FasL在缺血性卒中后的炎症作用及其病理机制探讨
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摘要
研究背景：
脑卒中因其发病率高、致残率高、死亡率高，严重危害人类健康和生命安全，然而目前临床上仍缺乏合适的神经保护剂减轻卒中脑损伤，因此寻找和发现缺血性卒中新的治疗靶点是当下脑血管病研究的热点。卒中后炎症反应是造成缺血性脑损伤的重要原因之一，在急性脑梗死时炎症反应首先表现为星形胶质细胞和小胶质细胞的大量激活，促进IL-1β，TNF-α等前炎症因子和MIP-1α，MCP-1等趋化因子水平增高，诱导外周淋巴细胞和中性粒细胞向中枢浸润，引起炎症级联放大效应而加剧脑损伤程度。另外卒中后免疫炎症反应与患者病情严重程度和预后亦直接相关，因此针对炎症反应作用机制的关键环节加以干预，有可能成为治疗脑梗死新的有效途径。
FasL是死亡受体Fas的配体，FasL与Fas结合后能诱导表达Fas的细胞凋亡。既往认为FasL是维持免疫豁免状态的重要分子，某些肿瘤细胞可通过高表达FasL逃避免疫攻击，因此有研究试图通过大量表达FasL来诱导异体移植术中的器官免疫耐受。但结果恰恰相反，过表达FasL不但没能抑制移植术后的免疫排斥反应，反而引起更严重的炎症反应和更快的的排斥反应。近年来研究逐渐发现除了传统的细胞凋亡，FasL还可直接诱导免疫炎症反应，如促进前炎症因子和趋化因子IL-6，MCP-1和IL-8的分泌；诱导中性粒细胞和/或T淋巴细胞聚集等；同时FasL本身还是T细胞激活增殖的一种协同/共刺激分子。业已发现FasL参与肺、心脏等多种器官的炎症反应，而FasL基因突变能显著改善上述器官的炎症状态，起到器官保护作用。因此FasL也被认为是免疫炎症反应的重要中介。
FasL亦参与中枢神经系统（central nervous system，CNS）免疫调节作用，在脑外伤、应激、缺血缺氧等情况下均发现患者病侧脑组织内FasL表达增高。然而，就FasL与缺血性卒中后免疫炎症反应的研究，无论是动物还是临床方面，都知之甚少，因为既往大多数FasL与脑缺血的研究均集中在FasL诱导细胞凋亡方面。既然免疫炎症反应是脑卒中发生发展的重要病因之一，而FasL又是诱导免疫炎症反应的重要分子，那么我们提出以下三个问题：1）FasL能否诱导卒中后免疫炎症反应加剧缺血性脑损伤？2）FasL诱导的免疫炎症反应是直接作用还是与其凋亡作用相关？3）FasL诱导卒中后炎症反应的病理机制以及哪些细胞内信号途径参与其中？该研究内容目前国内外少有相关报道，值得深入研究。
第一部分FasL诱导卒中后炎症反应加重缺血性脑损伤
目的：研究FasL在小鼠缺血性脑损伤中诱导免疫炎症的作用。
方法与材料：选取3月龄的FasL基因突变（FasL—/—，gld）和野生型（C57BL/6J，B6）雄性小鼠（体重约22-24克），应用线栓法建立一侧大脑中动脉梗死模型（MCAO），缺血为时间2h，再灌注时间点选取6h、24h、72h。在上述3个时间点进行神经功能缺陷评分（NSS），TTC染色观察脑梗死体积、干湿重法评估脑水肿程度。荧光定量PCR检测缺血侧皮层脑组织前炎症因子IL-1β，TNF-α，IFN-γ；趋化因子MCP-1，MIP-1α，MIP-2以及抗炎因子IL-4，IL-10的mRNA表达水平，ELISA法检测缺血皮层脑组织和外周血血清中前炎症因子IL-1β和TNF-α蛋白表达量，免疫组织化学法观察星形胶质细胞（GFAP+）、小胶质细胞（Iba1+）、中性粒细胞（MPO+）、淋巴细胞（CD3+）在B6和gld鼠缺血侧脑组织的表达量及其形态学改变。流式细胞术检测两组小鼠T淋巴细胞亚群CD4+，CD8+，Th1/Th2在缺血侧皮层脑组织和外周血中的变化。
结果：两种小鼠在缺血再灌注6h到72h时间窗均出现神经功能障碍及脑梗死。B6小鼠在缺血后上述时间窗神经功能评分低，脑梗死体积及脑水肿面积大，而FasL突变的gld小鼠能显著改善其神经功能障碍症状，脑水肿和脑梗死体积较B6小鼠均减小。在缺血再灌注24h时间点，两组小鼠的炎症因子mRNA表达均增高，但FasL突变的gld小鼠前炎症因子IL-1β、TNF-α、IFN-γ和趋化因子MCP-1、MIP-1α、MIP-2mRNA表达水平较B6小鼠降低。相应的，gld小鼠抗炎因子IL-10的mRNA水平较B6小鼠增高，而抗炎因子IL-4的mRNA水平在两组小鼠缺血皮层脑组织未见明显差异。在缺血24h时间点，gld小鼠CNS内激活的GFAP+星形胶质细胞，Iba1+小胶质细胞及外周浸润的MPO+中性粒细胞较B6小鼠均减少。虽然CD3+T淋巴细胞的总数在两组小鼠中并无显著差异，但T淋巴细胞亚群的比例发生变化。在缺血24h时间点两组小鼠均检测到向脑组织中浸润CD3+CD4+和CD3+CD8+T细胞，但FasL突变的gld小鼠在缺血侧脑组织和外周血中的CD8+T淋巴细胞浸润减少，并且CD4+T细胞Th1/Th2比例降低并向Th2偏移。
结论：FasL诱导卒中后免疫炎症反应，FasL突变可通过抑制炎症反应保护缺血性脑损伤。1）FasL突变降低炎症因子和趋化因子的mRNA与蛋白表达水平；2）FasL突变抑制星形胶质细胞和小胶质细胞的激活及外周中性粒细胞的浸润；3）FasL突变下调CD8+T细胞以及CD4+T细胞亚群Th1/Th2的比例。
第二部分FasL诱导的炎症作用不依赖其前凋亡作用
目的：证明FasL能直接诱导卒中后炎症反应，而非细胞凋亡的继发作用。
方法与材料：我们在MCAO术前24h给B6小鼠腹腔注射脂多糖（LPS i.p,0.5mg/kg），模拟和gld小鼠类似的缺血脑保护作用。比较MCAO术后24h三组小鼠（B6，gld，LPS预处理+B6组）的脑梗死体积及中性粒细胞浸润情况。AnnexinV染色检测细胞凋亡，双重免疫荧光染色分别观察gld和B6小鼠MCAO术后炎症细胞（星形胶质细胞、小胶质细胞、中性粒细胞、T细胞）和神经元的凋亡情况。分别给B6，gld小鼠侧脑室注射小剂量LPS（1ug）制备脑炎模型，荧光定量PCR检测其炎症因子和趋化因子的改变。免疫荧光观察在LPS诱导的脑炎模型中（不是MCAO）B6和gld小鼠细胞凋亡情况；免疫组化观察两组小鼠星形胶质细胞、小胶质细胞、中性粒细胞的表达。
结果：1)缺血后炎症反应的程度和梗死灶的体积密切相关，较小的梗死体积同时也伴随减轻的炎症反应。因此gld小鼠卒中后炎症反应减弱有可能是继发于脑梗死体积减小的结果而不是FasL突变直接抑制炎症的原因。为了阐明这个问题，我们增加了一组小鼠模型，即在B6小鼠MCAO术前24h给予腹腔注射LPS（i.p0.5mg/kg)，通过LPS预处理减小缺血后脑梗死体积，模拟和gld小鼠类似的缺血脑保护作用，再比较三组小鼠（B6，gld和LPS预处理+B6）在MCAO后24h的脑梗死体积以及中性粒细胞浸润情况。我们的结果表明LPS预处理+B6组小鼠减少缺血梗死体积的程度和gld小鼠类似，但是脑组织中性粒细胞浸润数量却比gld小鼠显著增多。对脑梗死体积与中性粒细胞浸润的拟合曲线研究发现，B6MCAO组与LPS预处理+B6MCAO两组小鼠的拟合曲线斜率基本一致，而gld MCAO组小鼠斜率降低，说明FasL突变抑制中性粒细胞浸润数量远超过脑梗死体积减少伴随的中性粒细胞减少。
2)由于卒中后缺血脑损伤程度与细胞凋亡密切相关，因此FasL突变的gld小鼠炎症反应减轻有可能是继发于细胞凋亡减少的结果，包括炎症细胞和神经元细胞凋亡减少而非FasL的直接抑炎作用。为了阐明这个问题，我们采用Annexin V染色代表细胞凋亡，NeuN染色代表神经元。免疫荧光双染显示Iba1，GFAP，MPO，CD3与Annexin V染色基本不重合，说明不论是B6还是gld小鼠MCAO后炎症细胞（小胶质细胞、星形胶质细胞、中性粒细胞和T淋巴细胞）并不发生显著凋亡。而Annexin V与NeuN部分重合说明缺血后神经元发生细胞凋亡，且FasL突变的gld鼠神经元凋亡较B6鼠减少。
3)上述结果表明B6小鼠缺血后神经元凋亡显著增多而gld小鼠神经元凋亡较少。为了排除FasL突变的gld小鼠炎症反应减轻是继发于神经元凋亡减少的这种可能性，我们采用了另一种脑组织炎症模型（不做MCAO），即侧脑室注射（ICV）小剂量LPS（1ug）。文献报道和我们的实验结果都证实ICV LPS诱导的脑炎模型能引起脑组织大量免疫炎症反应，却并没有显著的神经元细胞凋亡。在上述LPS脑炎模型中gld小鼠的炎症因子mRNA和炎症细胞浸润较B6鼠显著下降，这同第一部分MCAO模型的结果一致。
结论：FasL能直接诱导卒中后炎症反应，而非细胞凋亡的继发作用。1）FasL突变直接抑制外周中性粒细胞浸润，而非脑梗死体积减小的继发结果。2）FasL突变抑制卒中后炎症反应，此作用不依赖于炎症细胞凋亡。3）FasL突变抑制LPS诱导的脑组织免疫炎症反应，此作用与神经元凋亡无关。
第三部分FasL诱导卒中后炎症反应的病理机制
目的：探讨FasL诱导卒中后炎症反应的病理机制和细胞内信号通路，寻找卒中治疗的潜在靶点。
方法与材料：高速离心（100,000g，60min）法提取皮层脑组织胞质蛋白检测可溶性FasL（sFasL）；皮层脑组织全蛋白提取检测MAPK信号通路。免疫印迹Western blot法检测sFasL表达及JNK/ERK/P38蛋白及其磷酸化水平。免疫荧光双染检测缺血后Fas蛋白在星形胶质细胞上的表达情况。
结果：1）在缺血再灌注24h时间点，B6小鼠sFasL蛋白表达较假手术组上升至451.67%，而FasL突变的gld小鼠sFasL表达较B6小鼠下降了62%，说明sFasL可能参与FasL的炎症作用。2）MAPK信号通路的激活与缺血后炎症反应密切相关。我们研究发现磷酸化的JNK与ERK通路在缺血卒中的B6小鼠显著增加，而FasL突变仅能够抑制JNK通路的激活，并不影响磷酸化的ERK和p38。3）卒中后FasL诱导免疫炎症反应与JNK通路的激活相关，而JNK通路亦是FasL反向信号传导活化T淋巴细胞的重要信号通路。脑缺血时，T淋巴细胞脑组织浸润后首先遭遇到星形胶质细胞，星形胶质细胞是脑组织中最重要的抗原递呈细胞，也是表达Fas的主要细胞。因此我们接下来检测了脑缺血后Fas在星形胶质细胞上的表达，发现GFAP+星型胶质细表面Fas表达明显增多，提示脑缺血后星形胶质细胞高表达Fas可激活FasL反向信号促进T淋巴细胞的增殖活化，诱导缺血性卒中的免疫炎症反应。
结论：1）可溶性因子sFasL和磷酸化的SAPK/JNK蛋白在缺血侧皮层表达下降，提示FasL的促炎作用可能与sFasL的释放和JNK信号途径的激活相关。2）脑缺血后星形胶质细胞高表达Fas可能通过激活FasL的反向信号促进T淋巴细胞的增殖活化。3）临床上抑制FasL的表达可发挥抗炎作用改善卒中患者的预后，作为潜在的脑保护治疗的药物靶点。
综上所述，本研究的主要创新之处在于：
1、FasL诱导缺血性卒中后炎症反应，而FasL突变能抑制炎症反应保护缺血性脑损伤。
2、FasL直接诱导卒中后免疫炎症反应，包括上调炎症因子mRNA表达，激活中枢胶质细胞，趋化外周中性粒细胞浸润以及调节T淋巴细胞亚群等；且FasL诱导炎症反应的作用不依赖其前凋亡作用。
3、FasL诱导卒中后免疫炎症反应的分子机制可能与上调sFasL的表达以及激活磷酸化的MAPK/JNK通路相关。
Background:
Stroke is one of the main causes of morbidity and mortality worldwide. Untilnow there is no effective neuroprotectants against stroke damage. Identifying novelneuroprotectants that can protect the injured brain is becoming the most importantevent in the study of cerebrovascular diseases. Inflammation has been shown to be animportant player in the progression of ischemic brain damage. Cerebral inflammationis characterized by the activation of astrocytes and microglia, the accumulation ofinflammatory cells and subsequent production of inflammatory mediators, includingIL-1β, TNF-α, monocyte inflammatory protein-1α (MIP-1α), monocytechemoattractant protein-1(MCP-1) and other deleterious substances. The extent ofinflammatory response has been demonstrated to correlate with the severity of braininjury and long-term outcomes in stroke patients. Understanding the mechanism forpost-stroke inflammation will therefore provide potential targets for stroke management and functional recovery.
