Immunobiology of monocytes and macrophages during Chlamydia trachomatis infection

Infections caused by the intracellular bacterium Chlamydia trachomatis are a global health burden affecting more than 100 million people annually causing damaging long-lasting infections. In this review, we will present and discuss important aspects of the interaction between C. trachomatis and monocytes/macrophages.     

黄芪和酸应激对中华鳖幼鳖血清补体C3和C4含量的影响

首次探讨了黄芪和酸应激对中华鳖（Pelodiscus sinensis)幼鳖血清补体C3和C4含量的影响。实验设对照组和实验组,实验组在饵料中按5％添加黄芪原料粉。持续饲喂中华鳖幼鳖4周后取一半样,其余作酸处理24h后再取样,测定补体C3和C4的含量。结果表明,黄芪对补体C3和C4的合成有明显的促进作用;酸应激可导致血清补体C3和C4的含量下降,而黄芪能抵抗酸应激所致的下降。提示黄芪具有抗酸应激的作用。     

饵料维生素E含量对酸应激中华鳖幼鳖血清补体C3和C4的影响

为探讨维生素E(VE)对中华鳖(Pelodiscus sinensis)幼鳖血清补体C3和C4的影响及补体在酸应激下的变化,在6组(对照组,实验Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ组)幼鳖的饵料中依次添加0、50、250、500、1000和5000mg／kg的VE,喂食4周,每组取半数幼鳖经酸应激处理24h。取幼鳖血清,用透射比浊法测定血清补体C3和C4的含量。经和未经酸应激的实验Ⅱ、Ⅲ和Ⅳ组幼鳖血清补体C3的含量均明显高于对照组,实验Ⅰ和Ⅱ组C4含量也明显高于对照组;经酸应激的幼鳖与未经酸应激的比较,对照组和实验Ⅰ组C3和C4的含量显著下降,其余4组没有变化。分析说明,VE在一定剂量范围内能促进血清补体C3和C4的合成,酸应激能导致其下降;而高剂量的VE对酸应激导致的不利影响有拮抗作用。     

Complement Activation Products C3a and C4a as Endogenous Antimicrobial Peptides

All vertebrate species are constantly challenged by infectious agents and pathogens. In order to fight these infectious agents the human host has developed a sophisticated and powerful immune defense. The complement system, which represents the first defense line of innate immunity is activated immediately, within seconds. The activated immune system recognizes and damages an invading microbe, coordinates the host immune response and further orchestrates the adaptive immune response. Activation of the complement system leads to a rapid and amplified response which includes the generation of small peptides like C3a and C4a that display antimicrobial, anti-fungal and anaphylactic activity. Here we report how these antimicrobial peptides are generated during the immune response and summarize the functional mechanisms of these intrinsically generated anti microbial peptides.     

流行性出血热病毒体外直接激活补体C_3的研究

用蔗糖梯度纯化的4株不同来源的流行性出血热(EHF)病毒(陈株、H8205、ALC96、R22)抗原,在体外直接与健康人血清于37℃作用30分钟,用交叉免疫电泳检测血清中C_3激活情况。结果陈株、H8205、R22 3株病毒抗原,可以直接激活补体C_3,电泳后呈现典型降解峰,而ALC96与正常细胞抗原则均为阴性。病毒抗原激活C_3的活性,与病毒核衣壳蛋白(Nucleocapsid Protein)的含量及毒株对乳鼠的半数致死量有关,并可被特异性抗体(IgG)所中和。     

Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria

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维生素C和酸应激对中华鳖幼鳖血清补体C3和C4含量的影响

为研究维生素C对中华鳖(Pelodiscus sinensis)血清补体C3和C4的影响及其在酸应激条件下的变化,我们设置了6个实验组,饵料中维生素C的添加量依次为0、250、500、2500、5000和10000mg／kg,喂食4周后取其血清,用透射比浊法测定酸应激前后中华鳖血清补体C3和C4的含量。结果表明,维生素C添加量为250mg／kg时,血清补体C3的含量与对照组间没有明显不同;维生素C添加量为500、2500、5000和10000mg／kg的4组,血清补体C3的含量明显高于对照组和维生素C添加量为250mg／kg组;维生素C添加量为500mg／kg的一组,血清补体CA含量明显高于其它5组;维生素C添加量为250mg／kg组明显高于10000mg／kg组。酸应激后,补体C3的含量没有明显下降,将维生素C添加量为0、250和500mg／kg的三组并为一组处理,则应激后有明显下降。维生素C添加量为0、250和500mg／kg的3组,血清补体CA的含量在酸应激后明显下降,而维生素C添加量为2500、5000和10000mg／kg的3组,应激后血清补体C4没有明显变化。维生素C和酸应激对中华鳖血清补体C3和CA含量的影响没有交互作用。这说明,维生素C在一定剂量范围内,能提高中华鳖血清补体C3和CA的水平,酸应激能导致其含量降低,而高剂量的维生素C对其下降有颉颃作用[动物学报49(6)：769～774,2003]。     

Stability of C3 Convertase in the Rat Classical Complement Pathway

It has been reported that rat serum complement causes efficient hemolysis of antibody-sensitized sheep erythrocytes (EA) at 20 C but not at 37 C. In connection with this, we demonstrated that C3 convertase of rat complement was significantly unstable at 37 C using purified components of rat complement.     

