors.Annu Rev Immunol.2003;21:335-76.
12.Kim DD,Song WC.Membrane complement regulatory proteins.Clin Immunol.2006;118:127-36.
13.Suresh M,Molina H,Salvato MS,Mastellos D,Lambris JD,Sandor M. Complement component 3 is required for optimal expansion of CD8 T cells during a systemic viral infection.J Immunol.2003;170:788-94.
14.Kopf M,Abel B,Gallimore A,Carroll M,Bachmann MF.Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection.Nat Med.2002;8:373-8.
15.Schraufstatter IU,Trieu K,Sikora L,Sriramarao P,DiScipio R.Complement c3a and c5a induce different signal transduction cascades in endothelial cells.J Immunol.2002;169:2102-10.
16.Roth GA,Moser B,Roth-Walter F,Giacona MB,Harja E,Papapanou PN,Schmidt AM,Lalla E.Infection with a periodontal pathogen increases mononuclear cell adhesion to human aortic endothelial cells.Atherosclerosis.2007;190:271-81.
17.Kuroishi T,Tanaka Y,Sakai A,Sugawara Y,Komine K,Sugawara S.Human parotid saliva contains soluble toll-like receptor(TLR) 2 and modulates TLR2-mediated interleukin-8 production by monocytic cells.Mol Immunol.2007;44:1969-76.
18.Lundberg AM,Drexler SK,Monaco C,Williams LM,Sacre SM,Feldmann M,Foxwell BM.Key differences in TLR3/poiy I:C signaling and cytokine induction by human primary cells:a phenomenon absent from murine cell systems.Blood.2007;110:3245-52.
19.Hawlisch H,Belkaid Y,Baelder R,Hildeman D,Gerard C,Kohl J.C5a negatively regulates toll-like receptor 4-induced immune responses.Immunity.2005;22:415-26.
20.Braun MC,Lahey E,Kelsall BL.Selective suppression of IL-12 production by chemoattractants.J Immunol.2000;164:3009-17.
21.Karp CL,Wysocka M,Wahl LM,Ahearn JM,Cuomo PJ,Sherry B,Trinchieri G,Griffin DE.Mechanism of suppression of cell-mediated immunity by measles virus.Science.1996;273:228-31.
2.Hansson GK.Inflammation,atherosclerosis,and coronary artery disease.N Engl J Med.2005;352:1685-95.
3.Libby P.Inflammation in atherosclerosis.Nature.2002;420:868-74.
4.Curtiss LK.Reversing atherosclerosis? NEngl J Med.2009;360:1144-6.
5.Ait-Oufella H,Salomon BL,Potteaux S,Robertson AK,Gourdy P,Zoll J,Merval R,Esposito B,Cohen JL,Fisson S,Flavell RA,Hansson GK,Klatzmann D,Tedgui A,Mallat Z.Natural regulatory T cells control the development of atherosclerosis in mice.Nat Med.2006;12:178-80.
6.Bjorkbacka H,Kunjathoor VV,Moore K J,Koehn S,Ordija CM,Lee MA,Means T,Halmen K,Luster AD,Golenbock DT,Freeman MW.Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways.Nat Med.2004;10:416-21.
7.Yasojima K,Schwab C,McGeer EG,McGeer PL.Complement components,but not complement inhibitors,are upregulated in atherosclerotic plaques.Arterioscler Thromb Vasc Biol.2001;21:1214-9.
8.Oksjoki R,Kovanen PT,Pentikainen MO.Role of complement activation in atherosclerosis.Curr Opin Lipidol.2003;14:477-82.
9.Acosta J,Qin X,Halperin J.Complement and complement regulatory proteins as potential molecular targets for vascular diseases.Curr Pharm Des.2004;10:203-11.
10.McFarland HF,Martin R.Multiple sclerosis:a complicated picture of autoimmunity.Nat Immunol.2007;8:913-9.
11.Seifert PS,Hansson GK.Complement receptors and regulatory proteins in human atherosclerotic lesions.Arteriosclerosis.1989;9:802-11.
12.Bhakdi S,Torzewski M,Klouche M,Hemmes M.Complement and atherogenesis:binding of CRP to degraded,nonoxidized LDL enhances complement activation.Arterioscler Thromb Vasc Biol.1999;19:2348-54.
13.Li SH,Szrnitko PE,Weisel RD,Wang CH,Fedak PW,Li RK,Mickle DA,Verma S.C-reactive protein upregulates complement-inhibitory factors in endothelial cells.Circulation.2004;109:833-6.
14.Mason JC,Ahmed Z,Mankoff R,Lidington EA,Ahmad S,Bhatia V,Kinderlerer A,Randi AM,Haskard DO.Statin-induced expression of decay-accelerating factor protects vascular endothelium against complement-mediated injury.Circ Res.2002;91:696-703.
15.Kinderlerer AR,Ali F,Johns M,Lidington EA,Leung V,Boyle J J,Hamdulay SS,Evans PC,Haskard DO,Mason JC.KLF2-dependent,shear stress-induced expression of CD59:a novel cytoprotective mechanism against complement-mediated injury in the vasculature.J Biol Chem.2008;283:14636-44.
16.Sun X,Funk CD,Deng C;Sahu A,Lambris JD,Song WC.Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.Proc Natl Acad Sci U S A.1999;96:628-33.
17.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
18.Song WL,Lawson JA,Wang M,Zou H,FitzGerald GA.Noninvasive assessment of the role of cyclooxygenases in cardiovascular health:a detailed HPLC/MS/MS method.Methods Enzymol.2007;433:51-72.
19.Custodis F,Baumhakel M,Schlimmer N,List F,Gensch C,Bohm M,Laufs U.Heart rate reduction by ivabradine reduces oxidative stress,improves endothelial function,and prevents atherosclerosis in apolipoprotein E-deficient mice.Circulation.2008;117:2377-87.
20.Mehta JL,Sanada N,Hu CP,Chen J,Dandapat A,Sugawara F,Satoh H,Inoue K,Kawase Y,Jishage K,Suzuki H,Takeya M,Sehnaekenberg L,Beger R,Hermonat PL,Thomas M,Sawamura T.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res.2007;100:1634-42.
21.Wick G,Knoflach M,Xu Q.Autoimmune and inflammatory mechanisms in atherosclerosis.Annu Rev Immunol.2004;22:361-403.
22.Shah PK.Plaque disruption and thrombosis.Potential role of inflammation and infection.Cardiol Clin.1999;17:271-81.
23.Ross R.Atherosclerosis—an inflammatory disease.N Engl J Med.1999;340:115-26.
24.Nilsson J,Hansson GK.Autoimmunity in atherosclerosis:a protective response losing control? J Intern Med.2008;263:464-78.
25.Ridker PM,Silvertown JD.Inflammation,C-reactive protein,and atherothrombosis.J Periodontol.2008;79:1544-51.