FasL has been shown to be an apoptosis-inducing factor that triggers cell death insusceptible cells expressing the Fas receptor. In previous studies, FasL had beenthought to be a key component in the establishment of immune privilege. FasLexpression on cancer cells may render them refractory to immune attack and wasso-called “tumor counterattack”. This observation led to the hope that the artificialexpression of FasL on transplanted tissues would act as a local immunosuppressant.Several investigators have sought to mimic this natural immune privilege usingexpression of FasL as a strategy to protect engrafted tissue. However, FasLoverexpression in transplants induced accelerated destruction and rejection of thetransplanted organs, rather than conferring immune privilege. The unexpectedobservations suggested that, besides its pro-apoptotic property, FasL could induceimmuno-inflammatory response. Recently, FasL was suggested to be capable ofactivating signal transduction pathways that induce inflammatory responses. It hasbeen shown that FasL induces the production of pro-inflammatory cytokines andchemokines including IL-6, MCP-1and IL-8in various cells types, and recruitneutrophil and/or T lymphocyte accumulation. Meanwhile FasL is also regarded as anaccessory or costimulatory molecule for T cells activation. In fact, FasL has beeninvolved in many inflammatory response such as lung, heart and et al. FasL mutationcould significantly improve the function of these organs. Therefore, FasL plays ancritical role in the immuno-inflammatory system.
In central nervous system (CNS), FasL have been observed to be elevated incompromised brains in a variety of neurological disorders including brain trauma,ischemia stress and et al. The inflammatory role of FasL in ischemic stroke, however,is not fully understood. Most previous studies have associated the effect of FasL inischemic brain injury solely with its well-known pro-apoptotic property. Accepting that inflammatory reaction is one of the most important mechanisms after stroke, andFasL is considered a major modulator in the immune and inflammatory response, wemight ask the following questions:1) Does FasL exert an inflammatory effect in theonset and development of experimental stroke?2) Could FasL be able to induceinflammatory response directly by itself and independent of cell apoptosis?3）Howdose FasL promote inflammation during these processes, and which intracellularnon-apoptotic signaling pathways are triggered? Both of these important questions arecurrently unresolved and are desired further investigations.
PART I FasL induces brain inflammatory in experimental strokeand exacerbates brain damage
Aims: To determine the possible role of FasL in inducing inflammatory responseafter cerebral ischemia in mice.
Materials and Methods: Twelve to fourteen-week-old male FasL mutant (gld)mice (22–24g) and their wild type control C57BL/6J mice (B6) were chosen asexperimental animals. Focal cerebral ischemia was induced by transient middlecerebral artery occlusion (MCAO) operation for2h. The reperfusion time windowswere6h,24h and72h. The sensorimotor dysfunction was assessed by neurologicalseverity score (NSS), the infarct volume was measured by TTC staining and brainedema was obtained by water content formula. Real-time PCR (qPCR) was used todetect mRNA expressions of inflammatory cytokines (IL-1β, TNF-α, IFN-γ),anti-inflammatory cytokines (IL-4, IL-10) and chemokines (MCP-1, MIP-1α, MIP-2).Protein levels of IL-1β and TNF-α were detected by enzyme-linked immunosorbentassay (ELISA) in both ischemic cortex and blood serum. Immunostaining were usedto observe the expression of GFAP+, Iba1+, MPO+and CD3+in ischemic hemisphere.The T lymphocytes subpopulations CD4+, CD8+, Th1/Th2were assayed by flow cytometry (FACS) in the ischemic brain and peripheral blood.
Results:1) Both B6and gld mice showed neurological deficits and infarctvolumes from6h to72h time windows after ischemic stroke. Severe neurologicaldeficits and larger volumes were observed in B6mice. FasL mutation profoundlyimproved neurological performance and reduced brain edema and infarct volumes.2)The mRNA expressions of pro-infammatory cytokines (IL-1β, TNF-α, IFN-γ) andchemokines (MCP-1, MIP-1α, MIP-2) were attenuated in ischemic cortex of gld miceat24h after ischemia reperfusion. In contrast, the level of anti-inflammatory factorIL-10was up-regulated in gld mice, while the level of IL-4showed no significantchange between the two genotypes. At24h after cerebral ischemia, FasL mutationinhibited the activation of residential glial cells (Iba1+microglia and GFAP+astrocytes) and attenuated the recruitment of peripheral ifnammatory cells (MPO+neutrophils) into CNS. Although the total numbers of T cells infiltration are similar inboth gld and B6mice, the distributions of T cell subsets were different. FACSanalysis showed the infiltration of both CD3+CD4+and CD3+CD8+T cells into thebrain at24h after MCAO. Mutation of FasL in gld mice resulted in reducedinfiltration of CD8+T cells and turned the Th1/Th2balance towards Th2both in thebrain and peripheral blood after cerebral ischemia.
Conclusion: FasL exerts an inflammatory effect after stroke, FasL mutationcould inhibit inflammation and protect ischemia injury.1) FasL mutation reducespro-inflammatory cytokines and chemokines in ischemic brain.2) FasL mutationattenuates the cellular inflammatory reaction (astrocytes and microglia activation andneutrophils infiltration) associated with cerebral ischemia.3) FasL mutationmodulates T lymphocyte subsets (decreases CD8+T cell and CD4+Th1/Th2ratio)after cerebral ischemia.
Part II FasL contributes to inflammatory responseindependent of cell apoptosis
Aims: To prove that FasL could directly induce inflammation after cerebralischemia without significant cell apoptosis in FasL mutate gld mice.
Materials and Methods: We used intraperitoneal administration ofLipopolysaccharide (LPS i.p,0.5mg/kg)24h before MCAO in B6mice in order toprotect ischemia injury that similar to gld mice. The infarct volume and neutrophilinfiltration of the three groups mice (B6, gld, pretreatment of B6mice with LPS)were compared at24h after MCAO. Annexin V staining were used as a probe todetect apoptosis. Double immunofluorescence was used for identifying the apoptoticinflammatory cells (astrocytes, microglia, neutrophils, T cells) and apoptotic neurons.Then we used intracerebraventricular (ICV) LPS injection in a dose of1ug to inducebrain inflammation in B6and gld mice. Real-time PCR was used to detect mRNAexpressions of inflammatory cytokines and chemokines. Immunofluorescence wasused for apoptotic cells detection and immunohistochemical staining was taken forobservation of morphological changes and expressions of astrocytes, microglia andneutrophils in LPS-induced brain inflammation in B6and gld mice (without MCAO).
Results:1) As the intensity of the inflammatory reaction is related to the size ofischemic lesion, and small infarcts should have less inflammation than large infarcts.It is possible that the attenuation in brain inflammation in gld mice could be aconsequence of the reduced infarct size. To address this, we took an alternativestrategy. Pretreatment of B6mice with LPS (0.5mg/kg)24h before MCAO could leadto a reduced infarct volume that was similar in size to that seen in gld mice, but B6mice pretreatment of LPS had more neutrophil infiltration compared to gld mice. Todetermine whether the reduction in neutrophil infiltraiton in gld mice wascommensurate with the reduction in infarct size, a linear regression analysis was performed between the number of neutrophils at each rostrocaudal level and thecorresponding infarct area. In B6mice pretreatment of LPS, the slope of theregression line overlapped with that of untreated B6mice, indicating that thereduction in the number of neutrophils was proportional to the reduction in infarctsize. In contrast, the slope of the regression line in gld mice was lower than that of B6mice, indicating that the reduction in neutrophils was greater than anticipated fromthe smaller size of the infarct volume.
2) As the process of cerebral ischemia also associated with cell apoptosis, it ispossible that the attenuated inflammatory reaction in gld mice was a consequence ofdecreased cell apoptosis, especially neurons. To address this issue, we used AnnexinV as a probe for detecting early apoptosis. The lacking colocalization of Iba1, GFAP,MPO, CD3with Annexin V staining indicated that there was little apoptosis ofmicroglia, astrocytes, neutrophils or CD3+T cells in both the B6and gld brains afterMCAO. We further used neuronal nuclear antigen (NeuN) as a probe for detectingsurvival neurons, and the gld mice showed less neuron apoptosis than B6mice at24hafter MCAO.
3) Ischemic insult obviously increased neuron apoptosis in B6mice while gldmutation could partially abrogate this effect. In order to exclude the possibility thatless inflammatory response observed in gld mice after MCAO was simply due toreduced apoptotic loss of neurons, we took another inflammation model of LPSinjection (ICV,1ug) without MCAO to show that FasL had a direct role oninflammation. Both literature and our results confirmed that ICV LPS of small dose(1ug) could induce brain inflammation without triggering obvious neuron apoptosis.In contrast to cerebral ischemia, the molecular and cellular finammatory changesinduced by ICV LPS were also attenuated in gld mice.
Conclusion: FasL could directly exert an immuno-inflammatory effect in CNS after ischemic stroke, which is independent of cell apoptosis.1) In FasL mutate gld miceneutrohil infiltration was suppressed more than expected on the basis of the reductionin infarct size.2) FasL mutation attenuated cerebral inflammation independent ofinflammatory cells apoptosis in CNS after ischemic stroke.3) FasL mutationinhibited LPS induced inflammation in brain without being affected by apoptoticneuron death.
Part III The pathologic mechanisms of FasL mutation inpost-stroke inflammatory response
Aims: To investigate the intracellular molecular mechanisms and signalpathways of FasL in modulating inflammatory response associated with cerebralischemia and find the potential target for stroke treatment.
Materials and Methods: To detect sFasL, cytosolic protein fractions from braincortex were prepared using ultracentrifuge at100,000g for60min. Total proteins ofcortex were also extracted to detect phospho-SAPK/JNK, phospho-p38,phospho-p44/42. The protein expression of sFasL and activation of MAPK signalpathways (JNK, ERK and p38) after MCAO were detected by western blot. Doubleimmunofluorescence was used to examine the Fas expression in astrocytes aftercerebral ischemia.
Results:1) In the ischemic cortex of B6mice, the level of sFasL increased by451.67%over the sham-operated brain. The expression of sFasL decreased by62.7%in the gld cortex as compared to B6mice. These results suggested that gld mice hadless sFasL than B6mice that might contribute to its less inflammatory function afterMCAO.2) The activation of MAPK signal pathways (JNK, ERK and p38) has beenshown to contribute to cerebral inflammation in ischemic injury. Our study revealed that phosphorylation of JNK and ERK increased significantly in ischemic cortex ofB6mice. FasL mutation inhibited the phosphorylation of JNK, but did not affect thephosphorylation of p38or ERK.3) Our results suggested that JNK signaling might beinvolved in FasL induced inflammatory reaction after stroke, and JNK pathway wasalso reported to be very important in inducing FasL reverse signaling of T cellsactivation and proliferation. At the beginning of ischemia, T cells infiltration intobrain might encounter the activated astrocytes first, and astrocytes might be the mostsignificant antigen-presenting cell (APC) cells in CNS that express Fas protein. So wenext detected the Fas expression in astrocytes after ischemia, and the results showedthat B6mice had significantly increased Fas level in GFAP+astrocytes compared toB6mice. Our study revealed that the high level of Fas in GFAP+astrocytes mightactivate the FasL reverse signaling and contribute to T cells migration andproliferation, thus exacerbating the immuno-inflammatory response after ischemiastroke.
