C9-mediated killing of bacterial cells by transferred C5b-8 complexes: transferred C5b-9 complexes are nonbactericidal.

The formation of the C5b-9 complex on the outer membrane of complement-sensitive cells of Escherichia coli results in inhibition of inner membrane function and the death of the cell. Cells bearing a precursor of the C5b-9 site, the C5b-8 complex, suffer no loss in viability. Antibiotic-sensitive, complement-sensitive donor cells bearing precursor C5b-8 complexes were incubated with equal numbers of antibiotic-resistant, complement-sensitive acceptor cells that had not been exposed to a complement source. This cell mixture was incubated with 5 mM EDTA for 5 min and then with calcium chloride (20 mM) for various times. The excess calcium ion concentration was effectively reduced with additional EDTA, and the cell mixture was washed and resuspended in buffer. The viability of the acceptor cells was assayed by plating on antibiotic-containing media. C9 was added to the mixture, and the mixture was incubated for 10 min at 37 degrees C and then plated as described above. It was found that the acceptor cells were killed by the addition of purified C9 only after incubation with donor cells bearing C5b-8 sites during the transfer procedure. This indicates that precursor C5b-8 sites that support C9-mediated killing could be transferred between cells. No loss in viability was detected for acceptor cells subjected to the procedure described above in the presence of donor cells bearing complete C5b-9 complexes, formed prior to mixing with acceptor cells for the transfer procedure.     

Immunohistochemical Detection of the Membrane and Fluid-phase Terminal Complement Complexes C5b-9(m) and SC5b-9

Activation of the terminal pathway of complement on a membrane results in the generation of the membrane-damaging terminal C5b-9(m) complement complex, whereas the non-lytic water-soluble SC5b-9 complex is formed when complement is activated in the fluid phase. Both forms of the terminal complement complex (TCC) can be immunohistochemically detected, but not distinguished, by antibodies recognizing neoantigens in the complexes. By means of monoclonal antibodies against C9 neoantigens and against the S-protein, it was demonstrated that deposits of the TCC in tissue sections may be either in the form of C5b-9(m) or SC5b-9. The consequences of this for the interpretation of the histochemical data and the terminology of the two complexes are discussed.     

Sublytic C5b-9 complexes induce proliferative changes of glomerular mesangial cells in rat Thy-1 nephritis through TRAF6-mediated PI3K-dependent Akt1 activation

The proliferation of glomerular mesangial cells (GMCs) and secretion of extracellular matrix (ECM) in rat Thy-1 nephritis (Thy-1N), resembling human mesangioproliferative glomerulonephritis (MsPGN), have been studied for many years, but the mechanisms, especially the role of signalling pathway activation and its regulation in GMCs triggered by sublytic C5b-9 complexes in Thy-1N rats remain largely unclear. In the study, the proliferation of GMCs and production of ECM as well as the role of PI3K/Akt and its regulation, both in GMCs induced by sublytic C5b-9 (in vitro) and in the renal tissues of rats with Thy-1N (in vivo), were determined and the results revealed that GMCs proliferation and ECM secretion, both in vitro and in vivo, were notably increased, and that PI3K/Akt1 activation and its regulation, such as TNF receptor-associated factor 6 (TRAF6)-mediated Akt1 ubiquitination and PI3K-dependent Akt1 phosphorylation, were involved in the process of Thy-1N induction. On the other hand, silence of the TRAF6, PI3K or Akt1 genes could obviously diminish the proliferative damages and urinary protein secretion of Thy-1N rats. Together, these data implicated that sublytic C5b-9 complexes in Thy-1N rats could promote GMCs proliferation and ECM production through TRAF6-mediated PI3K-dependent Akt1 activation, in which the ubiquitination and phosphorylation of the Akt1 signal molecule played an important role in the initiation and development of the proliferative changes in the rats with Thy-1N.     

Complement C3 gene expression and regulation in human glomerular epithelial cells.