26.Robertson AK,Hansson GK.T cells in atherogenesis:for better or for worse?Arterioscler Thromb Vasc Biol.2006;26:2421-32.
27.Taleb S,Tedgui A,Mallat Z.Regulatory T-cell immunity and its relevance to atherosclerosis.J Intern Med.2008;263:489-99.
28.Udi N,Yehuda S.Intravenous immunoglobulin-indications and mechanisms in cardiovascular diseases.Autoimmun Rev.2008;7:445-52.
29.Medzhitov R.,Janeway C,Jr.Innate immunity.N Engl J Med.2000;343:338-44.
30.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
31.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
32.Yasojima K,Schwab C,McGeer EG,McGeer PL.Generation of C-reactive protein and complement components in atherosclerotic plaques.Am J Pathol.2001;158:1039-51.
33.Vlaicu R,Niculescu F,Rus HG,Cristea A.Immunohistochemical localization of the terminal C5b-9 cmplement complex in human aortic fibrous plaque.Atherosclerosis.1985;57:163-77.
34.Torzewski M,Torzewski J,Bowyer DE,Waltenberger J,Fitzsimmons C,Hombach V,Gabbert HE.Immunohistochemical colocalization of the terminal complex of human complement and smooth muscle cell alpha-actin in early atherosclerotic lesions.Arterioscler Thromb Vase Biol.1997;17:2448-52.
35.Torzewsld M,Klouche M,Hock J,Messner M,Dorweiler B,Torzewski J,Gabbert HE,Bhakdi S.Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion.Arterioscler Thromb Vase Biol.1998;18:369-78.
36.Seifert PS,Hugo F,Hansson GK,Bhakdi S.Prelesional complement activation in experimental atherosclerosis.Terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits.Lab Invest.1989;60:747-54.
37.Laine P,Pentikainen MO,Wurzner R,Penttila A,Paavonen T,Meri S,Kovanen PT.Evidence for complement activation in mptured coronary plaques in acute myocardial infarction.Am J Cardiol.2002;90:404-8.
38.Hammerschmidt DE,Greenberg CS,Yamada O,Craddock PR,Jacob HS.Cholesterol and atheroma lipids activate complement and stimulate granulocytes.A possible mechanism for amplification of ischemic injury in atherosclerotic states.J Lab Clin Med.1981;98:68-77.
39.Bhakdi S,Dorweiler B,Kirchmann R,Torzewsld J,Weise E,Tranum-Jensen J,Walev Ⅰ,Wieland E.On the pathogenesis of atherosclerosis:enzymatic transformation of human low density lipoprotein to an atherogenic moiety.J Exp Med.1995;182:1959-71.
40.Bhakdi S,Torzewski M,Paprotka K,Schmitt S,Barsoom H,Suriyaphol P,Han SR,Lackner K J,Husmann M.Possible protective role for C-reactive protein in atherogenesis:complement activation by modified lipoproteins halts before detrimental terminal sequence.Circulation.2004;109:1870-6.
41.Biro A,Thielens NM,Cervenak L,Prohaszka Z,Fnst G,Arlaud GJ.Modified low density lipoproteins differentially bind and activate the C1 complex of complement.Mol Immunol.2007;44:1169-77.
42.Ji H,Ohmura K,Mahmood U,Lee DM,Hofhuis FM,Boaekle SA,Takahashi K,Holers VM,Walport M,Gerard C,Ezekowitz A,Carroll MC,Brenner M,Weissleder R,Verbeek JS,Duehatelle V,Degott C,Benoist C,Mathis D.Arthritis critically dependent on innate immune system players.Immunity.2002;16:157-68.
43.Vlaicu R,Rus HG,Niculescu F,Cristea A.Quantitative determinations of immunoglobulins and complement components in human aortic atherosclerotic wall.Med Interne.1985;23:29-35.
44.Torzewski J,Torzewski M,Bowyer DE,Frohlich M,Koenig W,Waltenberger J,Fitzsimmons C,Hombach V.C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries.Arterioscler Thromb Vasc Biol.1998;18:1386-92.
45.Reynolds GD,Vance RP.C-reactive protein immunohistochemieal localization in normal and atherosclerotic human aortas.Arch Pathol Lab Med.1987;111:265-9.
46.Ji SR,Wu Y,Potempa LA,Liang YH,Zhao J.Effect of modified C-reactive protein on complement activation:a possible complement regulatory role of modified or monomeric C-reactive protein in atherosclerotic lesions.Arterioscler Thromb Vasc Biol.2006;26:935-41.
47.Rus HG,Niculescu F,Constantinescu E,Cristea A,Vlaicu R.Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque.Atherosclerosis.1986;61:35-42.
48.Seifert PS,Kazatchkine MD.Generation of complement anaphylatoxins and C5b-9 by crystalline cholesterol oxidation derivatives depends on hydroxyl group number and position.Mol Immunol.1987;24:1303-8.
49.Saito E,Fujioka T,Kanno H,Hata E,Ueno T,Matsumoto T,Takahashi Y, Tochihara T,Yasugi T.Complement receptors in atherosclerotic Iesions.Artery.1992;19:47-62.
50.Mevorach D,Mascarenhas JO,Gershov D,Elkon KB.Complement-dependent clearance of apoptotic cells by human macrophages.J Exp Med.1998;188:2313-20.
51.Aderem A,Underhill DM.Mechanisms of phagocytosis in macrophages.Annu Rev Immunol.1999;17:593-623.
52.Husemann J,Obstfeld A,Febbraio M,Kodama T,Silverstein SC.CD11b/CD18 mediates production of reactive oxygen species by mouse and human maerophages adherent to matrixes containing oxidized LDL.Arterioscler Thromb Vase Biol.2001;21:1301-5.
53.Helmy KY,Katschke KJ,Jr.,Gorgani NN,Kljavin NM,Elliott JM,Diehl L,Scales S J,Ghilardi N,van Lookeren Campagne M.CRIg:a maerophage complement receptor required for phagocytosis of circulating pathogens.Cell.2006;124:915-27.
54.Lee MY,Kim WJ,Kang YJ,Jung YM,Kang YM,Suk K,Park JE,Choi EM,Choi BK,Kwon BS,Lee WH.Z39Ig is expressed on maerophages and may mediate inflammatory reactions in arthritis and atherosclerosis.J Leukoc Biol.2006;80:922-8.
55.Niculescu F,Rus H.Mechanisms of signal transduction activated by sublytic assembly of terminal complement complexes on nucleated cells,Immunol Res.2001;24:191-9.
56.Seeger W,Suttorp N,Hellwig A,Bhakdi S.Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes.J Immunol.1986;137:1286-93.
57.Torzewski J,Oldroyd R,Lachmarm P,Fitzsimmons C,Proudfoot D,Bowyer D.Complement-induced release of monocyte chemotaetic protein-1 from human smooth muscle cells.A possible initiating event in atherosclerotic lesion formation.Arterioscler Thromb Vasc Biol.1996;16:673-7.
