短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡

目的:探讨短发夹状RNA基因沉默补体受体C5aR及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果。方法:构建针对大鼠补体受体C5aR基因编码区的短发夹状RNA(shRNA)真核表达载体质粒pRNAT-U6.1-C5aR shRNA,采用电穿孔的方法转染RK3E细胞,经G418筛选后,形成稳定的表达C5aR shRNA的细胞系。实验分为3组,①正常对照组:未转染的RK3E细胞;②阴性对照组:转染空载体pRNAT-U6.1的RK3E细胞系;③实验组:转染C5aR shRNA的RK3E细胞系。经脂多糖(LPS)孵育12小时后,流式细胞仪检测各组细胞凋亡率,RT-PCR检测mRNA的表达,γ计数仪测定125I标记的C5a与RK3E的结合情况。结果:与正常对照组和阴性对照组相比,实验组的细胞凋亡率显著降低(P0.01),C5aR mRNA水平显著降低(P0.01),125I标记的C5a与RK3E结合活性显著下降。结论:针对C5aR的特异性短发夹RNA可以明显引起靶基因的沉默,进而抑制LPS诱导的肾脏上皮细胞凋亡的发生。     

C5aR1促进神经干细胞增殖作用的实验研究

目的研究C5aR1在新生小鼠脑内及体外培养神经干细胞内的表达规律,探索C5aR1对神经干细胞增殖调节的作用。方法以免疫组化方法检测在新生小鼠脑内及体外培养的神经干细胞内C5aR1的表达;培养C5aR基因敲除小鼠来源的神经干细胞,同时利用C5aR拮抗剂处理正常小鼠来源的神经干细胞,分别观察神经干细胞的增殖状态,分析C5aR1对神经干细胞增殖的调节作用。结果 1)在新生小鼠脑内,C5aR1主要表达于海马和室下区等神经干细胞分布区域;2)C5aR1与nestin共表达于体外培养的神经干细胞内;3)C5aR特异性拮抗肽下调体外培养的神经干细胞内nestin表达并抑制其增殖;C5aR1基因敲除,神经干细胞的成球能力明显降低。结论 C5aR1在脑内以及神经干细胞内均有表达,对神经干细胞的增殖具有促进作用,其具体机制还有待进一步的实验研究。     

Functional identification of the stable transfection C5aR cell line Molt-4

The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Gcxi and Gtz16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERKI/2 phosphorylation, Ca＋＋ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR. Cellular ＆ Molecular Immunology.     

肾癌荷瘤小鼠组织中补体 C5a / C5aR 通路活化与肿瘤高凝状态 的关系及其机制研究

目的 研究肾癌荷瘤小鼠组织中补体 C5a / C5aR 通路活化与肿瘤高凝状态的关系及其机制。 方法 收集 2016 年 7 月至 2019 年 10 月于唐山市人民医院收治的 32 例经病理学活检确诊的肾癌患者的癌组织和 癌旁组织。 采用 Western 印迹法检测组织中 C5a / C5aR 通路活化相关蛋白表达情况。 于 BALB/ c-nu / nu 裸鼠 前肢腋窝皮下接种人 ACHN 肾癌细胞株, 接种成功后随机分为干预组、 模型组, 其中干预组尾静脉注射 C5a 联合 C5aR 阻断剂 (C5aR antagonist, C5aRA), 模型组等体积注射生理盐水, 另取 6 只空白小鼠作为对 照组。 观察小鼠肿瘤生长情况, 检测高凝状态相关指标水平。 脱颈处死小鼠, 收集癌组织, 采用 Western 印迹法检测组织中 C5a / C5aR 通路活化相关蛋白表达情况。 结果 两组小鼠肿瘤体积于荷瘤 6 ~ 21 d 差异具 有统计学意义, 且干预组的肿瘤体积明显低于模型组 ( P< 0. 05)。 3 组小鼠于荷瘤第 1 天的血清 D-D、 vWF、 TF 水平差异无统计学意义, 荷瘤第 14 天、 第 21 天干预组、 模型组血清凝血指标水平明显增加, 其 中干预组均明显低于模型组 (P< 0. 05)。 干预组癌组织中 C5aR、 C5b-9、 FGL2、 P38、 p-P38 的表达水平均 明显低于模型组 (P< 0. 05)。 肾癌患者癌组织中 C5aR、 C5b-9、 FGL2、 P38、 p-P38 的表达水平均明显高于 癌旁组织 (P< 0. 05)。 32 例肾癌患者中高凝状态患者 14 例, 非高凝状态患者 18 例, 高凝状态组癌组织中 C5aR、 C5b-9、 FGL2、 P38、 p-P38 的表达水平均明显高于非高凝状态组 (P< 0. 05)。 结论 补体 C5a / C5aR 通路可通过调控 FGL2 的表达而诱导肾癌的高凝状态, 进而参与肾癌的发病过程。     