Conclusion:1) FasL plays an infammatory role in cerebral ischemia. Themechanisms might relate to sFasL and the c-Jun N-terminal kinase (JNK) signalingpathway.2) The high expression of Fas in GFAP+astrocytes might activate the FasLreverse signaling and contribute to T cells migration and proliferation after cerebralischemia3) Neutralization of FasL may be a novel therapeutic strategy to suppresspost-stroke infammation and improve the long-term outcomes of stroke.
In summary, the novel findings of this study are as follows:
1. FasL is an important player in ischemic brain injury after stroke due to its potencyto provoke inflammation.
2. FasL could contribute to post-ischemia injury through activating residential CNS inflammatory cells, recruiting peripheral inflammatory cells, and modulating Tcell subpopulations.
3. sFasL and JNK signaling might contribute to FasL induced inflammatoryfunction.
脑卒中因其发病率高、致残率高、死亡率高，严重危害人类健康和生命安全，然而目前临床上仍缺乏合适的神经保护剂减轻卒中脑损伤，因此寻找和发现缺血性卒中新的治疗靶点是当下脑血管病研究的热点。卒中后炎症反应是造成缺血性脑损伤的重要原因之一，在急性脑梗死时炎症反应首先表现为星形胶质细胞和小胶质细胞的大量激活，促进IL-1β，TNF-α等前炎症因子和MIP-1α，MCP-1等趋化因子水平增高，诱导外周淋巴细胞和中性粒细胞向中枢浸润，引起炎症级联放大效应而加剧脑损伤程度。另外卒中后免疫炎症反应与患者病情严重程度和预后亦直接相关，因此针对炎症反应作用机制的关键环节加以干预，有可能成为治疗脑梗死新的有效途径。
FasL是死亡受体Fas的配体，FasL与Fas结合后能诱导表达Fas的细胞凋亡。既往认为FasL是维持免疫豁免状态的重要分子，某些肿瘤细胞可通过高表达FasL逃避免疫攻击，因此有研究试图通过大量表达FasL来诱导异体移植术中的器官免疫耐受。但结果恰恰相反，过表达FasL不但没能抑制移植术后的免疫排斥反应，反而引起更严重的炎症反应和更快的的排斥反应。近年来研究逐渐发现除了传统的细胞凋亡，FasL还可直接诱导免疫炎症反应，如促进前炎症因子和趋化因子IL-6，MCP-1和IL-8的分泌；诱导中性粒细胞和/或T淋巴细胞聚集等；同时FasL本身还是T细胞激活增殖的一种协同/共刺激分子。业已发现FasL参与肺、心脏等多种器官的炎症反应，而FasL基因突变能显著改善上述器官的炎症状态，起到器官保护作用。因此FasL也被认为是免疫炎症反应的重要中介。
FasL亦参与中枢神经系统（central nervous system，CNS）免疫调节作用，在脑外伤、应激、缺血缺氧等情况下均发现患者病侧脑组织内FasL表达增高。然而，就FasL与缺血性卒中后免疫炎症反应的研究，无论是动物还是临床方面，都知之甚少，因为既往大多数FasL与脑缺血的研究均集中在FasL诱导细胞凋亡方面。既然免疫炎症反应是脑卒中发生发展的重要病因之一，而FasL又是诱导免疫炎症反应的重要分子，那么我们提出以下三个问题：1）FasL能否诱导卒中后免疫炎症反应加剧缺血性脑损伤？2）FasL诱导的免疫炎症反应是直接作用还是与其凋亡作用相关？3）FasL诱导卒中后炎症反应的病理机制以及哪些细胞内信号途径参与其中？该研究内容目前国内外少有相关报道，值得深入研究。
第一部分FasL诱导卒中后炎症反应加重缺血性脑损伤
目的：研究FasL在小鼠缺血性脑损伤中诱导免疫炎症的作用。
方法与材料：选取3月龄的FasL基因突变（FasL—/—，gld）和野生型（C57BL/6J，B6）雄性小鼠（体重约22-24克），应用线栓法建立一侧大脑中动脉梗死模型（MCAO），缺血为时间2h，再灌注时间点选取6h、24h、72h。在上述3个时间点进行神经功能缺陷评分（NSS），TTC染色观察脑梗死体积、干湿重法评估脑水肿程度。荧光定量PCR检测缺血侧皮层脑组织前炎症因子IL-1β，TNF-α，IFN-γ；趋化因子MCP-1，MIP-1α，MIP-2以及抗炎因子IL-4，IL-10的mRNA表达水平，ELISA法检测缺血皮层脑组织和外周血血清中前炎症因子IL-1β和TNF-α蛋白表达量，免疫组织化学法观察星形胶质细胞（GFAP+）、小胶质细胞（Iba1+）、中性粒细胞（MPO+）、淋巴细胞（CD3+）在B6和gld鼠缺血侧脑组织的表达量及其形态学改变。流式细胞术检测两组小鼠T淋巴细胞亚群CD4+，CD8+，Th1/Th2在缺血侧皮层脑组织和外周血中的变化。
结果：两种小鼠在缺血再灌注6h到72h时间窗均出现神经功能障碍及脑梗死。B6小鼠在缺血后上述时间窗神经功能评分低，脑梗死体积及脑水肿面积大，而FasL突变的gld小鼠能显著改善其神经功能障碍症状，脑水肿和脑梗死体积较B6小鼠均减小。在缺血再灌注24h时间点，两组小鼠的炎症因子mRNA表达均增高，但FasL突变的gld小鼠前炎症因子IL-1β、TNF-α、IFN-γ和趋化因子MCP-1、MIP-1α、MIP-2mRNA表达水平较B6小鼠降低。相应的，gld小鼠抗炎因子IL-10的mRNA水平较B6小鼠增高，而抗炎因子IL-4的mRNA水平在两组小鼠缺血皮层脑组织未见明显差异。在缺血24h时间点，gld小鼠CNS内激活的GFAP+星形胶质细胞，Iba1+小胶质细胞及外周浸润的MPO+中性粒细胞较B6小鼠均减少。虽然CD3+T淋巴细胞的总数在两组小鼠中并无显著差异，但T淋巴细胞亚群的比例发生变化。在缺血24h时间点两组小鼠均检测到向脑组织中浸润CD3+CD4+和CD3+CD8+T细胞，但FasL突变的gld小鼠在缺血侧脑组织和外周血中的CD8+T淋巴细胞浸润减少，并且CD4+T细胞Th1/Th2比例降低并向Th2偏移。
结论：FasL诱导卒中后免疫炎症反应，FasL突变可通过抑制炎症反应保护缺血性脑损伤。1）FasL突变降低炎症因子和趋化因子的mRNA与蛋白表达水平；2）FasL突变抑制星形胶质细胞和小胶质细胞的激活及外周中性粒细胞的浸润；3）FasL突变下调CD8+T细胞以及CD4+T细胞亚群Th1/Th2的比例。
第二部分FasL诱导的炎症作用不依赖其前凋亡作用
目的：证明FasL能直接诱导卒中后炎症反应，而非细胞凋亡的继发作用。
方法与材料：我们在MCAO术前24h给B6小鼠腹腔注射脂多糖（LPS i.p,0.5mg/kg），模拟和gld小鼠类似的缺血脑保护作用。比较MCAO术后24h三组小鼠（B6，gld，LPS预处理+B6组）的脑梗死体积及中性粒细胞浸润情况。AnnexinV染色检测细胞凋亡，双重免疫荧光染色分别观察gld和B6小鼠MCAO术后炎症细胞（星形胶质细胞、小胶质细胞、中性粒细胞、T细胞）和神经元的凋亡情况。分别给B6，gld小鼠侧脑室注射小剂量LPS（1ug）制备脑炎模型，荧光定量PCR检测其炎症因子和趋化因子的改变。免疫荧光观察在LPS诱导的脑炎模型中（不是MCAO）B6和gld小鼠细胞凋亡情况；免疫组化观察两组小鼠星形胶质细胞、小胶质细胞、中性粒细胞的表达。
结果：1)缺血后炎症反应的程度和梗死灶的体积密切相关，较小的梗死体积同时也伴随减轻的炎症反应。因此gld小鼠卒中后炎症反应减弱有可能是继发于脑梗死体积减小的结果而不是FasL突变直接抑制炎症的原因。为了阐明这个问题，我们增加了一组小鼠模型，即在B6小鼠MCAO术前24h给予腹腔注射LPS（i.p0.5mg/kg)，通过LPS预处理减小缺血后脑梗死体积，模拟和gld小鼠类似的缺血脑保护作用，再比较三组小鼠（B6，gld和LPS预处理+B6）在MCAO后24h的脑梗死体积以及中性粒细胞浸润情况。我们的结果表明LPS预处理+B6组小鼠减少缺血梗死体积的程度和gld小鼠类似，但是脑组织中性粒细胞浸润数量却比gld小鼠显著增多。对脑梗死体积与中性粒细胞浸润的拟合曲线研究发现，B6MCAO组与LPS预处理+B6MCAO两组小鼠的拟合曲线斜率基本一致，而gld MCAO组小鼠斜率降低，说明FasL突变抑制中性粒细胞浸润数量远超过脑梗死体积减少伴随的中性粒细胞减少。
2)由于卒中后缺血脑损伤程度与细胞凋亡密切相关，因此FasL突变的gld小鼠炎症反应减轻有可能是继发于细胞凋亡减少的结果，包括炎症细胞和神经元细胞凋亡减少而非FasL的直接抑炎作用。为了阐明这个问题，我们采用Annexin V染色代表细胞凋亡，NeuN染色代表神经元。免疫荧光双染显示Iba1，GFAP，MPO，CD3与Annexin V染色基本不重合，说明不论是B6还是gld小鼠MCAO后炎症细胞（小胶质细胞、星形胶质细胞、中性粒细胞和T淋巴细胞）并不发生显著凋亡。而Annexin V与NeuN部分重合说明缺血后神经元发生细胞凋亡，且FasL突变的gld鼠神经元凋亡较B6鼠减少。
3)上述结果表明B6小鼠缺血后神经元凋亡显著增多而gld小鼠神经元凋亡较少。为了排除FasL突变的gld小鼠炎症反应减轻是继发于神经元凋亡减少的这种可能性，我们采用了另一种脑组织炎症模型（不做MCAO），即侧脑室注射（ICV）小剂量LPS（1ug）。文献报道和我们的实验结果都证实ICV LPS诱导的脑炎模型能引起脑组织大量免疫炎症反应，却并没有显著的神经元细胞凋亡。在上述LPS脑炎模型中gld小鼠的炎症因子mRNA和炎症细胞浸润较B6鼠显著下降，这同第一部分MCAO模型的结果一致。
结论：FasL能直接诱导卒中后炎症反应，而非细胞凋亡的继发作用。1）FasL突变直接抑制外周中性粒细胞浸润，而非脑梗死体积减小的继发结果。2）FasL突变抑制卒中后炎症反应，此作用不依赖于炎症细胞凋亡。3）FasL突变抑制LPS诱导的脑组织免疫炎症反应，此作用与神经元凋亡无关。
第三部分FasL诱导卒中后炎症反应的病理机制
目的：探讨FasL诱导卒中后炎症反应的病理机制和细胞内信号通路，寻找卒中治疗的潜在靶点。
方法与材料：高速离心（100,000g，60min）法提取皮层脑组织胞质蛋白检测可溶性FasL（sFasL）；皮层脑组织全蛋白提取检测MAPK信号通路。免疫印迹Western blot法检测sFasL表达及JNK/ERK/P38蛋白及其磷酸化水平。免疫荧光双染检测缺血后Fas蛋白在星形胶质细胞上的表达情况。
结果：1）在缺血再灌注24h时间点，B6小鼠sFasL蛋白表达较假手术组上升至451.67%，而FasL突变的gld小鼠sFasL表达较B6小鼠下降了62%，说明sFasL可能参与FasL的炎症作用。2）MAPK信号通路的激活与缺血后炎症反应密切相关。我们研究发现磷酸化的JNK与ERK通路在缺血卒中的B6小鼠显著增加，而FasL突变仅能够抑制JNK通路的激活，并不影响磷酸化的ERK和p38。3）卒中后FasL诱导免疫炎症反应与JNK通路的激活相关，而JNK通路亦是FasL反向信号传导活化T淋巴细胞的重要信号通路。脑缺血时，T淋巴细胞脑组织浸润后首先遭遇到星形胶质细胞，星形胶质细胞是脑组织中最重要的抗原递呈细胞，也是表达Fas的主要细胞。因此我们接下来检测了脑缺血后Fas在星形胶质细胞上的表达，发现GFAP+星型胶质细表面Fas表达明显增多，提示脑缺血后星形胶质细胞高表达Fas可激活FasL反向信号促进T淋巴细胞的增殖活化，诱导缺血性卒中的免疫炎症反应。
结论：1）可溶性因子sFasL和磷酸化的SAPK/JNK蛋白在缺血侧皮层表达下降，提示FasL的促炎作用可能与sFasL的释放和JNK信号途径的激活相关。2）脑缺血后星形胶质细胞高表达Fas可能通过激活FasL的反向信号促进T淋巴细胞的增殖活化。3）临床上抑制FasL的表达可发挥抗炎作用改善卒中患者的预后，作为潜在的脑保护治疗的药物靶点。
综上所述，本研究的主要创新之处在于：
1、FasL诱导缺血性卒中后炎症反应，而FasL突变能抑制炎症反应保护缺血性脑损伤。
2、FasL直接诱导卒中后免疫炎症反应，包括上调炎症因子mRNA表达，激活中枢胶质细胞，趋化外周中性粒细胞浸润以及调节T淋巴细胞亚群等；且FasL诱导炎症反应的作用不依赖其前凋亡作用。
3、FasL诱导卒中后免疫炎症反应的分子机制可能与上调sFasL的表达以及激活磷酸化的MAPK/JNK通路相关。
Background:
Stroke is one of the main causes of morbidity and mortality worldwide. Untilnow there is no effective neuroprotectants against stroke damage. Identifying novelneuroprotectants that can protect the injured brain is becoming the most importantevent in the study of cerebrovascular diseases. Inflammation has been shown to be animportant player in the progression of ischemic brain damage. Cerebral inflammationis characterized by the activation of astrocytes and microglia, the accumulation ofinflammatory cells and subsequent production of inflammatory mediators, includingIL-1β, TNF-α, monocyte inflammatory protein-1α (MIP-1α), monocytechemoattractant protein-1(MCP-1) and other deleterious substances. The extent ofinflammatory response has been demonstrated to correlate with the severity of braininjury and long-term outcomes in stroke patients. Understanding the mechanism forpost-stroke inflammation will therefore provide potential targets for stroke management and functional recovery.