58.Niculescu F,Badea T,Rus H.Sublytic C5b-9 induces proliferation of human aortic smooth muscle cells:role of mitogen activated protein kinase and phosphatidylinositol 3-kinase.Atherosclerosis.1999;142:47-56.
59.Benzaquen LR,Nicholson-Weller A,Halperin JA.Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells.J Exp Med.1994;179:985-92.
60.Kilgore KS,Schmid E,Shanley TP,Flory CM,Maheswari V,Tramontini NL,Cohen H,Ward PA,Friedl HP,Warren JS.Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.Am J Pathol.1997;150:2019-31.
61.Hattori R,Hamilton KK,McEver RP,Sims PJ.Complement proteins C5b-9induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.J IBiol Chem.1989;264:9053-60.
62.Tedesco F,Pause M,Nardon E,Introna M,Mantovani A,Dobrina A.The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.J Exp Med.1997;185:1619-27.
63.Kilgore KS,Shen JP,Miller BF,Ward PA,Warren JS.Enhancement by the complement membrane attack complex of tumor necrosis factor-alpha-induced endothelial cell expression of E-selectin and ICAM-1.J Immunol.1995;155:1434-41.
64.Oksjoki R,Laine P,Helske S,Vehmaan-Kreula P,Mayranpaa MI,Gasque P,Kovanen PT,Pentikainen MO.Receptors for the anaphylatoxins C3a and CSa are expressed in human atherosclerotic coronary plaques.Atheroselerosis.2007;195:90-9.
65.Arend WP,Massoni RJ,Niemann MA,Giclas PC.Absence of induction of IL-1 production in human monocytes by complement fragments.J Immunol.1989;142:173-8.
66.Murakami Y,Imarnichi T,Nagasawa S.Characterization of C3a anaphylatoxin receptor on guinea-pig macrophages.Immunology.1993;79:633-8.
67.Takabayashi T,Vannier E,Clark BD,Margolis NH,Dinarello CA,Burke JF,Gelfand JA.A new biologic role for C3a and C3a desArg:regulation of TNF-alpha and IL-1 beta synthesis.J Immunol.1996;156:3455-60.
68.Takabayashi T,Vannier E,Burke JF,Tompkins RG,Gelfand JA,Clark BD.Both C3a and C3a(desArg) regulate interleukin-6 synthesis in human peripheral blood mononuelear cells.J Infect Dis.1998;177:1622-8.
69.Verdeguer F,Castro C,Kubicek M,Pla D,Vila-Caballer M,Vinue A,Civeira F,Pocovi M,Calvete JJ,Andres V.Complement regulation in murine and human hypercholesterolemia and role in the control of macrophage and smooth muscle cell proliferation.Cardiovasc Res.2007;76:340-50.
70.Nataf S,Davoust N,Ames RS,Barnum SR.Human T cells express the C5a receptor and are chemoattracted to C5a.J Immunol.1999;162:4018-23.
71.Schulman ES,Post TJ,Henson PM,Giclas PC.Differential effects of the complement peptides,C5a and C5a des Arg on human basophil and lung mast cell histamine release.J Clin Invest.1988;81:918-23.
72.Foreman KE,Vaporciyan AA,Bonish BK,Jones ML,Johnson KJ,Glovsky MM,Eddy SM,Ward PA.C5a-induced expression of P-selectin in endothelial cells.J Clin Invest.1994;94:1147-55.
73.Okusawa S,Yancey KB,van der Meer JW,Endres S,Lonnemann G,Hefter K,Frank MM,Burke JF,Dinarello CA,Gelfand JA.C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro.Comparison with secretion of interleukin 1 beta and interleukin 1 alpha.J Exp Med.1988;168:443-8.
74.McCarthy K,Henson PM.Induction of lysosomal enzyme secretion by alveolar maerophages in response to the purified complement fragments C5a and C5a des-arg.J Immunol.1979;123:2511-7.
75.Kastl SP,Speidl WS,Kaun C,Rega G,Assadian A,Weiss TW,Valent P,Hagrnueller GW,Maurer G,Huber K,Wojta J.The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophagcs via NF-kappaB activation.J Thromb Haemost.2006;4:1790-7.
76.Kastl SP,Speidl WS,Kaun C,Katsaros KM,Rega G,Afonyushkin T,Bochkov VN,Valcnt P,Assadian A,Hagmueller GW,Hoeth M,de Martin R,Ma Y,Maurer G,Huber K,Wojta J.In human macrophages the complement component CSa induces the expression of oncostatin M via AP-1 activation.Arterioscler Thromb Vasc Biol.2008;28:498-503.
77.Schmiedt W,Kinseherf R,Deigner HP,Kamencic H,Nauen O,Kilo J,Oelert H,Metz J,Bhakdi S.Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits.Arterioscler Thromb Vasc Biol.1998;18:1790-5.
78.Patel S,Thelander EM,Hernandez M,Montenegro J,Hassing H,Burton C,Mundt S,Hermanowski-Vosatka A,Wright SD,Chao YS,Detmers PA.ApoE(-/-) mice develop atheroselerosis in the absence of complement component C5.Biochem Biophys Res Commun.2001;286:164-70.
79.Buono C,Come CE,Witztum JL,Maguire GF,Colmelly PW,Carroll M,Lichtman AH.Influence of C3 deficiency on atheroselerosis.Circulation.2002;105:3025-31.
80.Persson L,Boren J,Robertson AK,Wallenius V,Hansson GK,Pekna M.Lack of complement factor C3,but not factor B,increases hyperlipidemia and atheroselerosis in apolipoprotein E-/-low-density lipoprotein receptor-/-mice.Arterioscler Thromb Vasc Biol.2004;24:1062-7.
81.Bhatia Vk,Yun S,Leung V,Grimsditch DC,Benson GM,Botto MB,Boyle JJ,Haskard DO.Complement Clq reduces early atherosclerosis in low-density lipoprotein receptor-deficient mice.Am J Pathol.2007;170:416-26.
82.Molines TE,Song WC,Lambris JD.Complement in inflammatory tissue damage and disease.Trends Immunol.2002;23:61-4.
83.Miwa T,Song WC.Membrane complement regulatory proteins:insight from animal studies and relevance to human diseases,Int Immunopharmacol.2001;1:445-59.
84.Liszewski MK,Post TW,Atkinson JP.Membrane cofactor protein(MCP or CD46):newest member of the regulators of complement activation gene cluster. Annu Rev Immunol. 1991 ;9:431 -55.
85. Lublin DM, Atkinson JP. Decay-accelerating factor: biochemistry, molecular biology, and function. Annu Rev Immunol. 1989;7:35-58.
86. Ahearn JM, Fearon DT. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv Immunol. 1989;46:183-219.
87. Spicer AP, Seldin MF, Gendler SJ. Molecular cloning and chromosomal localization of the mouse decay-accelerating factor genes. Duplicated genes encode glycosylphosphatidylinositol-anchored and transmembrane forms. J Immunol 1995;155:3079-91.