Extra-hepatic synthesis of complement is thought to mediate local tissue inflammatory injury. To investigate this phenomenon in the glomerular epithelial cell (GEC), we examined the biosynthesis and regulation of gene expression of the third component of complement in isolated human GEC derived from normal tissue. Metabolic labelling and immunoprecipitation studies demonstrated that C3 protein was synthesized, processed and secreted by GEC under basal conditions. The secreted C3 alpha and beta polypeptide chains had identical electrophoretic mobilities with those of hepatic C3. Examination of cellular RNA using semi-quantitative polymerase chain reaction (PCR) showed that C3 gene expression was present in unstimulated GEC and was increased by stimulation with interferon-gamma (IFN-gamma) in a time- and dose-dependent manner. Tumour necrosis factor-alpha (TNF-alpha), while mediating an increase in monocyte U937 C3 expression, revealed no evidence of regulation of GEC C3 gene expression. These results indicate that human GEC spontaneously express the C3 gene and that increased gene expression is regulated by IFN-gamma. These observations may reflect part of a wider mechanism of protection against or mediation of local, immune-mediated tissue injury.     

Complement C5b-9 Induces Receptor Tyrosine Kinase Transactivation in Glomerular Epithelial Cells

In the passive Heymann nephritis (PHN) model of membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated via production of eicosanoids. Using rat GEC in culture, we demonstrated that sublytic C5b-9 induced tyrosine phosphorylation of the epidermal growth factor receptor (EGF-R), Neu, fibroblast growth factor receptor-2, and hepatocyte growth factor receptor. In addition, C5b-9 stimulated increases in tyrosine(204) phosphorylation of extracellular signal-regulated kinase-2 (ERK2), as well as free [(3)H]arachidonic acid (AA) and prostaglandin E(2) (PGE(2)). Phosphorylated EGF-R bound the adaptor protein, Grb2, and the EGF-R-selective tyrphostin, AG1478, blocked the C5b-9-induced ERK2 phosphorylation, [(3)H]AA release, and PGE(2) production by 45 to 65%, supporting a functional role for EGF-R kinase in mediating the activation of these pathways. Glomeruli isolated from rats with PHN demonstrated increases in ERK2 tyrosine(204) phosphorylation and PGE(2) production, as compared with glomeruli from control rats, and these increases were partially inhibited with AG1478. Thus, C5b-9 induces transactivation of receptor tyrosine kinases, in association with ERK2 activation, AA release, and PGE(2) production in cultured GEC and glomerulonephritis in vivo. Transactivated tyrosine kinases may serve as scaffolds for assembly and/or activation of proteins, which then lead to activation of the ERK2 cascade and AA metabolism.     

补体C5b-9复合物刺激大鼠肾小球系膜细胞合成NO的机制探讨

目的:探讨人C5b-9复合物刺激大鼠肾小球系膜细胞(MC)合成一氧化氮(NO)的机制。方法:用人C5b-9复合物刺激培养的大鼠肾小球MC诱生肿瘤坏死因子α(TNFα)和白细胞介素1β(IL-1β),并用抗TNFα或抗IL-1β单克隆抗体进行处理。在上述基础上,分析处理3 h、6 h及24 h时与NO升高有关的某些指标的变化。结果:经C5b-9复合物刺激6、24 h后的MC(C5b-9组)产生TNFα明显高于对照组,并能被TNFα单抗逆转。用C5b-9复合物刺激MC未见IL-1β的产生。另用C5b-9刺激3 h时可见MC表达iNOS mRNA,而在刺激6 h和24 h时,MCiNOSmRNA表达,MC内cGMP含量及培养上清液中NO³_-/NO²_-含量均显著高于对照组。不过,C5b-9刺激时加用TNFα单抗处理,这些指标在6 h、24 h时均较C5b-9组低。结论:C5b-9复合物早期(3 h)能诱导MC表达iNOS mRNA,而6h后NO的升高则与MC释放的TNFα作用有关。     

Induction of mediator release from human glomerular mesangial cells by the terminal complement components C5b-9.

Exposure of cultured human glomerular mesangial cells (GMC) to normal human serum and an activator of the complement system results in rapid uptake of the terminal complement proteins C5b-9 by the cells. This 'innocent bystander' complement attack, however, does not result in cell killing, but in the stimulation of the GMC to release prostaglandin E (PGE), interleukin 1 (Il-1) and tumor necrosis factor (TNF). Endogenously synthesized Il-1 in turn activates PGE release, indicating that the C5b-9 attack initiates an autocrine feedback stimulation. Together with the fact that C5b-9 is found in many forms of glomerulonephritis, the data point to a role of the terminal complement proteins in the initiation and perpetuation of an inflammatory response.     