FasL has been shown to be an apoptosis-inducing factor that triggers cell death insusceptible cells expressing the Fas receptor. In previous studies, FasL had beenthought to be a key component in the establishment of immune privilege. FasLexpression on cancer cells may render them refractory to immune attack and wasso-called “tumor counterattack”. This observation led to the hope that the artificialexpression of FasL on transplanted tissues would act as a local immunosuppressant.Several investigators have sought to mimic this natural immune privilege usingexpression of FasL as a strategy to protect engrafted tissue. However, FasLoverexpression in transplants induced accelerated destruction and rejection of thetransplanted organs, rather than conferring immune privilege. The unexpectedobservations suggested that, besides its pro-apoptotic property, FasL could induceimmuno-inflammatory response. Recently, FasL was suggested to be capable ofactivating signal transduction pathways that induce inflammatory responses. It hasbeen shown that FasL induces the production of pro-inflammatory cytokines andchemokines including IL-6, MCP-1and IL-8in various cells types, and recruitneutrophil and/or T lymphocyte accumulation. Meanwhile FasL is also regarded as anaccessory or costimulatory molecule for T cells activation. In fact, FasL has beeninvolved in many inflammatory response such as lung, heart and et al. FasL mutationcould significantly improve the function of these organs. Therefore, FasL plays ancritical role in the immuno-inflammatory system.
In central nervous system (CNS), FasL have been observed to be elevated incompromised brains in a variety of neurological disorders including brain trauma,ischemia stress and et al. The inflammatory role of FasL in ischemic stroke, however,is not fully understood. Most previous studies have associated the effect of FasL inischemic brain injury solely with its well-known pro-apoptotic property. Accepting that inflammatory reaction is one of the most important mechanisms after stroke, andFasL is considered a major modulator in the immune and inflammatory response, wemight ask the following questions:1) Does FasL exert an inflammatory effect in theonset and development of experimental stroke?2) Could FasL be able to induceinflammatory response directly by itself and independent of cell apoptosis?3）Howdose FasL promote inflammation during these processes, and which intracellularnon-apoptotic signaling pathways are triggered? Both of these important questions arecurrently unresolved and are desired further investigations.
PART I FasL induces brain inflammatory in experimental strokeand exacerbates brain damage
Aims: To determine the possible role of FasL in inducing inflammatory responseafter cerebral ischemia in mice.
Materials and Methods: Twelve to fourteen-week-old male FasL mutant (gld)mice (22–24g) and their wild type control C57BL/6J mice (B6) were chosen asexperimental animals. Focal cerebral ischemia was induced by transient middlecerebral artery occlusion (MCAO) operation for2h. The reperfusion time windowswere6h,24h and72h. The sensorimotor dysfunction was assessed by neurologicalseverity score (NSS), the infarct volume was measured by TTC staining and brainedema was obtained by water content formula. Real-time PCR (qPCR) was used todetect mRNA expressions of inflammatory cytokines (IL-1β, TNF-α, IFN-γ),anti-inflammatory cytokines (IL-4, IL-10) and chemokines (MCP-1, MIP-1α, MIP-2).Protein levels of IL-1β and TNF-α were detected by enzyme-linked immunosorbentassay (ELISA) in both ischemic cortex and blood serum. Immunostaining were usedto observe the expression of GFAP+, Iba1+, MPO+and CD3+in ischemic hemisphere.The T lymphocytes subpopulations CD4+, CD8+, Th1/Th2were assayed by flow cytometry (FACS) in the ischemic brain and peripheral blood.
Results:1) Both B6and gld mice showed neurological deficits and infarctvolumes from6h to72h time windows after ischemic stroke. Severe neurologicaldeficits and larger volumes were observed in B6mice. FasL mutation profoundlyimproved neurological performance and reduced brain edema and infarct volumes.2)The mRNA expressions of pro-infammatory cytokines (IL-1β, TNF-α, IFN-γ) andchemokines (MCP-1, MIP-1α, MIP-2) were attenuated in ischemic cortex of gld miceat24h after ischemia reperfusion. In contrast, the level of anti-inflammatory factorIL-10was up-regulated in gld mice, while the level of IL-4showed no significantchange between the two genotypes. At24h after cerebral ischemia, FasL mutationinhibited the activation of residential glial cells (Iba1+microglia and GFAP+astrocytes) and attenuated the recruitment of peripheral ifnammatory cells (MPO+neutrophils) into CNS. Although the total numbers of T cells infiltration are similar inboth gld and B6mice, the distributions of T cell subsets were different. FACSanalysis showed the infiltration of both CD3+CD4+and CD3+CD8+T cells into thebrain at24h after MCAO. Mutation of FasL in gld mice resulted in reducedinfiltration of CD8+T cells and turned the Th1/Th2balance towards Th2both in thebrain and peripheral blood after cerebral ischemia.
Conclusion: FasL exerts an inflammatory effect after stroke, FasL mutationcould inhibit inflammation and protect ischemia injury.1) FasL mutation reducespro-inflammatory cytokines and chemokines in ischemic brain.2) FasL mutationattenuates the cellular inflammatory reaction (astrocytes and microglia activation andneutrophils infiltration) associated with cerebral ischemia.3) FasL mutationmodulates T lymphocyte subsets (decreases CD8+T cell and CD4+Th1/Th2ratio)after cerebral ischemia.
Part II FasL contributes to inflammatory responseindependent of cell apoptosis
Aims: To prove that FasL could directly induce inflammation after cerebralischemia without significant cell apoptosis in FasL mutate gld mice.
Materials and Methods: We used intraperitoneal administration ofLipopolysaccharide (LPS i.p,0.5mg/kg)24h before MCAO in B6mice in order toprotect ischemia injury that similar to gld mice. The infarct volume and neutrophilinfiltration of the three groups mice (B6, gld, pretreatment of B6mice with LPS)were compared at24h after MCAO. Annexin V staining were used as a probe todetect apoptosis. Double immunofluorescence was used for identifying the apoptoticinflammatory cells (astrocytes, microglia, neutrophils, T cells) and apoptotic neurons.Then we used intracerebraventricular (ICV) LPS injection in a dose of1ug to inducebrain inflammation in B6and gld mice. Real-time PCR was used to detect mRNAexpressions of inflammatory cytokines and chemokines. Immunofluorescence wasused for apoptotic cells detection and immunohistochemical staining was taken forobservation of morphological changes and expressions of astrocytes, microglia andneutrophils in LPS-induced brain inflammation in B6and gld mice (without MCAO).
Results:1) As the intensity of the inflammatory reaction is related to the size ofischemic lesion, and small infarcts should have less inflammation than large infarcts.It is possible that the attenuation in brain inflammation in gld mice could be aconsequence of the reduced infarct size. To address this, we took an alternativestrategy. Pretreatment of B6mice with LPS (0.5mg/kg)24h before MCAO could leadto a reduced infarct volume that was similar in size to that seen in gld mice, but B6mice pretreatment of LPS had more neutrophil infiltration compared to gld mice. Todetermine whether the reduction in neutrophil infiltraiton in gld mice wascommensurate with the reduction in infarct size, a linear regression analysis was performed between the number of neutrophils at each rostrocaudal level and thecorresponding infarct area. In B6mice pretreatment of LPS, the slope of theregression line overlapped with that of untreated B6mice, indicating that thereduction in the number of neutrophils was proportional to the reduction in infarctsize. In contrast, the slope of the regression line in gld mice was lower than that of B6mice, indicating that the reduction in neutrophils was greater than anticipated fromthe smaller size of the infarct volume.
2) As the process of cerebral ischemia also associated with cell apoptosis, it ispossible that the attenuated inflammatory reaction in gld mice was a consequence ofdecreased cell apoptosis, especially neurons. To address this issue, we used AnnexinV as a probe for detecting early apoptosis. The lacking colocalization of Iba1, GFAP,MPO, CD3with Annexin V staining indicated that there was little apoptosis ofmicroglia, astrocytes, neutrophils or CD3+T cells in both the B6and gld brains afterMCAO. We further used neuronal nuclear antigen (NeuN) as a probe for detectingsurvival neurons, and the gld mice showed less neuron apoptosis than B6mice at24hafter MCAO.
3) Ischemic insult obviously increased neuron apoptosis in B6mice while gldmutation could partially abrogate this effect. In order to exclude the possibility thatless inflammatory response observed in gld mice after MCAO was simply due toreduced apoptotic loss of neurons, we took another inflammation model of LPSinjection (ICV,1ug) without MCAO to show that FasL had a direct role oninflammation. Both literature and our results confirmed that ICV LPS of small dose(1ug) could induce brain inflammation without triggering obvious neuron apoptosis.In contrast to cerebral ischemia, the molecular and cellular finammatory changesinduced by ICV LPS were also attenuated in gld mice.
Conclusion: FasL could directly exert an immuno-inflammatory effect in CNS after ischemic stroke, which is independent of cell apoptosis.1) In FasL mutate gld miceneutrohil infiltration was suppressed more than expected on the basis of the reductionin infarct size.2) FasL mutation attenuated cerebral inflammation independent ofinflammatory cells apoptosis in CNS after ischemic stroke.3) FasL mutationinhibited LPS induced inflammation in brain without being affected by apoptoticneuron death.
Part III The pathologic mechanisms of FasL mutation inpost-stroke inflammatory response
Aims: To investigate the intracellular molecular mechanisms and signalpathways of FasL in modulating inflammatory response associated with cerebralischemia and find the potential target for stroke treatment.
Materials and Methods: To detect sFasL, cytosolic protein fractions from braincortex were prepared using ultracentrifuge at100,000g for60min. Total proteins ofcortex were also extracted to detect phospho-SAPK/JNK, phospho-p38,phospho-p44/42. The protein expression of sFasL and activation of MAPK signalpathways (JNK, ERK and p38) after MCAO were detected by western blot. Doubleimmunofluorescence was used to examine the Fas expression in astrocytes aftercerebral ischemia.