88. Song WC, Deng C, Raszmann K, Moore R, Newbold R, McLachlan JA, Negishi M. Mouse decay-accelerating factor: selective and tissue-specific induction by estrogen of the gene encoding the glycosylphosphatidylinositol-anchored form. J Immunol. 1996; 157:4166-72.
89. Powell MB, Marchbank KJ, Rushmere NK, van den Berg CW, Morgan BP. Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59. J Immunol. 1997;158:1692-702.
90. Qian YM, Qin X, Miwa T, Sun X, Halperin JA, Song WC. Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59. J Immunol. 2000; 165:2528-34.
91. Nickells MW, Atkinson JP. Characterization of CR1- and membrane cofactor protein-like proteins of two primates. J Immunol. 1990; 144:4262-8.
92. Johnstone RW, Loveland BE, McKenzie IF. Identification and quantification of complement regulator CD46 on normal human tissues. Immunology. 1993;79:341-7.
93. Miwa T, Nonaka M, Okada N, Wakana S, Shiroishi T, Okada H. Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1. Immunogenetics. 1998;48:363-71.
94. Holers VM, Kinoshita T, Molina H. The evolution of mouse and human complement C3-binding proteins:divergence of form but conservation of function.Immunol Today.1992;13:231-6.
95.Li B,Sallee C,Dehoff M,Foley S,Molina H,Holers VM.Mouse Crry/p65.Characterization of monoclonal antibodies and the tissue distribution of a functional homologue of human MCP and DAF.J Immunol.1993;151:4295-305.
96.Song WC.Membrane complement regulatory proteins in autoimmune and inflammatory tissue injury.Curr Dir Autoimmun.2004;7:181-99.
97.Goodfellow RM,Williams AS,Levin JL,Williams BD,Morgan BP.Soluble complement receptor one(sCR1) inhibits the development and progression of rat collagen-induced arthritis.Clin Exp Immunol.2000;119:210-6.
98.Mizuno M,Nishikawa K,Morgan BP,Matsuo S.Comparison of the suppressive effects of soluble CR1 and C5a receptor antagonist in acute arthritis induced in rats by blocking of CD59.Clin Exp Immunol.2000;119:368-75.
99.Hamaeher J,Sadallah S,Schifferli JA,Villard J,Nicod LP.Soluble complement receptor type 1(CD35) in bronchoalveolar lavage of inflammatory lung diseases.Eur Respir J.1998;11:112-9.
100.Mulligan MS,Warner RL,Rittershaus CW,Thomas LJ,Ryan US,Foreman KE,Crouch LD,Till GO,Ward PA.Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewisx moieties.J Immunol.1999;162:4952-9.
101.Molina H,Kinoshita T,Inoue K,Carel JC,Holers VM.A molecular and immunochemical characterization of mouse CR2.Evidence for a single gene model of mouse complement receptors 1 and 2.J Immunol.1990;145:2974-83.
102.Kinoshita T,Takeda J,Hong K,Kozono H,Sakai H,Inoue K.Monoclonal antibodies to mouse complement receptor type 1(CR1).Their use in a distribution study showing that mouse erythrocytes and platelets are CR1-negative.J Immunol.1988;140:3066-72.
103.Holers VM,Boackle SA.Complement receptor 2 and autoimmunity.Curr Dir Autoimmun.2004;7:33-48.
104.Burge J,Nicholson-Weller A,Austen KF.Isolation of C4-binding protein from guinea pig plasma and demonstration of its function as a control protein of the classical complement pathway C3 convertase.J Immunol.1981;126:232-5.
105.Davitz MA,Low MG,Nussenzweig V.Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C(PIPLC).Selective modification of a complement regulatory protein.J Exp Med.1986;163:1150-61.
106.Kinoshita T,Rosenfeld SI,Nussenzweig V.A high m.w.form of decay-accelerating factor(DAF-2) exhibits size abnormalities in paroxysmal nocturnal hemoglobinuria erythrocytes.J Immunol.1987;138:2994-8.
107.Lublin DM,Lemons RS,Le Beau MM,Holers VM,Tykocinski ML,Medof ME,Atkinson JP.The gene encoding decay-accelerating factor(DAF) is located in the complement-regulatory locus on the long arm of chromosome 1.J Exp Med.1987;165:1731-6.
108.Medof ME,Lublin DM,Holers VM,Ayers DJ,Getty RR,Leykam JF,Atkinson JP,Tykocinski ML.Cloning and characterization of cDNAs encoding the complete sequence of decay-accelerating factor of human complement.Proc Natl Acad Sci USA.1987;84:2007-11.
109.Mizuno M,Donev RM,Harris CL,Morgan BP.CD55 in rat male reproductive tissue:differential expression in testis and expression of a unique truncated isoform on spermatozoa.Mol Immunol.2007;44:1613-22.
110.Yan J,Allendorf DJ,Li B,Yan R,Hansen R,Donev R.The role of membrane complement regulatory proteins in cancer immunotherapy.Adv Exp Med Biol.2008;632:159-74.
111.Pettigrew DM,Williams DT,Kerrigan D,Evans DJ,Lea SM,Bhella D.Structural and functional insights into the interaction of echoviruses and decay-accelerating factor.J Biol Chem.2006;281:5169-77.
112.Weeks C,Moratz C,Zacharia A,Stracener C,Egan R,Peckham R,Moore FD, Jr.,Tsokos GC.Decay-accelerating factor attenuates remote ischemia-reperfusion-initiated organ damage.Clin Immunol.2007;124:311-27.
113.Yamada K,Miwa T,Liu J,Nangaku M,Song WC.Critical protection from renal ischemia reperfusion injury by CD55 and CD59.J Immunol.2004;172:3869-75.
114.Pavlov V,Raedler H,Yuan S,Leisman S,Kwan WH,Lalli PN,Medof ME,Heeger PS.Donor deficiency of decay-accelerating factor accelerates murine T cell-mediated cardiac allograft rejection.J Immunol.2008;181:4580-9.
115.Barros MM,Yamamoto M,Figueiredo MS,Cancado R,Kimura EY,Langhi DM,Jr.,Chiattone CS,Bordin JO.Expression levels of CD47,CD35,CD55,and CD59 on red blood cells and signal-regulatory protein-alpha,beta on monocytes from patients with warm autoimmune hemolytic anemia.Transfusion.2009;49:154-60.
116.Parker C.Eculizumab for paroxysmal nocturnal haemoglobinuria.Lancet.2009;373:759-67.
117.Shichishirna T,Noji H.A new aspect of the molecular pathogenesis of paroxysmal nocturnal hemoglobinuria.Hematology.2002;7:211-27.
118.Sloand EM,Mainwaring L,Keyvanfar K,Chen J,Maciejewski J,Klein HG;Young NS.Transfer of glycosylphosphatidylinositol-anchored proteins to deficient cells after erythrocyte transfusion in paroxysmal nocturnal hemoglobinuria.Blood.2004;104:3782-8.