补体C5b-9复合物诱导大鼠肾系膜细胞iNOS mRNA表达的实验研究

观察人血清补体C5b 9复合物对大鼠肾小球系膜细胞 (MC)表达诱生性一氧化氮合酶 (iNOS)mRNA的影响。方法 :首先提取人血清补体C5b 9复合物 ,然后用人C5b 9复合物刺激培养的大鼠MC ,检测MC在受C5b 9复合物刺激后3、6、2 4和 48h时iNOSmRNA的表达情况。同时检测其培养上清液中一氧化氮 (NO)代谢产物———硝酸根 (NO3- )和亚硝酸根(NO2- )含量的变化。结果 :用人C5b 9复合物刺激培养大鼠的肾MC能使其表达iNOSmRNA ,培养上清液中NO3- NO2- 含量也明显升高。人C5b 9复合物对MC的刺激作用能部分被相应的抗人C5b 9复合物抗体和RNA合成抑制剂———放线菌素D所抑制。结论 :人补体C5b 9复合物具有刺激大鼠肾MC合成NO的作用。     

Complement Lysis: the Ultrastructure and Orientation of the C5b-9 Complex on Target Sheep Erythrocyte Membranes

The C5b-9 complex derived from human serum and assembled on target sheep erythrocyte membranes is a thin-walled cylinder rimmed by an annulus at one end. The total height of the cylinder is 150 A, towards which the annulus contributes 30 A. The cylinder has an apparently uniform internal diameter of 100 A. The external diameter of the annulus is 200 A. The classical complement 'rings' visualized on membranes after complement lysis represent such C5b-9 cylinders perpendicularly oriented on the membranes. The thin-walled cylinder is anchored in the membrane matrix and the annulus located in the exterior membrane glycocalyx. At the sites of attachment of the C5b-9 complexes, the continuity of the membrane bilayer is disturbed and the presence of trans-membrane pores is indicated. The data essentially support the 'doughnut' theory of complement lysis.     

Immunohistochemical detection of the terminal C5b-9 complement complex in children with glomerular diseases.

Kidney biopsies were obtained in 28 children with glomerular diseases and studied using indirect immunofluorescence and immunoperoxidase for IgG, IgA, IgM, C1q, C3c, C4, fibrinogen and the neoantigens of the terminal C5b-9 complement complex. C5b-9 deposits were detected in glomeruli of 14, in tubules of eight and in vessels of 12 cases. Patients with C5b-9 deposits fared worse than those without such deposits, even with the same histopathological type of glomerulonephritis. The C5b-9 complex suggests an in situ complement activation.     

The membrane attack complex, C5b-9, up regulates collagen gene expression in renal tubular epithelial cells

Evidence suggesting a direct role for proteinuria in the pathogenesis of renal tubulointerstitial fibrosis is accumulating. However the mechanism by which proteinuria leads to injury is unknown. In proteinuric states complement proteins are filtered through the glomerulus and could contribute to the tubular damage. The aim of this study was to investigate the role of complement activation in the progression of interstitial fibrosis. To determine whether complement activation may be responsible for the pro-fibrotic response that occurs in the tubulointerstitial compartment we stimulated primary cultures of proximal tubular epithelial cells with membrane attack complex, C5b-9. This led to increased mRNA concentrations of both collagen type IV and its intracellular chaperone, Heat Shock Protein 47 (HSP47). To determine whether this occurred in vivo Adriamycin was used to induce proteinuria in female Balb/c mice. The expression of collagen type IV and HSP47 was increased in proteinuric mice compared to control mice. In proteinuric mouse kidney, C3 was deposited at sites of tubulointerstitial injury and there was a relationship between C3 deposition and immunochemical staining for collagen type IV and HSP47. In situ hybridization suggested that the renal tubular epithelium was actively expressing HSP47 mRNA and, by implication, excess collagen. These observations support the hypothesis that complement activation on tubular epithelial cells can directly increase the pro-fibrotic process associated with tubulointerstitial damage.     

Quantitative measurement of SC5b-9 and C5b-9(m) in infarcted areas of human myocardium.