金黄色葡萄球菌分泌趋化抑制蛋白与C5aR、C5L2的相互作用

目的探讨金黄色葡萄球菌分泌的趋化抑制蛋白(CHIPs)与G蛋白偶联受体(GPCR)C5a受体(C5aR)或促酰化蛋白受体(C5L2))的相互作用。方法将纯化的CHIPs蛋白分别与表达C5aR、C5L2、CXC趋化因子受体3(CXCR3)的人肾胚胎上皮细胞293T分别孵育,用Western blot检测结合情况。结果CHIPs蛋白与C5aR相结合,与C5L2和CXCR3无明显结合。结论C5aR是CHIPs蛋白的受体,提示C5L2与C5aR在和CHIPs的结合作用上存在明显差异。     

Rapid‐onset clinical and mechanistic effects of anti‐C5aR treatment in the mouse collagen‐induced arthritis model

Preclinical evidence supports targeting the C5a receptor (C5aR) in rheumatoid arthritis (RA). To support ongoing clinical development of an anti‐C5aR monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti‐murine C5aR (anti‐mC5aR) surrogate antibody in mouse collagen‐induced arthritis (CIA). First, efficacy was demonstrated in a multiple‐dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti‐mC5aR‐treated mice. Then, the mechanism of action was examined by looking for early effects of anti‐mC5aR treatment in single‐dose treatment studies. We found that 48 h after single‐dose treatment with anti‐mC5aR, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose‐setting experiments supported a beneficial clinical effect of dosing above the C5aR saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5aR. The data have translational value in supporting the Novo Nordisk clinical trials of an anti‐C5aR antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5aR.     

Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4+ T cells

Complement activation is traditionally thought to occur in the extracellular space. However, it has been suggested that complement proteins are activated and function at additional locations. T cells contain intracellular stores of C3 and C5 that can be cleaved into C3a and C5a and bind to intracellular receptors, which have been shown to be of vital importance for the differentiation and function of these cells. However, whether the origin of the complement proteins located within T cells is derived from endogenous produced complement or from an uptake dependent mechanism is unknown.The presence of intracellular C3 in T cells from normal donors was investigated by fluorescence microscopy and flow cytometry. Moreover, mRNA expression levels of several genes encoding for complement proteins with primary focus on C3, C3aR, C5 and C5aR1 during resting state and upon activation of CD4⁺ T cells were investigated by a quantitative PCR technique. Furthermore, the gene expression level was evaluated at different time points.We confirmed the presence of intracellular C3 protein in normal T-cells. However, we could not see any increase in mRNA levels using any activation strategy tested. On the contrary, we observed a slight increase in C3 and C5aR1 mRNA only in the non-activated T-cells compared to the activated T cells, and a decrease in the activated T-cells at different incubation time points.Our results show that there is a baseline intracellular expression of the complement C3, C5, C3aR and C5aR1 genes in normal CD4⁺ T cells, but that expression is not increased during T-cell activation, but rather down regulated. Thus, the pool of intracellular complement in CD4⁺ T cells may either be due to accumulated complement due low-grade expression or arise from the circulation from an uptake dependent mechanism, but these possibilities are not mutually exclusive.     