119.Yin W,Ghebrehiwet B,Weksler B,Peerschke EI.Regulated complement deposition on the surface of human endothelial cells:effect of tobacco smoke and shear stress.Thromb Res.2008;122:221-8.
120.Liu J,Miwa T,Hilliard B,Chen Y,Lambris JD,Wells AD,Song WC.The complement inhibitory protein DAF(CD55) suppresses T cell immunity in vivo.J Exp Med.2005;201:567-77.Epub 2005 Feb 14.
121.Sogabe H,Nangaku M,Ishibashi Y,Wada T,Fujita T,Sun X,Miwa T,Madaio MP,Song WC.Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis,J Immunol. 2001;167:2791-7.
122.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
123.Molina H,Miwa T,Zhou L,Hilliard B,Mastellos D,Maldonado MA,Lambris JD,Song WC.Complement-mediated clearance of erythrocytes:mechanism and delineation of the regulatory roles of Crry and DAF.Decay-accelerating factor.Blood.2002;100:4544-9.
124.Kim DD,Miwa T,Kimttra Y,Schwendener RA,van Lookeren Campagne M,Song WC.Deficiency of DAF and Crry on murine platelets leads to complement-dependent clearance via the macrophage phagocytic receptor CRIg.Blood.2008.
125.Ratnoff WD,Knez JJ,Prince GM,Okada H,Lachmarm PJ,Medof ME.Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59.Clin Exp Immunol.1992;87:415-21.
126.Qin X,Miwa T,Aktas H,Gao M,Lee C,Qian YM,Morton CC,Shahsafaei A,Song WC,Halperin JA.Genomic structure,functional comparison,and tissue distribution of mouse Cd59a and Cd59b.Mamm Genome.2001;12:582-9.
127.Sawada R,Ohashi K,Anaguchi H,Okazaki H,Hattori M,Minato N,Naruto M.Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.DNA Cell Biol.1990;9:213-20.
128.Baalasubramanian S,Harris CL,Donev RM,Mizuno M,Omidvar N,Song WC,Morgan BP.CD59a is the primary regulator of membrane attack complex assembly in the mouse.J Immunol.2004;173:3684-92.
129.Longhi MP,Sivasankar B,Omidvar N,Morgan BP,Gallimore A.Cutting edge:routine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner.J Immunol.2005;175:7098-102.
130.Morgan BP,van den Berg CW,Davies EV,Hallett MB,Horejsi V. Cross-linking of CD59 and of other glycosyl phosphatidylinositol-anchored molecules on nentrophils triggers cell activation via tyrosine kinase.Eur J Immunol.1993;23:2841-50.
131.Murray EW,Robbins SM.Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement.J Biol Chem.1998;273:25279-84.
132.Seifert PS,Roth I,Schmiedt W,Oelert H,Okada N,Okada H,Bhakdi S.CD59(homologous restriction factor 20),a plasma membrane protein that protects against complement C5b-9 attack,in human atherosclerotic lesions.Atherosclerosis.1992;96:135-45.
133.Li W,Tada T,Miwa T,Okada N,Ito J,Okada H,Tateyama H,Eimoto T.mRNA expression of complement components and regulators in rat arterial smooth muscle cells.Microbiol Immunol.1999;43:585-93.
134.Kinderlerer AR,Steinberg R,Johns M,Harten SK,Lidington EA,Haskard DO,Maxwell PH,Mason JC.Statin-induced expression of CD59 on vascular endothelium in hypoxia:a potential mechanism for the anti-inflammatory actions of statins in rheumatoid arthritis.Arthritis Res Ther.2006;8:R130.
135.Thomas CM,Smart EJ.Gender as a regulator of atherosclerosis in routine models.Curr Drug Targets.2007;8:1172-80.
136.Patrono C,FitzGerald GA.Isoprostanes:potential markers of oxidant stress in atherothrombotic disease.Arterioscler Thromb Vasc Biol.1997;17:2309-15.
137.Pratico D,Tangirala RK,Rader DJ,Rokach J,FitzGerald GA.Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice.Nat Med.1998;4:1189-92.
138.Babaev VR,Patel MB,Semenkovich CF,Fazio S,Linton MF.Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice.J Biol Chem.2000;275:26293-9.
1.Anggard EE.The endothelium—the body's largest endocrine gland? J Endocrinol.1990;127:371-5.
2.Schmid D,Munz C.Innate and adaptive immunity through autophagy.Immunity.2007;27:11-21.
3.Medzhitov R.Recognition of microorganisms and activation of the immune response.Nature.2007;449:819-26.
4.Kirk P,Bazan JF.Pathogen recognition:TLRs throw us a curve:Immunity.2005;23:347-50.
5.Means TK,Wang S,Lien E,Yosbimura A,Golenboek DT,Fenton MJ.Human toll-like receptors mediate cellular activation by Mycobaetedum tuberculosis.J Immunol.1999;163:3920-7.
6.Sansonetti PJ.The innate signaling of dangers and the dangers of innate signaling.Nat Immunol.2006;7:1237-42.
7.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
8.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
9.Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol.2004;4:499-511.
10.Kawai T,Akira S.TLR signaling.CellDeath Differ.2006;13:816-25.
11.Zhang X,Kimura Y,Fang C,Zhou L,Sfyroera G,Larnbris JD,Wetsel RA,Miwa T,Song WC.Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo.Blood.2007;110:228-36.
12.Meneghin A,Hogaboam CM.Infectious disease,the innate immune response,and fibrosis.J Clin Invest.2007;117:530-8.
13.Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol.2004;5:987-95.
14.Takeda K,Kaisho T,Akira S.Toll-like receptors.Annu Rev Immunol. 2003;21:335-76.
15.Medzhitov R,Preston-Hurlburt P,Janeway CA,Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature.1997;388:394-7.
16.O'Neill LA.Signal transduction pathways activated by the IL-1receptor/toll-like receptor superfamily.Curr Top Microbiol Immunol.2002;270:47-61.
17.Homung V,Rothenfusser S,Britsch S,Krug A,Jahrsdorfer B,Giese T,Endres S,Hartmann G.Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides.J Immunol.2002;168:4531-7.
18.Kokkinopoulos I,Jordan WJ,Ritter MA.Toll-like receptor mRNA expression patterns in human dendritic cells and monocytes.Mol Immunol.2005;42:957-68.
19.Swantek JL,Tsen MF,Cobb MH,Thomas JA.IL-1 receptor-associated kinase modulates host responsiveness to endotoxin,J Immunol.2000;164:4301-6.
20.Poltorak A,Smirnova I,He X,Liu MY,Van Huffel C,McNally O,Birdwell D,Alejos E,Silva M,Du X,Thompson P,Chan EK,Ledesma J,Roe B,Clifton S,Vogel SN,Beutler B.Genetic and physical mapping of the Lps locus:identification of the toll-4 receptor as a candidate gene in the critical region.Blood Cells Mol Dis.1998;24:340-55.