Previous immunohistochemical work has indicated that terminal C5b-9 complement complexes are selectively deposited in infarcted areas of human myocardium. In the present study, we sought to quantify C5b-9 levels in myocardial tissue, and to differentiate between the membrane-bound C5b-9 (m) and the cytolytically inactive SC5b-9 complex. Paired tissue specimens from infarcted and non-infarcted myocardium were obtained from 36 autopsies. The homogenized and washed tissues were extracted with n-octyl-beta-D-glucopyranoside (octylglucoside) detergent, and the concentrations of C5b-9 in the extracts were determined by ELISA. Membrane-derived C5b-9 (m) and SC5b-9 were differentiated from each other on the basis of their characteristic sedimentation behaviour in sucrose density gradients. It was found that infarcted myocardial tissue contained on average an approximately three-fold higher concentration of C5b-9, compared with non-infarcted tissue. This increase was due in part to an increase in levels of C5b-9 (m). The results corroborate previous immunohistochemical data and show that complement activation occurs to completion with the generation of potentially cytotoxic C5b-9 complexes in infarcted myocardial tissues.     

Analysis of C5b-8 binding sites in the C9 molecule using monoclonal antibodies: participation of two separate epitopes of C9 in C5b-8 binding.

C5b-8 binding sites in C9 were examined using mAbs raised against C9. Among 16 mAbs, two, designated P40 and X197, blocked C9-mediated EAC1-8 lysis. C9 pretreated with the mAbs failed to bind to EAC1-8 at 4 degrees C. In addition, the mAbs became inaccessible to the C9 that had been incorporated into EAC1-8 at 4 degrees C. These findings suggest that C9 binding to EAC1-8, but not its membrane spanning or polymerization, is blocked by mAbs. By immunoblotting analysis using alpha-thrombin proteolytic fragments derived from C9 [a N-terminal fragment of mol. wt 25,000 (C9a) and a C-terminal one of mol. wt 37,000 (C9b)] and tryptic fragments of C9 (mol. wts 53,000 (C9a') and 20,000 (C9b')), the epitopes of P40 and X197 were mapped to the N-terminal and C-terminal regions of C9b, respectively. Both P40 and X197 bound to the C9 polymerized with Zn2+ in the fluid phase, whereas X197 but not P40 reacted with the membrane attack complex (MAC) formed on membranes. The results suggest that two distinct epitopes are involved in C9 binding to EAC1-8, and behave in a different manner for globular C9 bound to EAC1-8 at 4 degrees C, C9 assembled in MAC, or poly-C9 induced by Zn2+. These mAbs may be useful in clarifying the conformational states of C9 and in analyzing the molecular interaction between C9 and its inhibitors.     

Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement

Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5-C9 in pooled human serum by insulin or zymosan activation generates 220 +/- 40 micrograms/ml SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100-600 ng/ml SC5b-9, corresponding to 0.04-0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37 degrees C always led to spontaneous generation of SC5b-9; concentrations ranged from 490-4725 ng/ml after 60 min, 37 degrees C, with a mean of 1848 +/- 1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.     

Detection and Quantification of the Terminal C5b-9 Complex of Human Complement by a Sensitive Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay for detection and quantification of the terminal complexes (SC5b-9 and membrane attack complex) of human complement is described. We separate the complex from the native complement components, to use antibodies against the native components in a 'double-antibody sandwich' technique. It is thereby possible to detect the terminal complement complex in solution without the requirement of specific antibodies against the neoantigens. The results show that the assay is both sensitive and specific. Evidence is presented that a terminal complement complex occurs in a normal plasma pool. The terminal complement complex may be valuable for evaluating both the physiology and pathophysiology of the complement system in vivo.     

Sublytic complement C5b-9 complexes induce thrombospondin-1 production in rat glomerular mesangial cells via PI3-k/Akt: association with activation of latent transforming growth factor-beta1