A Revised Mechanism for the Activation of Complement C3 to C3b: A MOLECULAR EXPLANATION OF A DISEASE-ASSOCIATED POLYMORPHISM*

The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg¹⁰². In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg¹⁰²–Glu¹⁰³² salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg¹⁰²–Glu¹⁰³² salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg¹⁰²-Glu¹⁰³² salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg¹⁰²) and disease-linked C3F (Gly¹⁰²) allotypes of C3b were experimentally explained for the first time.     

Complement component 5a (C5a)

The 74 amino acid glycoprotein, complement component 5a (C5a), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less potent C5adesArg. Acting via a classical G protein-coupled receptor, CD88, C5a and C5adesArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C5a receptors throughout the body allows C5a to elicit a broad range of effects. Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy.     

Antithrombin III,but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting

In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting.Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27 ± 7 years) were pre-incubated with clinically relevant concentrations of AT-III (n = 11) and C1-INH (n = 12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3 h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n = 6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes.This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs.Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.     

中国广东省四个民族C6别型遗传特点——附三个新的罕见变异型报道

应用等电聚焦-免疫印迹法调查了广东省四个民族(汉、苗、黎和回族)C6遗传多态性。广州地区汉族C6等位基因频率分别为:C6*A0.4225,C6*B0.5288,C6*B2 0.0387和C6*R(M91,M92,M11,B21)0.0100。海南岛三个少数民族C6遗传特点与广州汉族相似,均处于Hardy-Weinberg平衡状态。共发现五个罕见基因的杂合子,其中三个等位基因为首次报道。     

Serglycin and secretion in human monocytes

The human monocytic cell line U-937 has been widely used as a model system for human monocytes. The subclone U-937-B has been adapted to serum-free conditions. This particular U-937 clone and its parent clone U-937-1 were used to investigate the role of the proteoglycan serglycin in human monocytes. For this purpose cells were treated with hexyl-β-D-thioxyloside to abrogate proteoglycan expression. U-937-B cells expressed and secreted exclusively chondroitin sulphate proteoglycans, and after treatment with this xyloside they only expressed and released free chondroitin sulphate chains. Western blotting showed that serglycin core protein was present in conditioned medium of control cells, but absent in medium from xyloside-treated cells. Also, serglycin core protein could be detected in the cell fractions of control cells, but not in the cell fractions from xyloside-treated cells. Furthermore, less proteoglycan-associated proteins could be detected in medium from cells incubated with xyloside, suggesting that the absence of secreted sergycin affects the secretion of such proteins. Cells incubated in the presence of xyloside were analyzed by transmission electron microscopy and shown to contain numerous large empty vesicles. The lack of serglycin, the dominant proteoglycan in U-937 monocyte-like cells, consequently, leads to effects on vesicle formation and secretion of some low molecular weight proteins, suggesting that this particular proteoglycan is of importance for secretory processes in human monocytes.     

补体第六成分（C6）在汉族5个群体中的多态分布

用聚丙烯酰胺等电聚焦技术和免疫酶标法,调查分析了汉族5个群体补体第六成分(C6)的遗传多态性,得出基因频率如下。郑州汉族:C6*A 0.4521、C6*B 0.5228、C6*B_2 0.0183、和C6*R 0.0068。兰州汉族:C6*A 0.4612、C6*B 0.5218和C6*B_2 0.0170。呼和浩特汉族:C6*A 0.4452、C6*B 0.5286、C6*B_2 0.0214和C6*R 0.0048。西安汉族:C6*A 0.4899、C6*B 0.4874、C6*B_2 0.0126和C6*R 0.0101。广东梅州客家人:C6*A 0.4569、C6*B 0.5152和C6*B_2 0.0279。C6*R为罕见等位基因之频率。     

A Molecular Switch Governs the Interaction between the Human Complement Protease C1s and Its Substrate,Complement C4

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492–499 and 573–580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.     