21.Michelsen KS,Wong MH,Shah PK,Zhang W,Yano J,Doherty TM,Akira S,Rajavashisth TB,Arditi M.Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E.Proc Natl Acad Sci U S A.2004;101:10679-84.
22.Xu XH,Shah PK,Faure E,Equils O,Thomas L,Fishbein MC,Luthringer D,Xu XP,Rajavashisth TB,Yano J,Kaul S,Arditi M.Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL.Circulation.2001;104:3103-8.
23.Ozinsky A,Underhill DM,Fontenot JD,Hajjar AM,Smith KD,Wilson CB,Schroeder L,Aderem A.The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.Proc Natl Acad Sci USA.2000;97:13766-71.
24.Jin MS,Lee JO.Structures of the toll-like receptor family and its ligand complexes.Immunity.2008;29:182-91.
25.Massari P,Hermeke P,Ho Y,Latz E,Golenbock DT,Wetzler LM.Cutting edge:Immune stimulation by neisserial porins is toll-like receptor 2 and MyD88 dependent.J Immunol.2002;168:1533-7.
26.Lee HK,Iwasald A.Innate control of adaptive immunity:dendritic cells and beyond.Semin Immunol.2007;19:48-55.
27.Alexopoulou L,Thomas V,Schnare M,Lobet Y,Anguita J,Schoen RT,Medzhitov R,Fikrig E,Flavell RA.Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1-and TLR2-deficient mice.Nat Med.2002;8:878-84.
28.Wooten RM,Ma Y,Yoder RA,Brown JP,Weis JH,Zachary JF,Kirschning CJ,Weis JJ.Toll-like receptor 2 is required for innate,but not acquired,host defense to Borrelia burgdorferi.J Immunol.2002;168:348-55.
29.Barton GM,Medzhitov R.Toll signaling:RIPping off the TNF pathway.Nat Immunol.2004;5:472-4.
30.Matsumoto M,Kikkawa S,Kohase M,Miyake K,Seya T.Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling.Biochem Biophys Res Commun.2002;293:1364-9.
31.Celia M,Salio M,Sakakibara Y,Langen H,Julkunen I,Lanzavecchia A.Maturation,activation,and protection of dendritic cells induced by double-stranded RNA.J Exp Med.1999;189:821-9.
32.Khvalevsky E,Rivkin L,Rachmilewitz J,Galun E,Giladi H.TLR3 signaling in a hepatoma cell line is skewed towards apoptosis,J Cell Biochem.2007;100:1301-12.
33.Schnare M,Holt AC,Takeda K,Akira S,Medzhitov R.Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88.Curr Biol.2000;10:1139-42.
34.Latz E,Schoenemeyer A,Visintin A,Fitzgerald KA,Monks BG,Knetter CF,Lien E,Nilsen NJ,Espevik T,Golenbock DT.TLR9 signals after translocating from the ER to CpG DNA in the lysosome.Nat Immunol.2004;5:190-8.
35.Stacey KJ,Sweet MJ,Hume DA.Macrophages ingest and are activated by bacterial DNA.J Immunol.1996;157:2116-22.
36.Sparwasser T,Koch ES,Vabulas RM,Heeg K,Lipford GB,Ellwart JW,Wagner H.Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells.Eur J Immunol.1998;28:2045-54.
37.Zuiderweg ER,Nettesheim DG,Mollison KW,Carter GW.Tertiary structure of human complement component C5a in solution from nuclear magnetic resonance data.Biochemistry.1989;28:172-85.
38.Kohl J,Lubbers B,Klos A,Bautsch W,Casaretto M.Evaluation of the C-terminal C5a effeetor site with short synthetic C5a analog peptides.Eur J Immunol.1993;23:646-52.
39.Foreman KE,Vaporciyan AA,Bonish BK,Jones ML,Johnson KJ,Glovsky MM,Eddy SM,Ward PA.C5a-induced expression of P-selectin in endothelial cells.J Clin Invest.1994;94:1147-55.
40.Mollnes TE,Brekke OL,Fung M,Fure H,Christiansen D,Bergseth G;Videm V,Lappegard KT,Kohl J,Lambris JD.Essential role of the C5a receptor in E coli-indueed oxidative burst and phagocytosis revealed by a novel lepirudin-based human whole blood model of inflammation.Blood.2002;100:1869-77.
41.Gutzmer R,Kother B,Zwirner J,Dijkstra D,Purwar R,Wittmann M,Werfel T.Human plasmacytoid dendritic cells express receptors for anaphylatoxins C3a and C5a and are chemoattracted to C3a and C5a.J Invest Dermatol.2006;126:2422-9.
42.Melendez AJ,Ibrahim FB.Antisense knockdown of sphingosine kinase 1 in human macrophages inhibits C5a receptor-dependent signal transduction,Ca2+ signals,enzyme release,cytokine production,and chemotaxis.J Immunol.2004;173:1596-603.
43.Kastl SP,Speidl WS,Kaun C,Rega G,Assadian A,Weiss TW,Valent P,Hagmueller GW,Maurer G,Huber K,Wojta J.The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-kappaB activation.J Thromb Haemost.2006;4:1790-7.
44.Malmsten M,Schmidtchen A.Antimicrobial C3a—biology,biophysics,and evolution.Adv Exp Med Biol.2007;598:141-58.
45.DiScipio RG,Schraufstatter IU.The role of the complement anaphylatoxins in the recruitment of eosinophils.Int Immunopharmacol.2007;7:1909-23.
46.Haas PJ,van Stdjp J.Anaphylatoxins:their role in bacterial infection and inflammation,Immunol Res.2007;37:161-75.
47.Fujita T,Matsushita M,Endo Y.The lectin-complement pathway—its role in innate immunity and evolution,Immunol Rev.2004;198:185-202.
48.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
49.Yamada K,Miwa T,Liu J,Nangaku M,Song WC.Critical protection from renal isehemia reperfusion injury by CD55 and CD59.J Immunol.2004;172:3869-75.
50.Triantafilou M,Triantafilou K.Lipopolysaccharide recognition:CD14,TLRs and the LPS-activation cluster.Trends Immunol.2002;23:301-4.
51.Coyne CB,Bergelson JM.Virus-induced Ab1 and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions.Cell.2006;124:119-31.
52.O'Brien DP,Israel DA,Krishna U,Romero-Gallo J,Nedrud J,Medof ME,Lin F,Redline R,Lublin DM,Nowicki BJ,Franco AT,Ogden S,Williams AD,Polk DB,Peek RM,Jr.The role of decay-accelerating factor as a receptor for Helicobacter pylori and a mediator of gastric inflammation.J Biol Chem.2006;281:13317-23.
1.Hansson GK.Inflammation,atherosclerosis,and coronary artery disease.N Engl J Med.2005;352:1685-95.