Results:1) In the ischemic cortex of B6mice, the level of sFasL increased by451.67%over the sham-operated brain. The expression of sFasL decreased by62.7%in the gld cortex as compared to B6mice. These results suggested that gld mice hadless sFasL than B6mice that might contribute to its less inflammatory function afterMCAO.2) The activation of MAPK signal pathways (JNK, ERK and p38) has beenshown to contribute to cerebral inflammation in ischemic injury. Our study revealed that phosphorylation of JNK and ERK increased significantly in ischemic cortex ofB6mice. FasL mutation inhibited the phosphorylation of JNK, but did not affect thephosphorylation of p38or ERK.3) Our results suggested that JNK signaling might beinvolved in FasL induced inflammatory reaction after stroke, and JNK pathway wasalso reported to be very important in inducing FasL reverse signaling of T cellsactivation and proliferation. At the beginning of ischemia, T cells infiltration intobrain might encounter the activated astrocytes first, and astrocytes might be the mostsignificant antigen-presenting cell (APC) cells in CNS that express Fas protein. So wenext detected the Fas expression in astrocytes after ischemia, and the results showedthat B6mice had significantly increased Fas level in GFAP+astrocytes compared toB6mice. Our study revealed that the high level of Fas in GFAP+astrocytes mightactivate the FasL reverse signaling and contribute to T cells migration andproliferation, thus exacerbating the immuno-inflammatory response after ischemiastroke.
Conclusion:1) FasL plays an infammatory role in cerebral ischemia. Themechanisms might relate to sFasL and the c-Jun N-terminal kinase (JNK) signalingpathway.2) The high expression of Fas in GFAP+astrocytes might activate the FasLreverse signaling and contribute to T cells migration and proliferation after cerebralischemia3) Neutralization of FasL may be a novel therapeutic strategy to suppresspost-stroke infammation and improve the long-term outcomes of stroke.
In summary, the novel findings of this study are as follows:
1. FasL is an important player in ischemic brain injury after stroke due to its potencyto provoke inflammation.
2. FasL could contribute to post-ischemia injury through activating residential CNS inflammatory cells, recruiting peripheral inflammatory cells, and modulating Tcell subpopulations.
3. sFasL and JNK signaling might contribute to FasL induced inflammatoryfunction.
引文
[1] Candelario-Jalil E. Injury and repair mechanisms in ischemic stroke:considerations for the development of novel neurotherapeutics. Curr OpinInvestig Drugs.2009.10(7):644-54.
[2] Mehta SL, Manhas N, Raghubir R. Molecular targets in cerebral ischemia fordeveloping novel therapeutics. Brain Res Rev.2007.54(1):34-66.
[3] Meisel C, Meisel A. Suppressing immunosuppression after stroke. N Engl JMed.2011.365(22):2134-6.
[4] Muir KW, Tyrrell P, Sattar N, Warburton E. Inflammation and ischaemicstroke. Curr Opin Neurol.2007.20(3):334-42.
[5] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. JNeuroimmunol.2007.184(1-2):53-68.
[6] Zaleska MM, Mercado ML, Chavez J, Feuerstein GZ, Pangalos MN, Wood A.The development of stroke therapeutics: promising mechanisms andtranslational challenges. Neuropharmacology.2009.56(2):329-41.
[7] Chamorro A, Hallenbeck J. The harms and benefits of inflammatory andimmune responses in vascular disease. Stroke.2006.37(2):291-3.
[8] Janssen O, Qian J, Linkermann A, Kabelitz D. CD95ligand--death factor andcostimulatory molecule. Cell Death Differ.2003.10(11):1215-25.
[9] Lettau M, Paulsen M, Kabelitz D, Janssen O. FasL expression and reversesignalling. Results Probl Cell Differ.2009.49:49-61.
[10] Askenasy N, Yolcu ES, Yaniv I, Shirwan H. Induction of tolerance using Fasligand: a double-edged immunomodulator. Blood.2005.105(4):1396-404.
[11] Strasser A, Jost PJ, Nagata S. The many roles of FAS receptor signaling in theimmune system. Immunity.2009.30(2):180-92.
[12] Miyagawa-Hayashino A, Tsuruyama T, Egawa H, et al. FasL expression inhepatic antigen-presenting cells and phagocytosis of apoptotic T cells byFasL+Kupffer cells are indicators of rejection activity in human liverallografts. Am J Pathol.2007.171(5):1499-508.
[13] Olson MR, Varga SM. Fas ligand is required for the development ofrespiratory syncytial virus vaccine-enhanced disease. J Immunol.2009.182(5):3024-31.
[14] de Oliveira GM, Diniz RL, Batista W, et al. Fas ligand-dependentinflammatory regulation in acute myocarditis induced by Trypanosoma cruziinfection. Am J Pathol.2007.171(1):79-86.
[15] Ahn JH, Park SM, Cho HS, et al. Non-apoptotic signaling pathways activatedby soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activatedprotein kinases and NF-kappaB-dependent gene expression. J Biol Chem.2001.276(50):47100-6.
[16] Choi C, Benveniste EN. Fas ligand/Fas system in the brain: regulator ofimmune and apoptotic responses. Brain Res Brain Res Rev.2004.44(1):65-81.
[17] Guo Z, Zhang M, Tang H, Cao X. Fas signal links innate and adaptiveimmunity by promoting dendritic-cell secretion of CC and CXC chemokines.Blood.2005.106(6):2033-41.
[18] Lettau M, Paulsen M, Kabelitz D, Janssen O. FasL expression and reversesignalling. Results Probl Cell Differ.2009.49:49-61.
[19] Voss M, Lettau M, Paulsen M, Janssen O. Posttranslational regulation of Fasligand function. Cell Commun Signal.2008.6:11.
[20] Letellier E, Kumar S, Sancho-Martinez I, et al. CD95-ligand on peripheralmyeloid cells activates Syk kinase to trigger their recruitment to theinflammatory site. Immunity.2010.32(2):240-52.
[21] Liu L, Kim JY, Koike MA, et al. FasL shedding is reduced by hypothermia inexperimental stroke. J Neurochem.2008.106(2):541-50.
[22] Tian L, Rauvala H, Gahmberg CG. Neuronal regulation of immune responsesin the central nervous system. Trends Immunol.2009.30(2):91-9.
[23] Martin-Villalba A, Hahne M, Kleber S, et al. Therapeutic neutralization ofCD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ.2001.8(7):679-86.
[24] Dupont PJ, Warrens AN. Fas ligand exerts its pro-inflammatory effects vianeutrophil recruitment but not activation. Immunology.2007.120(1):133-9.
[25] Blanco-Colio LM, Martin-Ventura JL, Tunon J, Garcia-Camarero T,Berrazueta JR, Egido J. Soluble Fas ligand plasma levels are associated withforearm reactive hyperemia in subjects with coronary artery disease: a novelbiomarker of endothelial function. Atherosclerosis.2008.201(2):407-12.
[26]陈彤,张秋业,曲少连等.新生儿缺氧缺血性脑病患儿血清sFas／sFasL水平的变化.实用儿科临床杂志.2006.21(2):84-86.
[27]于本章.新生儿缺氧缺血性脑病血清中sFas/sFasl水平变化的结果研究.医学检验与临床.2008.(3):-.
[28]徐沙贝,许峰,杨业新,唐洲平.脑出血急性期外周血sFas和sFas配体水平的观察.华中科技大学学报：医学版.2008.37(5):672-674.
[29] Tian L, Rauvala H, Gahmberg CG. Neuronal regulation of immune responsesin the central nervous system. Trends Immunol.2009.30(2):91-9.
[30] Sun M, Fink PJ. A new class of reverse signaling costimulators belongs to theTNF family. J Immunol.2007.179(7):4307-12.
[31] Sun M, Ames KT, Suzuki I, Fink PJ. The cytoplasmic domain of Fas ligandcostimulates TCR signals. J Immunol.2006.177(3):1481-91.
[32] Sun M, Fink PJ. A new class of reverse signaling costimulators belongs to theTNF family. J Immunol.2007.179(7):4307-12.
[33] Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, HurnPD. Experimental stroke induces massive, rapid activation of the peripheralimmune system. J Cereb Blood Flow Metab.2006.26(5):654-65.
[34] Worthmann H, Tryc AB, Goldbecker A, et al. The temporal profile ofinflammatory markers and mediators in blood after acute ischemic strokediffers depending on stroke outcome. Cerebrovasc Dis.2010.30(1):85-92.
[35] Choi C, Benveniste EN. Fas ligand/Fas system in the brain: regulator ofimmune and apoptotic responses. Brain Res Brain Res Rev.2004.44(1):65-81.
[36] Kunes P, Krejsek J, Brtko M, et al. Neutrophil apoptosis by Fas/FasL: harmfulor advantageous in cardiac surgery. Thorac Cardiovasc Surg.2009.57(1):1-6.
[37] Borges VM, Falcao H, Leite-Junior JH, et al. Fas ligand triggers pulmonarysilicosis. J Exp Med.2001.194(2):155-64.
[38] Seko Y, Kayagaki N, Seino K, Yagita H, Okumura K, Nagai R. Role ofFas/FasL pathway in the activation of infiltrating cells in murine acutemyocarditis caused by Coxsackievirus B3. J Am Coll Cardiol.2002.39(8):1399-403.
[39] Tian L, Rauvala H, Gahmberg CG. Neuronal regulation of immune responsesin the central nervous system. Trends Immunol.2009.30(2):91-9.
[40] Durukan A, Tatlisumak T. Acute ischemic stroke: overview of majorexperimental rodent models, pathophysiology, and therapy of focal cerebralischemia. Pharmacol Biochem Behav.2007.87(1):179-97.
[41] Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administrationof bone marrow stromal cells after cerebral ischemia in rats. Stroke.2001.32(4):1005-11.
[42] Zhao MZ, Nonoguchi N, Ikeda N, et al. Novel therapeutic strategy for strokein rats by bone marrow stromal cells and ex vivo HGF gene transfer withHSV-1vector. J Cereb Blood Flow Metab.2006.26(9):1176-88.
[43] Pitcher C, Honing S, Fingerhut A, Bowers K, Marsh M. Cluster ofdifferentiation antigen4(CD4) endocytosis and adaptor complex bindingrequire activation of the CD4endocytosis signal by serine phosphorylation.Mol Biol Cell.1999.10(3):677-91.
[44] Rosenbaum DM, Gupta G, D'Amore J, et al. Fas (CD95/APO-1) plays a rolein the pathophysiology of focal cerebral ischemia. J Neurosci Res.2000.61(6):686-92.
[45] Kerschensteiner M, Meinl E, Hohlfeld R. Neuro-immune crosstalk in CNSdiseases. Neuroscience.2009.158(3):1122-32.
[46] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. JNeuroimmunol.2007.184(1-2):53-68.
[47] Takahashi T, Tanaka M, Brannan CI, et al. Generalized lymphoproliferativedisease in mice, caused by a point mutation in the Fas ligand. Cell.1994.76(6):969-76.
[48] Martin-Villalba A, Hahne M, Kleber S, et al. Therapeutic neutralization ofCD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ.2001.8(7):679-86.
[49] Sairanen T, Karjalainen-Lindsberg ML, Paetau A, Ijas P, Lindsberg PJ.Apoptosis dominant in the periinfarct area of human ischaemic stroke--apossible target of antiapoptotic treatments. Brain.2006.129(Pt1):189-99.
[50] Kaur G, Han SJ, Yang I, Crane C. Microglia and central nervous systemimmunity. Neurosurg Clin N Am.2010.21(1):43-51.
[51] Jack CS, Arbour N, Manusow J, et al. TLR signaling tailors innate immuneresponses in human microglia and astrocytes. J Immunol.2005.175(7):4320-30.
[52] Deng H, Han HS, Cheng D, Sun GH, Yenari MA. Mild hypothermia inhibitsinflammation after experimental stroke and brain inflammation. Stroke.2003.34(10):2495-501.
[53] Arumugam TV, Granger DN, Mattson MP. Stroke and T-cells.Neuromolecular Med.2005.7(3):229-42.
[54] Yilmaz G, Granger DN. Leukocyte recruitment and ischemic brain injury.Neuromolecular Med.2010.12(2):193-204.