Mesangial cell proliferation is a common cellular response to a variety of different types of glomerular injury. Complement C5b-9 is a prime candidate to mediate mesangial cell proliferation, especially sublytic C5b-9, which can induce the production of multiple inflammatory factors and cytokines. Transforming growth factor (TGF)-beta1 plays a major role in the accumulation of extracellular matrix (ECM), while thrombospondin (TSP)-1 has been identified as an activator of latent TGF-beta1 in an in vitro system. Using rat glomerular mesangial cells (GMCs) as a model system, we assessed the effect of sublytic C5b-9 on the expression of TSP-1 and TGF-beta1 and explored the relevant pathway of signal transduction. First, we ensured the concentrations of anti-Thy1 antibody and complement, which were regarded as a sublytic C5b-9 dose, and examined whether the sublytic C5b-9 induced expression of TSP-1 in rat GMCs which, in turn, activated latent TGF-beta1 by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, we investigated the role of the PI3-k/Akt pathway in sublytic C5b-9-induced TSP-1 production in rat GMCs by Western blot analysis. The addition of sublytic C5b-9 (5% anti-Thy1 antibody and 4% normal serum) to rat GMCs induced activation of latent TGF-beta1 via TSP-1. The addition of sublytic C5b-9 apparently increased the protein of Akt phosphorylation, whereas PI3-k inhibitor LY294002 could clearly reduce the increase of TSP-1 induced by sublytic C5b-9. These results indicate that TSP-1 is an activator of latent TGF-beta1 in sublytic C5b-9-induced rat GMCs; furthermore, the PI3-k/Akt signal transduction pathway may play a key role in sublytic C5b-9-induced TSP-1 production.     

补体C5b-9复合物促进树突状细胞成熟

探讨补体C5b-9复合物对树突状细胞成熟及免疫学功能的影响。rhGM-CSF+rhIL-4体外诱导单核细胞分化为不成熟树突状细胞,体外于不成熟树突状细胞表面用补体蛋白纯品组装CSb-9,37℃,5%CO_2温育96 h,流式分析细胞表型及抗原捕获能力;与同种异体CD4~+和CD8~+T细胞共培养检测其免疫刺激功能;ELISA检测细胞因子分泌。结果显示,亚溶解型C5b-9处理DC表面标志CD83、HLA、CD80、CD86、B7-H1、B7-H3、B7-H4以及BTLA等表达上调;DC分泌IL-12及TNF-α上调,抗原捕获能力降低;C5b-9处理DC刺激CD4~+T细胞活化及分泌IFN-γ、IL-2能力增强。结论:补体C5b-9复合物可以促进树突状细胞成熟,衔接天然免疫和特异性免疫。     

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization

Sublytic C5b‐9 has been described as a pro‐inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen‐specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b‐9 in DC function has not been described. In this report, we use in vitro assembled functional C5b‐9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b‐9. This was demonstrated by up‐regulation of CD83, HLA‐antigens and costimulatory molecules, including CD80, D86, B7‐H1, B7‐H3, B7‐H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)‐12 and tumor necrosis factor‐α was increased while the capacity for antigen uptake (FITC‐Dextran and Lucifer Yellow) was reduced in C5b‐9‐treated DC. Mixed lymphocyte reactions indicated that C5b‐9‐activated DC acted as stimulators that significantly promoted CD4⁺ T cell activation and elicited production of cytokines, including interferon‐γ and IL‐2. Interestingly, C5b‐9‐treated DC also orient CD4⁺CD45RA⁺ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b‐9, suggesting that C5b‐9 bridges innate and acquired immunity by inducing DC maturation.     

C5b-7 and C5b-8 precursors of the membrane attack complex (C5b-9) are effective killers of E. Coli J5 during serum incubation

The finding that C9-deficient sera (C9D) can kill serum sensitive strains of Gram-negative bacteria by us and other investigators, questions the role of C9 in the membrane attack complex as necessary for cell death. In these studies we have demonstrated that C5b-8 complexes generated on E. coli J5 during incubation in C9-depleted and C9-neutralized sera are effective in killing Gram-negative bacteria. In the same study, we extended our investigations to show that the deposition of C5b-7 complexes (from C8-deficient [C8D], C8 depleted and C8-neutralized sera) is also effective in killing Gram-negative bacteria. In all cases, these studies demonstrated that when E. coli J5 was incubated with C8D, C9D and pooled normal human serum [PNHS], deposited C5b-9 complexes from PNHS produced more killing than C5b-7 or C5b-8 complexes alone. These experiments clearly demonstrated that C5b-7 and C5b-8 complexes are bactericidal and that multimeric C9 within C5b-9 is not an absolute requirment for inner membrane damage and cell death of Gram-negative bacteria.