Use of monoclonal antibodies to assess expression of anaphylatoxin receptors in rat and murine models of lung inflammation

The anaphylatoxins C3a and C5a are involved in the pathophysiology of microbial as well as allergic inflammation in the lungs. Besides their expression in leukocytes, receptors for C3a and C5a (C3aR and C5aR) have been noted in alveolar and bronchial epithelial cells, bronchial smooth muscle cells as well as in vascular endothelial and smooth muscle cells of normal and inflamed human and murine lungs. Recently, however, expression of anaphylatoxin receptors in parenchymal cells of the lung (and kidney) has been challenged. Using well-characterized monoclonal antibodies (mabs) against murine and rat anaphylatoxin receptors, we reexamined the pulmonary distribution of C3aR and C5aR. Immunohistochemistry was performed on frozen sections of lung tissues from normal mice and rats as well as from animals subjected to lipopolysaccharide (LPS)-induced inflammation or from MRL/lpr mice suffering from autoimmune disease. Furthermore, ovalbumin (OVA)-induced models of allergic asthma in the rat and mouse were investigated. Prominent expression of both anaphylatoxin receptors was detectable in resident as well as infiltrating leukocytes. No C3aR protein was observed in alveolar macrophages. Upon LPS- and OVA-challenge as well as in autoimmune inflammation, numbers of infiltrating leukocytes expressing prominent amounts of anaphylatoxin receptors increased. Even under these highly inflammatory conditions, however, expression of C3aR and C5aR was not inducible in parenchymal cells. Thus, our findings identify infiltrating leukocytes as a prominent source of anaphylatoxin receptors in inflamed lungs. A direct involvement of parenchymal cells in anaphylatoxin-mediated pulmonary inflammation is unlikely.     

C5a/C5aR通路对约氏疟原虫增殖和小鼠生存的影响

目的观察C5a/C5aR通路对约氏疟原虫17XL（P.y17XL）的增殖及其对BALB/c小鼠致死率的影响。结论将实验小鼠分为正常BALB/c组和C5aR-/-BALB/c组,每组5只,相同剂量的P.y17XL（2×105）分别感染正常BALB/c组小鼠和C5aR-/-BALB/c组小鼠,于感染后0、2、4和6d观察两组小鼠原虫血症和存活率的变化,并且采用ELISA检测感染P.y17XLBALB/c小鼠血清中C5a的水平。结果与正常组小鼠相比,C5aR-/-组小鼠生存期缩短,P.y17XL在C5aR-/-组小鼠的原虫血症较野生株小鼠高（P〈0.05）,而P.y17XL感染小鼠的血清中C5a含量低于未感染的小鼠。结论 C5a/C5aR能够抑制P.y17XL的增殖,并能延长感染小鼠的生存期。     

人视网膜前膜组织及培养的人RPE细胞上C5a受体的表达

目的：检测手术中取出的增生性玻璃体视网膜(PVR)病变患者的视网膜前膜(ERM)组织及培养的人视网膜色素上皮细胞(RPE)中的C5a受体(C5aR)的表达及分布，阐明C5a受体在发生PVR的病理过程中可能的作用。方法：手术取出ERM组织，常规制备石腊切片。另取新鲜人眼球，常规分离、培养人RPE细胞。以小鼠抗人C5aR单克隆抗体作为检测抗体。对PVR患者的FRM组织及培养的人RPE细胞做细胞免疫化学ABC染色，检测C5aR的表达。结果：PVR患者的ERM组织及培养的人RPE细胞上均有C5aR的表达：结论：C5a作为一种前炎性因子，与RPE细胞上的C5aR结合后，可能介导PVR的发生，即PVR的发生可能与C5a有关。     