[55] Osawa H, Maruyama K, Streilein JW. CD95ligand expression on cornealepithelium and endothelium influences the fates of orthotopic and heterotopiccorneal allografts in mice. Invest Ophthalmol Vis Sci.2004.45(6):1908-15.
[56] Sano Y, Yamada J, Ishino Y, et al. Non-cleavable mutant Fas ligandtransfection of donor cornea abrogates ocular immune privilege. Exp Eye Res.2002.75(4):475-83.
[57] Allison J, Georgiou HM, Strasser A, Vaux DL. Transgenic expression ofCD95ligand on islet beta cells induces a granulocytic infiltration but does notconfer immune privilege upon islet allografts. Proc Natl Acad Sci U S A.1997.94(8):3943-7.
[58] Yagita H, Seino K, Kayagaki N, Okumura K. CD95ligand in graft rejection.Nature.1996.379(6567):682.
[59] Dosreis GA, Borges VM, Zin WA. The central role of Fas-ligand cellsignaling in inflammatory lung diseases. J Cell Mol Med.2004.8(3):285-93.
[60] Gee JM, Kalil A, Shea C, Becker KJ. Lymphocytes: potential mediators ofpostischemic injury and neuroprotection. Stroke.2007.38(2Suppl):783-8.
[61] Suzuki I, Martin S, Boursalian TE, Beers C, Fink PJ. Fas ligand costimulatesthe in vivo proliferation of CD8+T cells. J Immunol.2000.165(10):5537-43.
[62] Martin-Villalba A, Herr I, Jeremias I, et al. CD95ligand (Fas-L/APO-1L) andtumor necrosis factor-related apoptosis-inducing ligand mediateischemia-induced apoptosis in neurons. J Neurosci.1999.19(10):3809-17.
[63] Kunz A, Park L, Abe T, et al. Neurovascular protection by ischemic tolerance:role of nitric oxide and reactive oxygen species. J Neurosci.2007.27(27):7083-93.
[64] Lehnardt S, Massillon L, Follett P, et al. Activation of innate immunity in theCNS triggers neurodegeneration through a Toll-like receptor4-dependentpathway. Proc Natl Acad Sci U S A.2003.100(14):8514-9.
[65] Rivest S. Molecular insights on the cerebral innate immune system. BrainBehav Immun.2003.17(1):13-9.
[66] Milatovic D, Zaja-Milatovic S, Montine KS, Horner PJ, Montine TJ.Pharmacologic suppression of neuronal oxidative damage and dendriticdegeneration following direct activation of glial innate immunity in mousecerebrum. J Neurochem.2003.87(6):1518-26.
[67] Yin W, Signore AP, Iwai M, et al. Preconditioning suppresses inflammation inneonatal hypoxic ischemia via Akt activation. Stroke.2007.38(3):1017-24.
[68] Acosta REV, Zuniga E, Montes CL, Gruppi A. Interleukin-4biasesdifferentiation of B cells from Trypanosoma cruzi-infected mice and restrainstheir fratricide: role of Fas ligand down-regulation and MHC classII-transactivator up-regulation. J Leukoc Biol.2003.73(1):127-36.
[69] Chakour R, Allenbach C, Desgranges F, et al. A new function of the Fas-FasLpathway in macrophage activation. J Leukoc Biol.2009.86(1):81-90.
[70] Wagner DC, Riegelsberger UM, Michalk S, Hartig W, Kranz A, Boltze J.Cleaved caspase-3expression after experimental stroke exhibits differentphenotypes and is predominantly non-apoptotic. Brain Res.2011.1381:237-42.
[71] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. JNeuroimmunol.2007.184(1-2):53-68.
[72] Song JH, Bellail A, Tse MC, Yong VW, Hao C. Human astrocytes are resistantto Fas ligand and tumor necrosis factor-related apoptosis-inducingligand-induced apoptosis. J Neurosci.2006.26(12):3299-308.
[73] Luo HR, Loison F. Constitutive neutrophil apoptosis: mechanisms andregulation. Am J Hematol.2008.83(4):288-95.
[74] Suzuki Y, Nagai N, Yamakawa K, Kawakami J, Lijnen HR, Umemura K.Tissue-type plasminogen activator (t-PA) induces stromelysin-1(MMP-3) inendothelial cells through activation of lipoprotein receptor-related protein.Blood.2009.114(15):3352-8.
[75] Song JH, Bellail A, Tse MC, Yong VW, Hao C. Human astrocytes are resistantto Fas ligand and tumor necrosis factor-related apoptosis-inducingligand-induced apoptosis. J Neurosci.2006.26(12):3299-308.
[76] Chen ZB, Huang DQ, Niu FN, Zhang X, Li EG, Xu Y. Human urinarykallidinogenase suppresses cerebral inflammation in experimental stroke anddownregulates nuclear factor-kappaB. J Cereb Blood Flow Metab.2010.30(7):1356-65.
[77] Dupont PJ, Warrens AN. Fas ligand exerts its pro-inflammatory effects vianeutrophil recruitment but not activation. Immunology.2007.120(1):133-9.
[78] Sun M, Ames KT, Suzuki I, Fink PJ. The cytoplasmic domain of Fas ligandcostimulates TCR signals. J Immunol.2006.177(3):1481-91.
[79] Kaminska B, Gozdz A, Zawadzka M, Ellert-Miklaszewska A, Lipko M.MAPK signal transduction underlying brain inflammation and gliosis astherapeutic target. Anat Rec (Hoboken).2009.292(12):1902-13.
[80] Matsumoto N, Imamura R, Suda T. Caspase-8-and JNK-dependent AP-1activation is required for Fas ligand-induced IL-8production. FEBS J.2007.274(9):2376-84.
[1] O' RLA, Tai L, Lee L, et al. Membrane-bound Fas ligand only is essential forFas-induced apoptosis. Nature.2009.461(7264):659-63.
[2] Janssen O, Qian J, Linkermann A, Kabelitz D. CD95ligand--death factor andcostimulatory molecule. Cell Death Differ.2003.10(11):1215-25.
[3] Green DR, Ferguson TA. The role of Fas ligand in immune privilege. Nat RevMol Cell Biol.2001.2(12):917-24.
[4] Lettau M, Paulsen M, Kabelitz D, Janssen O. FasL expression and reversesignalling. Results Probl Cell Differ.2009.49:49-61.
[5] Lettau M, Paulsen M, Kabelitz D, Janssen O. Storage, expression and functionof Fas ligand, the key death factor of immune cells. Curr Med Chem.2008.15(17):1684-96.
[6] Russell JH, Ley TJ. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol.2002.20:323-70.
[7] Barry M, Bleackley RC. Cytotoxic T lymphocytes: all roads lead to death. NatRev Immunol.2002.2(6):401-9.
[8] Brunner T, Wasem C, Torgler R, Cima I, Jakob S, Corazza N. Fas(CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediatedcytotoxicity and immune pathology. Semin Immunol.2003.15(3):167-76.
[9] Krammer PH. CD95's deadly mission in the immune system. Nature.2000.407(6805):789-95.
[10] Hutcheson J, Scatizzi JC, Siddiqui AM, et al. Combined deficiency ofproapoptotic regulators Bim and Fas results in the early onset of systemicautoimmunity. Immunity.2008.28(2):206-17.
[11] Niederkorn JY. See no evil, hear no evil, do no evil: the lessons of immuneprivilege. Nat Immunol.2006.7(4):354-9.
[12] Andreola G, Rivoltini L, Castelli C, et al. Induction of lymphocyte apoptosisby tumor cell secretion of FasL-bearing microvesicles. J Exp Med.2002.195(10):1303-16.
[13] Wada A, Tada Y, Kawamura K, et al. The effects of FasL on inflammation andtumor survival are dependent on its expression levels. Cancer Gene Ther.2007.14(3):262-7.
[14] Boursalian TE, Fink PJ. Mutation in fas ligand impairs maturation ofthymocytes bearing moderate affinity T cell receptors. J Exp Med.2003.198(2):349-60.
[15] Sun M, Ames KT, Suzuki I, Fink PJ. The cytoplasmic domain of Fas ligandcostimulates TCR signals. J Immunol.2006.177(3):1481-91.
[16] Sun M, Fink PJ. A new class of reverse signaling costimulators belongs to theTNF family. J Immunol.2007.179(7):4307-12.
[17] Suzuki I, Martin S, Boursalian TE, Beers C, Fink PJ. Fas ligand costimulatesthe in vivo proliferation of CD8+T cells. J Immunol.2000.165(10):5537-43.
[18] Paulsen M, Mathew B, Qian J, Lettau M, Kabelitz D, Janssen O. FasLcross-linking inhibits activation of human peripheral T cells. Int Immunol.2009.21(5):587-98.
[19] Rieux-Laucat F, Le DF, Fischer A. Autoimmune lymphoproliferativesyndromes: genetic defects of apoptosis pathways. Cell Death Differ.2003.10(1):124-33.
[20] Del-Rey M, Ruiz-Contreras J, Bosque A, et al. A homozygous Fas ligand genemutation in a patient causes a new type of autoimmune lymphoproliferativesyndrome. Blood.2006.108(4):1306-12.
[21] Dosreis GA, Borges VM, Zin WA. The central role of Fas-ligand cellsignaling in inflammatory lung diseases. J Cell Mol Med.2004.8(3):285-93.
[22] Torgler R, Jakob S, Ontsouka E, et al. Regulation of activation-induced Fas(CD95/Apo-1) ligand expression in T cells by the cyclin B1/Cdk1complex. JBiol Chem.2004.279(36):37334-42.
[23] Dzialo-Hatton R, Milbrandt J, Hockett RD Jr, Weaver CT. Differentialexpression of Fas ligand in Th1and Th2cells is regulated by early growthresponse gene and NF-AT family members. J Immunol.2001.166(7):4534-42.
[24] Crist SA, Griffith TS, Ratliff TL. Structure/function analysis of the murineCD95L promoter reveals the identification of a novel transcriptional repressorand functional CD28response element. J Biol Chem.2003.278(38):35950-8.
[25] Lettau M, Schmidt H, Kabelitz D, Janssen O. Secretory lysosomes and theircargo in T and NK cells. Immunol Lett.2007.108(1):10-9.
[26] He JS, Ostergaard HL. CTLs contain and use intracellular stores of FasLdistinct from cytolytic granules. J Immunol.2007.179(4):2339-48.
[27] Ethell DW, Kinloch R, Green DR. Metalloproteinase shedding of Fas ligandregulates beta-amyloid neurotoxicity. Curr Biol.2002.12(18):1595-600.
[28] Linkermann A, Qian J, Lettau M, Kabelitz D, Janssen O. Considering Fasligand as a target for therapy. Expert Opin Ther Targets.2005.9(1):119-34.
[29] Strand S, Vollmer P, den Abeelen L v, et al. Cleavage of CD95by matrixmetalloproteinase-7induces apoptosis resistance in tumour cells. Oncogene.2004.23(20):3732-6.
[30] Schulte M, Reiss K, Lettau M, et al. ADAM10regulates FasL cell surfaceexpression and modulates FasL-induced cytotoxicity and activation-inducedcell death. Cell Death Differ.2007.14(5):1040-9.
[31] Kirkin V, Cahuzac N, Guardiola-Serrano F, et al. The Fas ligand intracellulardomain is released by ADAM10and SPPL2a cleavage in T-cells. Cell DeathDiffer.2007.14(9):1678-87.
[32] Simons K, Ikonen E. Functional rafts in cell membranes. Nature.1997.387(6633):569-72.
[33] Legler DF, Micheau O, Doucey MA, Tschopp J, Bron C. Recruitment of TNFreceptor1to lipid rafts is essential for TNFalpha-mediated NF-kappaBactivation. Immunity.2003.18(5):655-64.
[34] Doan JE, Windmiller DA, Riches DW. Differential regulation of TNF-R1signaling: lipid raft dependency of p42mapk/erk2activation, but notNF-kappaB activation. J Immunol.2004.172(12):7654-60.
[35] Muppidi JR, Siegel RM. Ligand-independent redistribution of Fas (CD95)into lipid rafts mediates clonotypic T cell death. Nat Immunol.2004.5(2):182-9.
[36] Nachbur U, Kassahn D, Yousefi S, Legler DF, Brunner T. Posttranscriptionalregulation of Fas (CD95) ligand killing activity by lipid rafts. Blood.2006.107(7):2790-6.
[37] Cahuzac N, Baum W, Kirkin V, et al. Fas ligand is localized to membrane rafts,where it displays increased cell death-inducing activity. Blood.2006.107(6):2384-91.
[38] Schmidt H, Gelhaus C, Nebendahl M, et al.2-D DIGE analyses of enrichedsecretory lysosomes reveal heterogeneous profiles of functionally relevantproteins in leukemic and activated human NK cells. Proteomics.2008.8(14):2911-25.
[39] Zuccato E, Blott EJ, Holt O, et al. Sorting of Fas ligand to secretorylysosomes is regulated by mono-ubiquitylation and phosphorylation. J CellSci.2007.120(Pt1):191-9.
[40] Chitu V, Stanley ER. Pombe Cdc15homology (PCH) proteins: coordinators ofmembrane-cytoskeletal interactions. Trends Cell Biol.2007.17(3):145-56.
[41] Qian J, Chen W, Lettau M, et al. Regulation of FasL expression: a SH3domain containing protein family involved in the lysosomal association ofFasL. Cell Signal.2006.18(8):1327-37.
[42] Lettau M, Qian J, Linkermann A, et al. The adaptor protein Nck interacts withFas ligand: Guiding the death factor to the cytotoxic immunological synapse.Proc Natl Acad Sci U S A.2006.103(15):5911-6.
[2] Mehta SL, Manhas N, Raghubir R. Molecular targets in cerebral ischemia fordeveloping novel therapeutics. Brain Res Rev.2007.54(1):34-66.
[3] Meisel C, Meisel A. Suppressing immunosuppression after stroke. N Engl JMed.2011.365(22):2134-6.
[4] Muir KW, Tyrrell P, Sattar N, Warburton E. Inflammation and ischaemicstroke. Curr Opin Neurol.2007.20(3):334-42.
[5] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. JNeuroimmunol.2007.184(1-2):53-68.
[6] Zaleska MM, Mercado ML, Chavez J, Feuerstein GZ, Pangalos MN, Wood A.The development of stroke therapeutics: promising mechanisms andtranslational challenges. Neuropharmacology.2009.56(2):329-41.
[7] Chamorro A, Hallenbeck J. The harms and benefits of inflammatory andimmune responses in vascular disease. Stroke.2006.37(2):291-3.
[8] Janssen O, Qian J, Linkermann A, Kabelitz D. CD95ligand--death factor andcostimulatory molecule. Cell Death Differ.2003.10(11):1215-25.
[9] Lettau M, Paulsen M, Kabelitz D, Janssen O. FasL expression and reversesignalling. Results Probl Cell Differ.2009.49:49-61.
[10] Askenasy N, Yolcu ES, Yaniv I, Shirwan H. Induction of tolerance using Fasligand: a double-edged immunomodulator. Blood.2005.105(4):1396-404.
[11] Strasser A, Jost PJ, Nagata S. The many roles of FAS receptor signaling in theimmune system. Immunity.2009.30(2):180-92.
[12] Miyagawa-Hayashino A, Tsuruyama T, Egawa H, et al. FasL expression inhepatic antigen-presenting cells and phagocytosis of apoptotic T cells byFasL+Kupffer cells are indicators of rejection activity in human liverallografts. Am J Pathol.2007.171(5):1499-508.
[13] Olson MR, Varga SM. Fas ligand is required for the development ofrespiratory syncytial virus vaccine-enhanced disease. J Immunol.2009.182(5):3024-31.
[14] de Oliveira GM, Diniz RL, Batista W, et al. Fas ligand-dependentinflammatory regulation in acute myocarditis induced by Trypanosoma cruziinfection. Am J Pathol.2007.171(1):79-86.
[15] Ahn JH, Park SM, Cho HS, et al. Non-apoptotic signaling pathways activatedby soluble Fas ligand in serum-starved human fibroblasts. Mitogen-activatedprotein kinases and NF-kappaB-dependent gene expression. J Biol Chem.2001.276(50):47100-6.
[16] Choi C, Benveniste EN. Fas ligand/Fas system in the brain: regulator ofimmune and apoptotic responses. Brain Res Brain Res Rev.2004.44(1):65-81.
[17] Guo Z, Zhang M, Tang H, Cao X. Fas signal links innate and adaptiveimmunity by promoting dendritic-cell secretion of CC and CXC chemokines.Blood.2005.106(6):2033-41.
[18] Lettau M, Paulsen M, Kabelitz D, Janssen O. FasL expression and reversesignalling. Results Probl Cell Differ.2009.49:49-61.
[19] Voss M, Lettau M, Paulsen M, Janssen O. Posttranslational regulation of Fasligand function. Cell Commun Signal.2008.6:11.
[20] Letellier E, Kumar S, Sancho-Martinez I, et al. CD95-ligand on peripheralmyeloid cells activates Syk kinase to trigger their recruitment to theinflammatory site. Immunity.2010.32(2):240-52.
[21] Liu L, Kim JY, Koike MA, et al. FasL shedding is reduced by hypothermia inexperimental stroke. J Neurochem.2008.106(2):541-50.
[22] Tian L, Rauvala H, Gahmberg CG. Neuronal regulation of immune responsesin the central nervous system. Trends Immunol.2009.30(2):91-9.
[23] Martin-Villalba A, Hahne M, Kleber S, et al. Therapeutic neutralization ofCD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ.2001.8(7):679-86.
[24] Dupont PJ, Warrens AN. Fas ligand exerts its pro-inflammatory effects vianeutrophil recruitment but not activation. Immunology.2007.120(1):133-9.
[25] Blanco-Colio LM, Martin-Ventura JL, Tunon J, Garcia-Camarero T,Berrazueta JR, Egido J. Soluble Fas ligand plasma levels are associated withforearm reactive hyperemia in subjects with coronary artery disease: a novelbiomarker of endothelial function. Atherosclerosis.2008.201(2):407-12.
[26]陈彤,张秋业,曲少连等.新生儿缺氧缺血性脑病患儿血清sFas／sFasL水平的变化.实用儿科临床杂志.2006.21(2):84-86.
[27]于本章.新生儿缺氧缺血性脑病血清中sFas/sFasl水平变化的结果研究.医学检验与临床.2008.(3):-.
[28]徐沙贝,许峰,杨业新,唐洲平.脑出血急性期外周血sFas和sFas配体水平的观察.华中科技大学学报：医学版.2008.37(5):672-674.
[29] Tian L, Rauvala H, Gahmberg CG. Neuronal regulation of immune responsesin the central nervous system. Trends Immunol.2009.30(2):91-9.
[30] Sun M, Fink PJ. A new class of reverse signaling costimulators belongs to theTNF family. J Immunol.2007.179(7):4307-12.
[31] Sun M, Ames KT, Suzuki I, Fink PJ. The cytoplasmic domain of Fas ligandcostimulates TCR signals. J Immunol.2006.177(3):1481-91.
[32] Sun M, Fink PJ. A new class of reverse signaling costimulators belongs to theTNF family. J Immunol.2007.179(7):4307-12.
[33] Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, HurnPD. Experimental stroke induces massive, rapid activation of the peripheralimmune system. J Cereb Blood Flow Metab.2006.26(5):654-65.
[34] Worthmann H, Tryc AB, Goldbecker A, et al. The temporal profile ofinflammatory markers and mediators in blood after acute ischemic strokediffers depending on stroke outcome. Cerebrovasc Dis.2010.30(1):85-92.
[35] Choi C, Benveniste EN. Fas ligand/Fas system in the brain: regulator ofimmune and apoptotic responses. Brain Res Brain Res Rev.2004.44(1):65-81.
[36] Kunes P, Krejsek J, Brtko M, et al. Neutrophil apoptosis by Fas/FasL: harmfulor advantageous in cardiac surgery. Thorac Cardiovasc Surg.2009.57(1):1-6.
[37] Borges VM, Falcao H, Leite-Junior JH, et al. Fas ligand triggers pulmonarysilicosis. J Exp Med.2001.194(2):155-64.
[38] Seko Y, Kayagaki N, Seino K, Yagita H, Okumura K, Nagai R. Role ofFas/FasL pathway in the activation of infiltrating cells in murine acutemyocarditis caused by Coxsackievirus B3. J Am Coll Cardiol.2002.39(8):1399-403.
[39] Tian L, Rauvala H, Gahmberg CG. Neuronal regulation of immune responsesin the central nervous system. Trends Immunol.2009.30(2):91-9.
[40] Durukan A, Tatlisumak T. Acute ischemic stroke: overview of majorexperimental rodent models, pathophysiology, and therapy of focal cerebralischemia. Pharmacol Biochem Behav.2007.87(1):179-97.
[41] Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administrationof bone marrow stromal cells after cerebral ischemia in rats. Stroke.2001.32(4):1005-11.
[42] Zhao MZ, Nonoguchi N, Ikeda N, et al. Novel therapeutic strategy for strokein rats by bone marrow stromal cells and ex vivo HGF gene transfer withHSV-1vector. J Cereb Blood Flow Metab.2006.26(9):1176-88.
[43] Pitcher C, Honing S, Fingerhut A, Bowers K, Marsh M. Cluster ofdifferentiation antigen4(CD4) endocytosis and adaptor complex bindingrequire activation of the CD4endocytosis signal by serine phosphorylation.Mol Biol Cell.1999.10(3):677-91.
[44] Rosenbaum DM, Gupta G, D'Amore J, et al. Fas (CD95/APO-1) plays a rolein the pathophysiology of focal cerebral ischemia. J Neurosci Res.2000.61(6):686-92.
[45] Kerschensteiner M, Meinl E, Hohlfeld R. Neuro-immune crosstalk in CNSdiseases. Neuroscience.2009.158(3):1122-32.
[46] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. JNeuroimmunol.2007.184(1-2):53-68.
[47] Takahashi T, Tanaka M, Brannan CI, et al. Generalized lymphoproliferativedisease in mice, caused by a point mutation in the Fas ligand. Cell.1994.76(6):969-76.
[48] Martin-Villalba A, Hahne M, Kleber S, et al. Therapeutic neutralization ofCD95-ligand and TNF attenuates brain damage in stroke. Cell Death Differ.2001.8(7):679-86.
[49] Sairanen T, Karjalainen-Lindsberg ML, Paetau A, Ijas P, Lindsberg PJ.Apoptosis dominant in the periinfarct area of human ischaemic stroke--apossible target of antiapoptotic treatments. Brain.2006.129(Pt1):189-99.
[50] Kaur G, Han SJ, Yang I, Crane C. Microglia and central nervous systemimmunity. Neurosurg Clin N Am.2010.21(1):43-51.
[51] Jack CS, Arbour N, Manusow J, et al. TLR signaling tailors innate immuneresponses in human microglia and astrocytes. J Immunol.2005.175(7):4320-30.
[52] Deng H, Han HS, Cheng D, Sun GH, Yenari MA. Mild hypothermia inhibitsinflammation after experimental stroke and brain inflammation. Stroke.2003.34(10):2495-501.
[53] Arumugam TV, Granger DN, Mattson MP. Stroke and T-cells.Neuromolecular Med.2005.7(3):229-42.
[54] Yilmaz G, Granger DN. Leukocyte recruitment and ischemic brain injury.Neuromolecular Med.2010.12(2):193-204.
[55] Osawa H, Maruyama K, Streilein JW. CD95ligand expression on cornealepithelium and endothelium influences the fates of orthotopic and heterotopiccorneal allografts in mice. Invest Ophthalmol Vis Sci.2004.45(6):1908-15.
[56] Sano Y, Yamada J, Ishino Y, et al. Non-cleavable mutant Fas ligandtransfection of donor cornea abrogates ocular immune privilege. Exp Eye Res.2002.75(4):475-83.
[57] Allison J, Georgiou HM, Strasser A, Vaux DL. Transgenic expression ofCD95ligand on islet beta cells induces a granulocytic infiltration but does notconfer immune privilege upon islet allografts. Proc Natl Acad Sci U S A.1997.94(8):3943-7.
[58] Yagita H, Seino K, Kayagaki N, Okumura K. CD95ligand in graft rejection.Nature.1996.379(6567):682.
[59] Dosreis GA, Borges VM, Zin WA. The central role of Fas-ligand cellsignaling in inflammatory lung diseases. J Cell Mol Med.2004.8(3):285-93.
[60] Gee JM, Kalil A, Shea C, Becker KJ. Lymphocytes: potential mediators ofpostischemic injury and neuroprotection. Stroke.2007.38(2Suppl):783-8.