2.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
3.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
4.Song WC,Sarrias MR,Lambris JD.Complement and innate immunity.Immunopharmacology.2000;49:187-98.
5.Mollnes TE,Song WC,Lambris JD.Complement in inflammatory tissue damage and disease.Trends Immunol.2002;23:61-4.
6.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
7.Lublin DM,Atkinson JP.Decay-accelerating factor:biochemistry,molecular biology,and function.Annu Rev Immunol.1989;7:35-58.
8.Nicholson-Weller A,Burge J,Fearon DT,Weller PF,Austen KF.Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system.J Immunol.1982;129:184-9.
9.Okada N,Harada R,Fujita T,Okada H.A novel membrane glycoprotein capable of inhibiting membrane attack by homologous complement.Int Immunol.1989;1:205-8.
10.Rollins SA,Sims PJ.The complement-inhibitory activity of CD59 resides in its capacity to block incorporation of C9 into membrane C5b-9.J Immunol.1990;144:3478-83.
11.Oksjoki R,Kovanen PT,Pentikainen MO.Role of complement activation in atherosclerosis.Curr Opin Lipidol.2003;14:477-82.
12.Vlaicu R,Rus HG,Niculescu F,Cristea A.Immunoglobulins and complement components in human aortic atherosclerotic intima.Atherosclerosis.1985;55:35-50.
13.Seifert PS,Hansson GK.Complement receptors and regulatory proteins in human atherosclerotic lesions.Arteriosclerosis.1989;9:802-11.
14.Vlaicu R,Rus HG,Niculescu F,Cristea A.Quantitative determinations of immunoglobulins and complement components in human aortic atherosclerotic wall.Med Interne.1985;23:29-35.
15.Niculescu F,Rus H.The role of complement activation in atherosclerosis.Immunol Res.2004;30:73-80.
16.Bhatia VK,Yun S,Leung V,Grimsditch DC,Benson GM,Botto MB,Boyle JJ,Haskard DO.Complement C1q reduces early atherosclerosis in low-density lipoprotein receptor-deficient mice.Am J Pathol.2007;170:416-26.
17.Schmiedt W,Kinscherf R,Deigner HP,Kamencic H,Nauen O,Kilo J,Oelert H,Metz J,Bhakdi S.Complement C6 deficiency protects against diet-induced atherosclerosis in rabbits.Arterioscler Thromb Vasc Biol.1998;18:1790-5.
18.Patel S,Thelander EM,Hernandez M,Montenegro J,Hassing H,Burton C,Mundt S,Hermanowski-Vosatka A,Wright SD,Chao YS,Detmers PA.ApoE(-/-) mice develop atherosclerosis in the absence of complement component C5.Biochem Biophys Res Commun.2001;286:164-70.
19.Buono C,Coffee CE,Witztum JL,Maguire GF,Connelly PW,Carroll M,Lichtman AH.Influence of C3 deficiency on atherosclerosis.Circulation.2002;105:3025-31.
20.Persson L,Boren J,Robertson AK,Wallenius V,Hansson GK,Pekna M.Lack of complement factor C3,but not factor B,increases hyperlipidemia and atherosclerosis in apolipoprotein E-/- low-density lipoprotein receptor-/- mice.Arterioscler Thromb Vasc Biol.2004;24:1062-7.
21.Seifert PS,Hansson GK.Decay-accelerating factor is expressed on vascular smooth muscle cells in human atherosclerotic lesions.J Clin Invest.1989;84:597-604.
22.Niculescu F,Pus HG,Vlaicu R.Decay-accelerating factor regulates complement-mediated damage in the human atherosclerotic wall.Immunol Lett.1990;26:17-23.
23.Niculescu F,Rus HG,Vlaicu R.Immunohistochemical localization of C5b-9, S-protein,C3d and apolipoprotein B in human arterial tissues with atherosclerosis.Atherosclerosis.1987;65:1-11.
24.Mackness B,Hunt R,Durrington PN,Mackness MI.Increased immunolocalization of paraoxonase,clusterin,and apolipoprotein A-I in the human artery wall with the progression of atherosclerosis.Arterioscler Thromb Vasc Biol.1997;17:1233-8.
25.Li SH,Szmitko PE,Weisel RD,Wang CH,Fedak PW,Li RK,Mickle DA,Verma S.C-reactive protein upregulates complement-inhibitory factors in endothelial cells.Circulation.2004;109:833-6.
26.Bhakdi S,Torzewski M,Klouche M,Hemmes M.Complement and atherogenesis:binding of CRP to degraded,nonoxidized LDL enhances complement activation.Arterioscler Thromb Vase Biol.1999;19:2348-54.
27.Mason JC,Ahmed Z,Mankoff R,Lidington EA,Ahmad S,Bhatia V,Kinderlerer A,Randi AM,Haskard DO.Statin-induced expression of decay-accelerating factor protects vascular endothelium against complement-mediated injury.Circ Res.2002;91:696-703.
28.Sun X,Funk CD,Deng C,Sahu A,Lambris JD,Song WC.Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting.Proc Natl Acad Sci U S A.1999;96:628-33.
29.Miwa T,Zhou L,Hilliard B,Molina H,Song WC.Crry,but not CD59 and DAF,is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack.Blood.2002;99:3707-16.
30.Tangirala RK,Rubin EM,Palinski W.Quantitation of atherosclerosis in murine models:correlation between lesions in the aortic origin and in the entire aorta,and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.J Lipid Res.1995;36:2320-8.
31.Nicoletti A,Kaveri S,Caligiuri G,Bariety J,Hansson GK.Immunoglobulin treatment reduces atherosclerosis in apo E knockout mice.J Clin lnvest. 1998;102:910-8.
32.Wang M,Lee E,Song W,Ricciotti E,Rader DJ,Lawson JA,Pure E,FitzGerald GA.Microsomal prostaglandin E synthase-1 deletion suppresses oxidative stress and angiotensin Ⅱ-induced abdominal aortic aneurysm formation.Circulation.2008;117:1302-9.
33.Song WL,Lawson JA,Wang M,Zou H,FitzGerald GA.Noninvasive assessment of the role of cyclooxygenases in cardiovascular health:a detailed HPLC/MS/MS method.Methods Enzymol.2007;433:51-72.
34.Niculescu F,Rus H.Complement activation and atherosclerosis.Mol Immunol.1999;36:949-55.
35.Yun S,Leung VW,Botto M,Boyle JJ,Haskard DO.Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice Lacking the Membrane-Bound Complement Regulator CD59.Arterioscler Thromb Vasc Biol.2008.
36.Liu J,Miwa T,Hilliard B,Chen Y,Lambris JD,Wells AD,Song WC.The complement inhibitory protein DAF(CD55) suppresses T cell immunity in vivo.J Exp Med.2005;201:567-77.Epub 2005 Feb 14.