C5aR在慢性移植物抗宿主病中的表达及其作用机制

目的:探讨补体5a(C5a)及其受体(C5aR)在慢性移植物抗宿主病(cGVHD)中的表达及其作用机制。方法:用流式细胞术检测20例cGVHD患者及9例健康供者外周血淋巴细胞中C5aR的表达及CD4~+CD25~+Foxp3~+调节性T细胞(Tregs)在CD4~+T细胞中的比例,并分析两者的相关性;将体外分离培养小鼠脾细胞分为对照组及重组小鼠C5a蛋白(rmC 5a)刺激组,用流式细胞术检测2组Tregs在CD4~+T细胞中的表达比例;另外,提取患者外周血单个核细胞进行体外培养,分为空白对照组及C5aR拮抗剂(C5aRA)组,用流式细胞术检测2组Tregs在CD4~+T细胞中的表达比例。结果:cGVHD患者外周血淋巴细胞表面C5aR的表达明显增多,而Tregs在CD4~+T细胞中的比例明显减少,两者呈显著负性相关(P0.05);体外培养小鼠脾细胞结果显示C5a下调Tregs在CD4~+T细胞中的比例;而体外培养患者外周血单个核细胞显示阻断C5aR可上调Tregs在CD4~+T细胞中的比例。结论:C5a/C5aR可能通过抑制Tregs的分化来介导cGVHD的发生发展。     

Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by "G protein-coupled receptor kinases.

Continuous stimulation of anaphylatoxin receptors C3aR and C5aR with their cognate ligands engenders, within minutes, diminished responsiveness of these receptors. We tested the hypothesis that agonist-induced desensitization involves C3aR and C5aR phosphorylation by G protein-coupled receptor kinases (GRK). When expressed in rat basophilic leukemia cells and exposed to C3a, the C3aR underwent rapid (t(1/2) approximately 15 s), dose-dependent (EC50 approximately 10 nM) and reversible phosphorylation by a kinase refractory to the effects of PKC inhibitors. Phosphoamino acid analysis revealed that the C3aR is phosphorylated on serine and threonine, but not on tyrosine residues. Overexpression of GRK2, GRK3, GRK5 or GRK6 together with C3aR in COS-7 cells enhanced the C3a-induced C3aR phosphorylation 1.5 - 1.9-fold (p < 0.05), but each kinase reduced ligand-stimulated phospholipase C activity differently. Conversely, antibody-mediated inhibition of endogenous GRK2 and GRK3 significantly inhibited C3aR phosphorylation in permeabilized cells. GRK overexpression in cells which co-expressed C5aR and were exposed to C5a resulted in the hyperphosphorylation of the C5aR. These findings are of physiological relevance, since we observed anaphylatoxin-induced phosphorylation of C3aR and C5aR endogenously expressed in human mast cells (HMC-1) which contain significant intracellular levels of GRK2 and GRK3.     

Role of complement anaphylatoxin receptors (C3aR, C5aR) in the development of the rat cerebellum

There is now strong evidence for non-immune or inflammatory functions of complement, notably in the central nervous system. In particular, it has been recently reported that the anaphylatoxin receptors C3aR and C5aR are transiently expressed in the cerebellar cortex of newborn rat, suggesting that anaphylatoxins are involved in the histogenesis of the cerebellum. In the present study, we have investigated the effects of C3aR and C5aR agonists and antagonists on the development of the cerebellum of 11-12-day-old rats in vivo and in vitro. Sub-dural injection of C3aR and C5aR agonists at the surface of the cerebellum transiently modified the thickness of the cortical layers. The C5aR agonist provoked an enlargement of the external granule cell layer (EGL) that was due to increased proliferation of immature granule neurons. Conversely, the C3aR agonist decreased the thickness of the EGL and increased the thickness of the internal granule cell layer (IGL), suggesting that C3a accelerates the migration process of granule cells from the EGL to the IGL. Video-microscopy examination of cultured granule neurons confirmed the role of C3aR in cell motility. These results provide clear evidence for the involvement of anaphylatoxin receptors in the histogenesis of the cerebellar cortex.     