[61] Suzuki I, Martin S, Boursalian TE, Beers C, Fink PJ. Fas ligand costimulatesthe in vivo proliferation of CD8+T cells. J Immunol.2000.165(10):5537-43.
[62] Martin-Villalba A, Herr I, Jeremias I, et al. CD95ligand (Fas-L/APO-1L) andtumor necrosis factor-related apoptosis-inducing ligand mediateischemia-induced apoptosis in neurons. J Neurosci.1999.19(10):3809-17.
[63] Kunz A, Park L, Abe T, et al. Neurovascular protection by ischemic tolerance:role of nitric oxide and reactive oxygen species. J Neurosci.2007.27(27):7083-93.
[64] Lehnardt S, Massillon L, Follett P, et al. Activation of innate immunity in theCNS triggers neurodegeneration through a Toll-like receptor4-dependentpathway. Proc Natl Acad Sci U S A.2003.100(14):8514-9.
[65] Rivest S. Molecular insights on the cerebral innate immune system. BrainBehav Immun.2003.17(1):13-9.
[66] Milatovic D, Zaja-Milatovic S, Montine KS, Horner PJ, Montine TJ.Pharmacologic suppression of neuronal oxidative damage and dendriticdegeneration following direct activation of glial innate immunity in mousecerebrum. J Neurochem.2003.87(6):1518-26.
[67] Yin W, Signore AP, Iwai M, et al. Preconditioning suppresses inflammation inneonatal hypoxic ischemia via Akt activation. Stroke.2007.38(3):1017-24.
[68] Acosta REV, Zuniga E, Montes CL, Gruppi A. Interleukin-4biasesdifferentiation of B cells from Trypanosoma cruzi-infected mice and restrainstheir fratricide: role of Fas ligand down-regulation and MHC classII-transactivator up-regulation. J Leukoc Biol.2003.73(1):127-36.
[69] Chakour R, Allenbach C, Desgranges F, et al. A new function of the Fas-FasLpathway in macrophage activation. J Leukoc Biol.2009.86(1):81-90.
[70] Wagner DC, Riegelsberger UM, Michalk S, Hartig W, Kranz A, Boltze J.Cleaved caspase-3expression after experimental stroke exhibits differentphenotypes and is predominantly non-apoptotic. Brain Res.2011.1381:237-42.
[71] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. JNeuroimmunol.2007.184(1-2):53-68.
[72] Song JH, Bellail A, Tse MC, Yong VW, Hao C. Human astrocytes are resistantto Fas ligand and tumor necrosis factor-related apoptosis-inducingligand-induced apoptosis. J Neurosci.2006.26(12):3299-308.
[73] Luo HR, Loison F. Constitutive neutrophil apoptosis: mechanisms andregulation. Am J Hematol.2008.83(4):288-95.
[74] Suzuki Y, Nagai N, Yamakawa K, Kawakami J, Lijnen HR, Umemura K.Tissue-type plasminogen activator (t-PA) induces stromelysin-1(MMP-3) inendothelial cells through activation of lipoprotein receptor-related protein.Blood.2009.114(15):3352-8.
[75] Song JH, Bellail A, Tse MC, Yong VW, Hao C. Human astrocytes are resistantto Fas ligand and tumor necrosis factor-related apoptosis-inducingligand-induced apoptosis. J Neurosci.2006.26(12):3299-308.
[76] Chen ZB, Huang DQ, Niu FN, Zhang X, Li EG, Xu Y. Human urinarykallidinogenase suppresses cerebral inflammation in experimental stroke anddownregulates nuclear factor-kappaB. J Cereb Blood Flow Metab.2010.30(7):1356-65.
[77] Dupont PJ, Warrens AN. Fas ligand exerts its pro-inflammatory effects vianeutrophil recruitment but not activation. Immunology.2007.120(1):133-9.
[78] Sun M, Ames KT, Suzuki I, Fink PJ. The cytoplasmic domain of Fas ligandcostimulates TCR signals. J Immunol.2006.177(3):1481-91.
[79] Kaminska B, Gozdz A, Zawadzka M, Ellert-Miklaszewska A, Lipko M.MAPK signal transduction underlying brain inflammation and gliosis astherapeutic target. Anat Rec (Hoboken).2009.292(12):1902-13.
[80] Matsumoto N, Imamura R, Suda T. Caspase-8-and JNK-dependent AP-1activation is required for Fas ligand-induced IL-8production. FEBS J.2007.274(9):2376-84.
[1] O' RLA, Tai L, Lee L, et al. Membrane-bound Fas ligand only is essential forFas-induced apoptosis. Nature.2009.461(7264):659-63.
[2] Janssen O, Qian J, Linkermann A, Kabelitz D. CD95ligand--death factor andcostimulatory molecule. Cell Death Differ.2003.10(11):1215-25.
[3] Green DR, Ferguson TA. The role of Fas ligand in immune privilege. Nat RevMol Cell Biol.2001.2(12):917-24.
[4] Lettau M, Paulsen M, Kabelitz D, Janssen O. FasL expression and reversesignalling. Results Probl Cell Differ.2009.49:49-61.
[5] Lettau M, Paulsen M, Kabelitz D, Janssen O. Storage, expression and functionof Fas ligand, the key death factor of immune cells. Curr Med Chem.2008.15(17):1684-96.
[6] Russell JH, Ley TJ. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol.2002.20:323-70.
[7] Barry M, Bleackley RC. Cytotoxic T lymphocytes: all roads lead to death. NatRev Immunol.2002.2(6):401-9.
[8] Brunner T, Wasem C, Torgler R, Cima I, Jakob S, Corazza N. Fas(CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediatedcytotoxicity and immune pathology. Semin Immunol.2003.15(3):167-76.
[9] Krammer PH. CD95's deadly mission in the immune system. Nature.2000.407(6805):789-95.
[10] Hutcheson J, Scatizzi JC, Siddiqui AM, et al. Combined deficiency ofproapoptotic regulators Bim and Fas results in the early onset of systemicautoimmunity. Immunity.2008.28(2):206-17.
[11] Niederkorn JY. See no evil, hear no evil, do no evil: the lessons of immuneprivilege. Nat Immunol.2006.7(4):354-9.
[12] Andreola G, Rivoltini L, Castelli C, et al. Induction of lymphocyte apoptosisby tumor cell secretion of FasL-bearing microvesicles. J Exp Med.2002.195(10):1303-16.
[13] Wada A, Tada Y, Kawamura K, et al. The effects of FasL on inflammation andtumor survival are dependent on its expression levels. Cancer Gene Ther.2007.14(3):262-7.
[14] Boursalian TE, Fink PJ. Mutation in fas ligand impairs maturation ofthymocytes bearing moderate affinity T cell receptors. J Exp Med.2003.198(2):349-60.
[15] Sun M, Ames KT, Suzuki I, Fink PJ. The cytoplasmic domain of Fas ligandcostimulates TCR signals. J Immunol.2006.177(3):1481-91.
[16] Sun M, Fink PJ. A new class of reverse signaling costimulators belongs to theTNF family. J Immunol.2007.179(7):4307-12.
[17] Suzuki I, Martin S, Boursalian TE, Beers C, Fink PJ. Fas ligand costimulatesthe in vivo proliferation of CD8+T cells. J Immunol.2000.165(10):5537-43.
[18] Paulsen M, Mathew B, Qian J, Lettau M, Kabelitz D, Janssen O. FasLcross-linking inhibits activation of human peripheral T cells. Int Immunol.2009.21(5):587-98.
[19] Rieux-Laucat F, Le DF, Fischer A. Autoimmune lymphoproliferativesyndromes: genetic defects of apoptosis pathways. Cell Death Differ.2003.10(1):124-33.
[20] Del-Rey M, Ruiz-Contreras J, Bosque A, et al. A homozygous Fas ligand genemutation in a patient causes a new type of autoimmune lymphoproliferativesyndrome. Blood.2006.108(4):1306-12.
[21] Dosreis GA, Borges VM, Zin WA. The central role of Fas-ligand cellsignaling in inflammatory lung diseases. J Cell Mol Med.2004.8(3):285-93.
[22] Torgler R, Jakob S, Ontsouka E, et al. Regulation of activation-induced Fas(CD95/Apo-1) ligand expression in T cells by the cyclin B1/Cdk1complex. JBiol Chem.2004.279(36):37334-42.
[23] Dzialo-Hatton R, Milbrandt J, Hockett RD Jr, Weaver CT. Differentialexpression of Fas ligand in Th1and Th2cells is regulated by early growthresponse gene and NF-AT family members. J Immunol.2001.166(7):4534-42.
[24] Crist SA, Griffith TS, Ratliff TL. Structure/function analysis of the murineCD95L promoter reveals the identification of a novel transcriptional repressorand functional CD28response element. J Biol Chem.2003.278(38):35950-8.
[25] Lettau M, Schmidt H, Kabelitz D, Janssen O. Secretory lysosomes and theircargo in T and NK cells. Immunol Lett.2007.108(1):10-9.
[26] He JS, Ostergaard HL. CTLs contain and use intracellular stores of FasLdistinct from cytolytic granules. J Immunol.2007.179(4):2339-48.
[27] Ethell DW, Kinloch R, Green DR. Metalloproteinase shedding of Fas ligandregulates beta-amyloid neurotoxicity. Curr Biol.2002.12(18):1595-600.
[28] Linkermann A, Qian J, Lettau M, Kabelitz D, Janssen O. Considering Fasligand as a target for therapy. Expert Opin Ther Targets.2005.9(1):119-34.
[29] Strand S, Vollmer P, den Abeelen L v, et al. Cleavage of CD95by matrixmetalloproteinase-7induces apoptosis resistance in tumour cells. Oncogene.2004.23(20):3732-6.
[30] Schulte M, Reiss K, Lettau M, et al. ADAM10regulates FasL cell surfaceexpression and modulates FasL-induced cytotoxicity and activation-inducedcell death. Cell Death Differ.2007.14(5):1040-9.
[31] Kirkin V, Cahuzac N, Guardiola-Serrano F, et al. The Fas ligand intracellulardomain is released by ADAM10and SPPL2a cleavage in T-cells. Cell DeathDiffer.2007.14(9):1678-87.
[32] Simons K, Ikonen E. Functional rafts in cell membranes. Nature.1997.387(6633):569-72.
[33] Legler DF, Micheau O, Doucey MA, Tschopp J, Bron C. Recruitment of TNFreceptor1to lipid rafts is essential for TNFalpha-mediated NF-kappaBactivation. Immunity.2003.18(5):655-64.
[34] Doan JE, Windmiller DA, Riches DW. Differential regulation of TNF-R1signaling: lipid raft dependency of p42mapk/erk2activation, but notNF-kappaB activation. J Immunol.2004.172(12):7654-60.
[35] Muppidi JR, Siegel RM. Ligand-independent redistribution of Fas (CD95)into lipid rafts mediates clonotypic T cell death. Nat Immunol.2004.5(2):182-9.
[36] Nachbur U, Kassahn D, Yousefi S, Legler DF, Brunner T. Posttranscriptionalregulation of Fas (CD95) ligand killing activity by lipid rafts. Blood.2006.107(7):2790-6.
[37] Cahuzac N, Baum W, Kirkin V, et al. Fas ligand is localized to membrane rafts,where it displays increased cell death-inducing activity. Blood.2006.107(6):2384-91.
[38] Schmidt H, Gelhaus C, Nebendahl M, et al.2-D DIGE analyses of enrichedsecretory lysosomes reveal heterogeneous profiles of functionally relevantproteins in leukemic and activated human NK cells. Proteomics.2008.8(14):2911-25.
[39] Zuccato E, Blott EJ, Holt O, et al. Sorting of Fas ligand to secretorylysosomes is regulated by mono-ubiquitylation and phosphorylation. J CellSci.2007.120(Pt1):191-9.
[40] Chitu V, Stanley ER. Pombe Cdc15homology (PCH) proteins: coordinators ofmembrane-cytoskeletal interactions. Trends Cell Biol.2007.17(3):145-56.
[41] Qian J, Chen W, Lettau M, et al. Regulation of FasL expression: a SH3domain containing protein family involved in the lysosomal association ofFasL. Cell Signal.2006.18(8):1327-37.
[42] Lettau M, Qian J, Linkermann A, et al. The adaptor protein Nck interacts withFas ligand: Guiding the death factor to the cytotoxic immunological synapse.Proc Natl Acad Sci U S A.2006.103(15):5911-6.