37.Sogabe H,Nangaku M,Ishibashi Y,Wada T,Fujita T,Sun X,Miwa T,Madaio MP,Song WC.Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.J Immunol.2001;167:2791-7.
38.Miwa T,Maldonado MA,Zhou L,Sun X,Luo HY,Cai D,Werth VP,Madaio MP,Eisenberg RA,Song WC.Deletion of decay-accelerating factor(CD55)exacerbates autoimmune disease development in MRL/lpr mice.Am J Pathol.2002;161:1077-86.
39.Molina H,Miwa T,Zhou L,Hilliard B,Mastellos D,Maldonado MA,Lambris JD,Song WC.Complement-mediated clearance of erythrocytes:mechanism and delineation of the regulatory roles of Crry and DAF.Decay-accelerating factor.Blood.2002;100:4544-9.
40.Kim DD,Miwa T,Kimura Y,Schwendener RA,van Lookeren Campagne M, Song WC.Deficiency of DAF and Crry on murine platelets leads to complement-dependent clearance via the macrophage phagocytic receptor CRIg.Blood.2008.
41.Babaev VR,Patel MB,Semenkovich CF,Fazio S,Linton MF.Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice.J Biol Chem.2000;275:26293-9.
42.Patrono C,FitzGerald GA.Isoprostanes:potential markers of oxidant stress in atherothrombotic disease.Arterioscler Thromb Vasc Biol.1997;17:2309-15.
43.Pratico D,Iuliano L,Mauriello A,Spagnoli L,Lawson JA,Rokach J,Maclouf J,Violi F,FitzGerald GA.Localization of distinct F2-isoprostanes in human atherosclerotic lesions.J Clin Invest.1997;100:2028-34.
44.Rokach J,Khanapure SP,Hwang SW,Adiyaman M,Lawson JA,FitzGerald GA.Nomenclature of isoprostanes:a proposal.Prostaglandins.1997;54:853-73.
45.Rudic RD,Brinster D,Cheng Y,Fries S,Song WL,Austin S,Coffman TM,FitzGerald GA.COX-2-derived prostacyclin modulates vascular remodeling.Circ Res.2005;96:1240-7.
46.Benzaquen LR,Nicholson-Weller A,Halperin JA.Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells.J Exp Med.1994;179:985-92.
47.Halperin JA,Taratuska A,Nicholson-Weller A.Terminal complement complex C5b-9 stimulates mitogenesis in 3T3 cells.J Clin Invest.1993;91:1974-8.
48.Morgan BP,van den Berg CW,Davies EV,Hallett MB,Horejsi V.Cross-linking of CD59 and of other glycosyl phosphatidylinositol-anchored molecules on neutrophils triggers cell activation via tyrosine kinase.Eur J Immunol.1993;23:2841-50.
49.Murray EW,Robbins SM.Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement.J Biol Chem.1998;273:25279-84.
50.Longhi MP,Sivasankar B,Omidvar N,Morgan BP,GaUimore A.Cutting edge:routine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner.J Immunol.2005;175:7098-102.
51.Longhi MP,Harris CL,Morgan BP,Gallimore A.Holding T cells in check—a new role for complement regulators? Trends Immunol.2006;27:102-8.
1.Akira S,Uematsu S,Takeuchi O.Pathogen recognition and innate immunity.Cell.2006;124:783-801.
2.Carroll MC.The complement system in regulation of adaptive immunity.Nat Immunol.2004;5:981-6.
3.Iwasaki A,Medzhitov R.Toll-like receptor control of the adaptive immune responses.Nat Immunol.2004;5:987-95.
4.Medzhitov R,Preston-Hurlburt P,Janeway CA,Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature.1997;388:394-7.
5.Walport MJ.Complement.First of two parts.N Engl J Med.2001;344:1058-66.
6.Walport MJ.Complement.Second of two parts.N Engl J Med.2001;344:1140-4.
7.Akira S,Takeda K.Toll-like receptor signalling.Nat Rev Immunol.2004;4:499-511.
8.Fujita T,Matsushita M,Endo Y.The lectin-complement pathway—its role in innate immunity and evolution.Immunol Rev.2004;198:185-202.
9.Zhang X,Kimura Y,Fang C,Zhou L,Sfyroera G,Lambris JD,Wetsel RA,Miwa T,Song WC.Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo.Blood.2007;110:228-36.
10.Lee H,Zahra D,Vogelzang A,Newton R,Thatcher J,Quan A,So T,Zwirner J,Koentgen F,Padkjaer SB,Mackay F,Whitfeld PL,Mackay CR.Human C5aR knock-in mice facilitate the production and assessment of anti-inflammatory monoclonal antibodies.Nat Biotechnol.2006;24:1279-84.
11.Takeda K,Kaisho T,Akira S.Toll-like receptors.Annu Rev Immunol.2003;21:335-76.
12.Kim DD,Song WC.Membrane complement regulatory proteins.Clin Immunol.2006;118:127-36.
13.Suresh M,Molina H,Salvato MS,Mastellos D,Lambris JD,Sandor M. Complement component 3 is required for optimal expansion of CD8 T cells during a systemic viral infection.J Immunol.2003;170:788-94.
14.Kopf M,Abel B,Gallimore A,Carroll M,Bachmann MF.Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection.Nat Med.2002;8:373-8.
15.Schraufstatter IU,Trieu K,Sikora L,Sriramarao P,DiScipio R.Complement c3a and c5a induce different signal transduction cascades in endothelial cells.J Immunol.2002;169:2102-10.
16.Roth GA,Moser B,Roth-Walter F,Giacona MB,Harja E,Papapanou PN,Schmidt AM,Lalla E.Infection with a periodontal pathogen increases mononuclear cell adhesion to human aortic endothelial cells.Atherosclerosis.2007;190:271-81.
17.Kuroishi T,Tanaka Y,Sakai A,Sugawara Y,Komine K,Sugawara S.Human parotid saliva contains soluble toll-like receptor(TLR) 2 and modulates TLR2-mediated interleukin-8 production by monocytic cells.Mol Immunol.2007;44:1969-76.
18.Lundberg AM,Drexler SK,Monaco C,Williams LM,Sacre SM,Feldmann M,Foxwell BM.Key differences in TLR3/poiy I:C signaling and cytokine induction by human primary cells:a phenomenon absent from murine cell systems.Blood.2007;110:3245-52.
19.Hawlisch H,Belkaid Y,Baelder R,Hildeman D,Gerard C,Kohl J.C5a negatively regulates toll-like receptor 4-induced immune responses.Immunity.2005;22:415-26.
20.Braun MC,Lahey E,Kelsall BL.Selective suppression of IL-12 production by chemoattractants.J Immunol.2000;164:3009-17.
21.Karp CL,Wysocka M,Wahl LM,Ahearn JM,Cuomo PJ,Sherry B,Trinchieri G,Griffin DE.Mechanism of suppression of cell-mediated immunity by measles virus.Science.1996;273:228-31.