Novel insights in localization and expression levels of C5aR and C5L2 under native and post-transplant conditions in the kidney

AIMS: The complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown. MATERIALS AND RESULTS: Renal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA. C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions, C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation. CONCLUSIONS: This study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a-C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells.     

C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

To study the molecular interplay between TLRs and complement representing ancient danger-sensing mechanisms, we examined the regulation of the C3a/anaphylatoxin C3a receptor (C3aR) axis in normal human epidermal keratinocytes (NHEKs) by treatment with different TLR ligands. Protein staining followed by flow cytometry revealed highly constitutive intracellular expression levels of the C3aR in NHEKs. Stimulation with Poly I:C up-regulated C3aR mRNA and intra- and extracellular expression in NHEKs which showed functional relevance by up-regulating CXCL10 and down-regulating C3 expression in response to C3a. mRNA and protein levels of C3 and protease cathepsin L (CTSL) that can cleave C3 were up-regulated by the TLR3 ligand Poly I:C. Enhanced intracellular expression levels of the biologically active C3 fragment (C3a), in response to TLR3 stimulation were also detectable in NHEKs. Cathelicidin antimicrobial peptide LL-37 potentiated Poly I:C-induced C3aR, C3, and CTSL up-regulation. In conclusion, we point to a role of TLR3 to promote up-regulation of C3aR, C3, and CTSL expression levels and generation of C3a. Our data provide evidence that local generation and activation of complement components as described for T cells or myeloid cells represent a scenario which may take place in a similar way in NHEKs.     

Modulation of C5aR expression on human neutrophils by encapsulated and acapsular Cryptococcus neoformans

Cryptococcus neoformans and cryptococcal surface polysaccharides influenced C5aR expression on human polymorphonuclear neutrophils (PMN). Encapsulated and acapsular strains produced dramatically different effects. Treatment of PMN with acapsular cryptococci up-regulated C5aR expression; whereas treatment with encapsulated cells suppressed C5aR expression. Glucuronoxylomannan (GXM), the principal constituent of the cryptococcal capsule, was responsible for such inhibition. Increased C5aR expression following treatment with acapsular cryptococci was accompanied by increased binding of C5a to PMN, increased superoxide production in response to stimulation with C5a, and an increased chemotactic response to C5a. Conversely, decreased C5aR expression following treatment with encapsulated cryptococci or acapsular cryptococci that had been pretreated with GXM was accompanied by decreased binding of C5a to PMN and a decreased chemotactic response to C5a. Our results raise the possibility that the down-regulation of C5aR expression by encapsulated cryptococci might alter PMN function at the site of cryptococcal infection.     

C5a对中性粒细胞和肾小球内皮细胞粘附行为的影响

目的：为揭示Ｃ５ａ和Ｃ５ａＲ对肾小球内皮细胞（ＧＶＥＣ）－中性粒细胞（ＰＭＮ）粘附的影响及其影响程度。方法：采用免疫组化胶体金银染色检测Ｃ５ａＲ在人ＧＶＥＣ上的表达，并用微管吸吮技术定量测定了经Ｃ５ａ诱导后，ＧＶＥＣ和ＰＭＮ细胞对间的粘附力，同时通过测定粘附的ＰＭＮ中髓过氧化物酶（ＭＰＯ）含量变化，观察不同剂量Ｃ５ａ对粘附的影响。结果：发现ＧＶＥＣ上分布Ｃ５ａＲ，经２０００ｎｇ／ｍｌＣ５ａ刺激，Ｇ     

Novel insights in localization and expression levels of C5aR and C5L2 under native and post-transplant conditions in the kidney

AimsThe complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown.Materials and resultsRenal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA.C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions, C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation.ConclusionsThis study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a–C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells.