Thromboxane B2 synthesis in human platelets induced by the late complement components C5b-9

Sublytic doses of purified C5b6, C7, C8 and C9 induce the release of thromboxane B2 (TXB2) in human platelets. In the present study, we attempted to analyze the signal by which C5b-9 triggers the prostanoid synthesis in platelets. TXB2 release was accompanied by liberation of Ca++ from intracellular stores. Influx of Ca++ was not observed, indicating that C5b-9 did not form a channel large enough to permit passage of Ca++ ions. Therefore, channel formation is apparently not required for cell stimulation.     

Role of complement C9 and calcium in the generation of arachidonic acid and its metabolites from rat polymorphonuclear leukocytes

We have previously shown that antibody-sensitized mouse peritoneal macrophages release arachidonic acid (C20:4) and its oxygenated derivatives when treated with complement, and that the major part of the release depended on the terminal complement complexes (TCC). To further delineate the process(es) responsible for this release we have extended our studies to rat peritoneal poly-morphonuclear leukocytes (PMNs). Experiments were performed with antibody-sensitized rat PMNs labeled with [³H]C20:4 and carrying the TCC, C5b-7, C5b-8 or C5b-9. In contrast to the results of other studies, production of leukotriene B₄ (LTB₄), the major radiolabeled derivative, was strictly dependent on the presence of C9. However, low levels of C20:4 and prostaglandins (PGs) were produced prior to the C5b-9 stage. Kinetic studies demonstrated that release of LTB₄ was rapid; the initial release occurred within 4–6 min and a second rise in release coincided with cell death. Virtually all the LTB₄ produced was released as we found no evidence of retention of intracellular LTB₄ at either the C5b-8 or C5b-9 stages. In the absence of extracellular calcium, the release of LTB₄ was completely abolished and the release of C20:4 and PGs was drastically reduced. [³H]C20:4-labeled PMNs carrying C5b-9 did release substantial amounts of radiolabeled material in the presence of EGTA; however, the majority of this lipid was in the form of intact phospholipid and triglyceride. These results indicate that release of C20:4 and its oxygenated derivatives from rat PMNs is (1) dependent on the participation ofC9 in the preexisting C5b-8 complex in the cell membrane, and (2) largely dependent on the presence of calcium.     

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。     

Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.     

Formation of the Membrane Attack Complex of Complement (MAC) on Erythrocytes from Monocyte-Produced Terminal Complement Components

By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.     

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.     

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.     

Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.     

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.     

Prelesional complement activation in experimental atherosclerosis. Terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits

Cholesterol accumulation in the tunica intima of large and medium sized arteries is a characteristic of atherosclerotic lesion development. Cholesterol activates the complement system in vitro generating C5b-9 complexes, and C5b-9 antigens have been observed in atherosclerotic lesions. We therefore investigated whether complement activation correlates temporally with accumulation of intimal cholesterol in vivo. Frozen sections of aortic tissue from rabbits fed a 0.3% cholesterol diet for 0, 2, 6, and 10 weeks were stained with antibodies directed against neoantigens of the C5b-9 complex. Lipids were detected with oil red O, filipin, and antibodies against apoprotein B. C5b-9 complexes were observed to colocalize with extracellular lipid in the subendothelial space beginning at 2 weeks on the diet and extending past 10 weeks. Quantitation of aorta extracted C5b-9 complexes by an enzyme-linked immunosorbent assay revealed a progressive increase in aortic C5b-9 content as a function of time on the cholesterol diet. An approximately 30-fold increase in aortic C5b-9 occurred between 0 and 10 weeks on the diet, whereas plasma C5b-9 remained at normal levels throughout this time. Aortic cholesterol and triglyceride levels approximately doubled between 0 and 10 weeks on the diet and plasma cholesterol increased nearly 50-fold. Intimal C5b-9 deposition preceded monocyte infiltration and foam cell development. The data indicates that early cholesterol accumulation in the aortic intima results in complement activation. Since complement activation products are chemotactic for monocytes and promote their adhesion to the endothelium, complement may provide a mechanism by which monocytes are attracted to arterial regions of lipid accumulation.     

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。     

Quantitation of activation of the human terminal complement pathway by ELISA

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigenic determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.     

Complement S-protein (vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes.

It has been assumed that S-protein (vitronectin) associates with terminal C5b-9 complement complexes only when the latter fail to attach to target lipid bilayers, thereby forming inactive fluid-phase SC5b-9 complexes. Using monoclonal anti-S-protein antibodies, we show here that a minor portion of C5b-9 complexes associated with both homologous and heterologous cells contain S-protein. This conclusion derives from Western blot analyses, from the sedimentation behaviour of solubilized S-protein, and from the fact that the protein co-immunoprecipitates with C5b-9(m). Association of S-protein with C5b-9(m) takes place primarily at the stage of C9-binding. An average of less than or equal to 0.4 moles of S-protein are estimated to be present per mole C5b-9(m). Hence, only a fraction of C5b-9 complexes contain S-protein. The function of cell-bound S-protein is unknown. Haemolytic titrations with purified components failed to demonstrate any protective effect of S-protein on the lysis of sheep or human erythrocytes by C5b-9. S-protein bound to complement-lysed homologous or heterologous cells is readily detectable by conventional immunocytochemical staining. We conclude that differentiation between tissue-deposited fluid-phase C5b-9 and membrane C5b-9 complexes cannot be made on the basis of immunohistological stainings for S-protein alone.     

Does complement kill E. coli by producing transmural pores?