Thrombin regulates matrix metalloproteinase-9 expression in human monocytes

We investigated whether thrombin, the final activator of coagulation cascade, regulates expression of matrix metalloproteinases (MMP)-9 in human monocytes.We show that thrombin stimulation induced MMP-9 secretion of monocytes dose- and time-dependently as revealed by gelatin zymography. Real-time RT-PCR and Western blot analysis demonstrated that thrombin up-regulated mRNA and protein levels of MMP-9. Pre-incubation with anti-protease-activated receptor (PAR)-1 or anti-PAR-3 antibody partially inhibited the thrombin-induced MMP-9 secretion. Simultaneous incubation with both showed synergistic effect, indicating the involvement of both receptors in this thrombin effect. BAPTA, a Ca²⁺ chelator, abolished the thrombin-induced MMP-9 secretion, indicating the requirement of Ca²⁺ mobilization in this process. Inhibition of thrombin-induced MMP-9 secretion by either MEK inhibitor or p38 kinase inhibitor revealed that the thrombin effect was mediated by both ERK1/2 and p38 pathways. The activation of NFκB by thrombin as demonstrated by electromobility shift assay was also shown to be critical to the thrombin-induced MMP-9 up-regulation.     

Expression of a Functional Anaphylatoxin C3a Receptor by Astrocytes

Abstract: Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of ¹²⁵I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000–80,000 sites per cell) with an estimated K _D of 1–2 n M . Low-affinity binding sites with a K _D of 209 n M were largely expressed ( n ≥ 4 × 10⁶ sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca²⁺] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca²⁺] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.     

Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disease resulting in progressive cognitive decline. Amyloid plaque deposits consisting specifically of β-amyloid peptides that have formed fibrils displaying β-pleated sheet conformation are associated with activated microglia and astrocytes, are colocalized with C1q and other complement activation products, and appear at the time of cognitive decline in AD. Amyloid precursor protein (APP) transgenic mouse models of AD that lack the ability to activate the classical complement pathway display less neuropathology than do the APPQ+/+ mice, consistent with the hypothesis that complement activation and the resultant inflammation may play a role in the pathogenesis of AD. Further investigation of the presence of complement proteins C3 and C4 in the brain of these mice demonstrate that both C3 and C4 deposition increase with age in APPQ+/+ transgenic mice, as expected with the age-dependent increase in fibrillar β-amyloid deposition. In addition, while C4 is predominantly localized on the plaques and/or associated with oligodendrocytes in APPQ+/+ mice, little C4 is detected in APPQ−/− brains consistent with a lack of classical complement pathway activation because of the absence of C1q in these mice. In contrast, plaque and cell associated C3 immunoreactivity is seen in both animal models and, surprisingly, is higher in APPQ−/− than in APPQ+/+ mice, providing evidence for alternative pathway activation. The unexpected increase in C3 levels in the APPQ−/− mice coincident with decreased neuropathology provides support for the hypothesis that complement can mediate protective events as well as detrimental events in this disease. Finally, induced expression of C3 in a subset of astrocytes suggests the existence of differential activation states of these cells.     

艾拉莫德联合沙利度胺对强直性脊柱炎患者免疫球蛋白及ESR、C3、C4的影响

摘要 目的：探讨艾拉莫德联合沙利度胺对强直性脊柱炎患者免疫球蛋白及血沉（ESR）、补体C3（C3）、补体C4（C4）的影响。方法：选择2016年11月到2019年12月在我院接受治疗的149例强直性脊柱炎患者,采用随机数表法分为联合组（n=75）和单药组（n=74）。单药组给予沙利度胺治疗,联合组在单药组的基础上给予艾拉莫德治疗。比较两组临床疗效、免疫球蛋白、ESR、C3、C4、肿瘤坏死因子（TNF-α）、C反应蛋白（CRP）、临床症状情况及不良反应发生情况。结果：治疗后,两组总有效率比较差异显著（P<0.05）;与治疗前比较,联合组和单药组免疫球蛋白水平检验结果比较无显著差异;治疗后,联合组和单药组IgA、IgG及IgM水平均随着时间的推移而下降,且联合组低于单药组,差异显著（P<0.05）;与治疗前比较,联合组和单药组ESR、C3、C4水平检验结果比较无显著差异;治疗后,联合组和单药组ESR、C3及C4水平均随着时间的推移而下降,且联合组低于单药组,差异显著（P<0.05）;与治疗前比较,联合组和单药组炎症因子水平检验结果比较无显著差异;治疗后,联合组和单药组TNF-α、CRP水平均随着时间的推移而下降,且联合组低于单药组,差异显著（P<0.05）;与治疗前比较,联合组和单药组临床症状比较无显著差异;治疗后,联合组和单药组关节肿胀、晨僵时间均随着时间的推移而下降,且联合组低于单药组,差异显著（P<0.05）;联合组与单药组患者不良反应总发生率相比,差异无统计学意义（P>0.05）。结论：在强直性脊柱炎患者中应用艾拉莫德联合沙利度胺效果显著,可有效改善患者免疫球蛋白及ESR、C3、C4水平,且不增加不良反应。