Three lines of evidence are presented to indicate that C5b-9 kills serum-sensitive E. coli K 12 cells by generating functional pores across the outer and inner bacterial membrane. First, viable cells carrying C5b-8 complexes are impermeable to o-nitrophenyl-beta-D-galactoside (ONPG), but lose viability and become permeable to this marker upon post-treatment with purified C9 in the absence of lysozyme. Cells killed with colicin E1 or gentamicin are also impermeable to ONPG but take up the marker if they are post-treated with lysozyme-free serum. Second, killing by C5b-9 is highly effective, deposition of only a small number of complexes being lethal. This has been demonstrated in experiments where viable cells carrying 2000-4000 C5b-7 complexes per CFU were permitted to multiply in broth culture, and the daughter generations subsequently treated with purified C8 and C9. Fifty percent killing was observed in the fifth to sixth generation, corresponding to a dilution of C5b-7 complexes to 50-100 molecules/CFU. In the presence of 2 mM EDTA, further dilution of C5b-7 down to 8-30 complexes/CFU still caused 50% killing of daughter cells. Third, treatment of C5b-7 cells with purified CC8 and C9 results in the release of intracellular K+, which commences immediately after addition of C8/C9. This was shown in experiments where C5b-7 cells were packed to high density in saline, post-treated with C8 + C9, and K+ directly measured in the cell supernatants. Based on these results, we propose that C5b-9 pores deposited in the outer bacterial membrane periodically fuse with the inner membrane, the transmural pores thus generated permitting rapid K+ efflux, with cell death ensuing through the collapse of membrane potential.     

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins.     

Stimulation of human rheumatoid synovial cells by non-lethal complement membrane attack.

The effects of non-lethal complement attack on cultured human rheumatoid synovial cells have been investigated by measuring a variety of parameters. Within 3-4 min of initiating non-lethal complement membrane attack there was a rise in reactive oxygen metabolite release from cultured synovial cells, which slowly returned to basal levels over a period of 45 min. The response was dependent on the formation of the complete C5b-9 complex. Prostaglandin E2 was also released during non-lethal attack in a biphasic manner, an early phase of release occurring within the first hour and a second, larger phase commencing at 4 hr and rising to levels of over 1000 ng/10(6) cells at 24 hr, compared to control levels at this time of less than 100 ng/10(6) cells. This response was dependent on the formation of the C5b-8 complex but did not require C9. Removal of extracellular calcium reduced release of prostaglandin E2 to background levels, and inclusion of an inhibitor of protein synthesis abolished the second phase of release but not the first phase. Non-lethal attack caused release of small amounts of leukotriene B4 but no detectable release of tumour necrosis factor.     

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.     

Immunohistochemical study of complement S protein (Vitronectin) in normal and diseased human kidneys: relationship to neoantigens of the C5b-9 terminal complex.

The localization of S protein (Vitronectin) antigen was studied by indirect immunofluorescence and immunoelectron microscopy in normal adult human kidneys and in biopsy specimens from patients with a wide range of renal diseases, and compared with that of neoantigens of the C5b-9 terminal complement complex. S protein antigen was diffusely present in arteriolar perimyocytic matrices, the glomerular basement membrane and mesangial matrix, and tubular basement membranes in the cortex of normal and diseased kidneys without superimposable staining for C5b-9 neoantigens. Cell remnants embedded in normal and sclerotic extracellular matrices expressed S protein antigen and also stained for C5b-9 neoantigens. Several lines of evidence suggested that S protein present in connective matrices most likely represents S protein or C5b-9 complexes trapped from the circulation. Glomerular immune deposits and arteriolar hyalin deposits which contained C5b-9 neoantigens also contained S protein antigen in the same location. In a few specimens from patients with membranous nephritis stage I and IgA nephropathy, immune deposits contained neither detectable C5b-9 neoantigens nor S protein. The observed strong co-staining of immune deposits for S-protein and C5b-9 caution against the generalization that C5b-9 within glomerular immune deposits represent membrane-bound cytolytic complement complexes.     

Effect of osmotic protection on nucleated cell killing by C5b-9: cell death is not affected by the prevention of cell swelling

Formation of C5b-9 channels in the plasma membrane can lead to erythrocyte lysis or nucleated cell death. Lysis of erythrocytes by complement occurs as a result of colloid osmotic swelling and rupture of the plasma membrane, due to the unregulated flux of ions and water through C5b-9 channels. This colloid osmotic mechanism of lysis is largely based on the evidence that the extent of hemolysis is reduced, when macromolecules are placed in the medium to balance the osmotic gradient created by intracellular macromolecules, which are too large to diffuse through complement channels. The role of colloid osmotic deregulation, as a cause of nucleated cell killing by C5b-9, however, has been recently questioned [Kim S., Carney D. F. and Shin M. L. J. Immun. 138, 1530 (1987)]. In the present study, we investigated the effect of osmotic protection, with an 81,000 mol. wt dextran or bovine serum albumin, on Ehrlich cell killing by complement channels. The results indicated that prevention of cell swelling by dextran did not reduce the extent or rate of nucleated cell killing by either small (C5b-9l), or large (C5b-9m), complement channels when assessed by vital dye stain. The release of cytoplasmic lactate dehydrogenase as an alternative measure of cell death, however, was retarded and/or reduced, in the presence of dextran or albumin, at concns that prevented cell swelling. These results indicate that C5b-9 can kill nucleated cells effectively, in the absence of colloidal osmotic cell swelling, and that release of cytoplasmic macromolecules may not be a reliable indicator of cell death, when osmotic protectants are employed.