Fluid-phase Complement Activation Research Articles

Autoantibodies against complement factor B in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder affecting the joints. Many patients carry anti-citrullinated protein autoantibodies (ACPA). Overactivation of the complement system seems to be part of the pathogenesis of RA, and autoantibodies against the pathway initiators C1q and MBL, and the regulator of the complement alternative pathway, factor H (FH), were previously reported. Our aim was to analyze the presence and role of autoantibodies against complement proteins in a Hungarian RA cohort. To this end, serum samples of 97 ACPA-positive RA patients and 117 healthy controls were analyzed for autoantibodies against FH, factor B (FB), C3b, C3-convertase (C3bBbP), C1q, MBL and factor I. In this cohort, we did not detect any patient with FH autoantibodies but detected C1q autoantibodies in four patients, MBL autoantibodies in two patients and FB autoantibodies in five patients. Since the latter autoantibodies were previously reported in patients with kidney diseases but not in RA, we set out to further characterize such FB autoantibodies. The isotypes of the analyzed autoantibodies were IgG2, IgG3, IgGκ, IgGλ and their binding site was localized in the Bb part of FB. We detected in vivo formed FB-autoanti-FB complexes by Western blot. The effect of the autoantibodies on the formation, activity and FH-mediated decay of the C3 convertase in solid phase convertase assays was determined. In order to investigate the effect of the autoantibodies on complement functions, hemolysis assays and fluid phase complement activation assays were performed. The autoantibodies partially inhibited the complement-mediated hemolysis of rabbit red blood cells, inhibited the activity of the solid phase C3-convertase and C3 and C5b-9 deposition on complement activating surfaces. In summary, in ACPA-positive RA patients we identified FB autoantibodies. The characterized FB autoantibodies did not enhance complement activation, rather, they had inhibitory effect on complement. These results support the involvement of the complement system in the pathomechanism of RA and raise the possibility that protective autoantibodies may be generated in some patients against the alternative pathway C3 convertase. However, further analyses are needed to assess the exact role of such autoantibodies.

Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement

PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system.In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products.The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes.Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs.Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins.

Atypical hemolytic uremic syndrome associated with a factor B genetic variant and fluid-phase complement activation: an exception to the rule?

Gain-of-function variants in CFB encoding factor B (FB), a component of the alternative pathway C3 convertase, have been reported in a minority of patients with aHUS and result in massive C3 activation. Zhang etal. describe the functional characterization of a novel FB variant (p.Ser367Arg) that they identified in 2 unrelated aHUS pedigrees who had undetectable C3 levels. The mutant FB caused strong C3 cleavage in fluid-phase but also C3 deposition on cell surface. This commentary addresses the implications of these findings for understanding the complexity of complement-related genetic renal diseases.

Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex-Mediated Membranoproliferative GN.

Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement-mediated C3 glomerulopathy (C3G) and immune complex-mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1-3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment.

Alginate microsphere compositions dictate different mechanisms of complement activation with consequences for cytokine release and leukocyte activation

The inflammatory potential of 12 types of alginate-based microspheres was assessed in a human whole blood model. The inflammatory potential could be categorized from low to high based on the four main alginate microsphere types; alginate microbeads, liquefied core poly-l-ornithine (PLO)-containing microcapsules, liquefied core poly-l-lysine (PLL)-containing microcapsules, and solid core PLL-containing microcapsules. No complement or inflammatory cytokine activation was detected for the Ca/Ba alginate microbeads. Liquefied core PLO- and PLL-containing microcapsules induced significant fluid phase complement activation (TCC), but with low complement surface deposition (anti-C3c), and a low proinflammatory cytokine secretion, with exception of an elevated MCP-1(CCL2) secretion. The solid core PLL-containing microcapsules generated lower TCC but a marked complement surface deposition and significant induction of the proinflammatory cytokines interleukin (IL-1)β, TNF, IL-6, the chemokines IL-8 (CXCL8), and MIP-1α (CCL3) and MCP-1(CCL2). Inhibition with compstatin (C3 inhibitor) completely abolished complement surface deposition, leukocyte adhesion and the proinflammatory cytokines. The C5 inhibitions partly lead to a reduction of the proinflammatory cytokines. The leukocyte adhesion was abolished by inhibitory antibodies against CD18 and partly reduced by CD11b, but not by CD11c. Anti-CD18 significantly reduced the (IL-1)β, TNF, IL-6 and MIP-1α and anti-CD11b significantly reduced the IL-6 and VEGF secretion. MCP-1 was strongly activated by anti-CD18 and anti-CD11b. In conclusion the initial proinflammatory cytokine responses are driven by the microspheres potential to trigger complement C3 (C3b/iC3b) deposition, leukocyte activation and binding through complement receptor CR3 (CD11b/CD18). MCP-1 is one exception dependent on the fluid phase complement activation mediated through CR3.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

C-reactive protein in myocardial infarction binds to circulating microparticles but is not associated with complement activation

C-reactive protein (CRP) is elevated in patients with acute myocardial infarction (AMI). When CRP binds to membrane phospholipids or Fc receptors, it activates the complement system. Recent studies show that CRP can be exposed on cell-derived microparticles (MP) and is associated complement activation. We studied complement activation on circulating MP in AMI patients and healthy controls. MP were isolated from plasma of AMI patients (n=21) and sex- and age-matched healthy individuals (n=10), and analyzed by flow cytometry for bound complement components (C1q, C4, C3) and complement inhibitor and activator molecules (C4bp, CRP, serum amyloid P component, immunoglobulins IgM and IgG). Concurrently, the levels of fluid phase complement activation products and inhibitor and activator molecules were determined. Fluid phase CRP, MP with bound CRP (CRP + MP), and C3 activation products were elevated in AMI patients compared to controls (P=0.032, P=0.031 and P=0.023, respectively), and fluid phase CRP correlated with CRP+ MP (r=0.84, P<0.001). Although CRP+ MP were elevated, they were not associated with C1q+ MP (r=0.32, P=0.174). In contrast, IgG+ MP were associated with C1q+ MP (r=0.73, P<0.001), C4+ MP and C3+ MP (r=0.78 and r=0.87, respectively; both P<0.001), and C4bp (r=0.63, P=0.004). In healthy individuals, CRP+ MP were strongly associated with C1q+ MP (r=0.82, P=0.007), which in turn were associated with C4+ MP and C3+ MP (r=0.68, P=0.032 and r=0.68, P=0.031, respectively). Despite CRP-associated complement activation on the surface of MP in healthy individuals and a strong correlation between MP-bound CRP and fluid phase CRP in AMI patients, the MP-associated complement activation is IgG- but not CRP-dependent in AMI patients.

C3b deposition on human erythrocytes induces the formation of a membrane skeleton–linked protein complex

Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used single-particle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, alpha-spectrin, beta-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeleton-linked DAF-C3b-GPA-band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation.

Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets.

Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets. Here we test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase. Thrombin receptor-activating peptide (TRAP)-6 was used to activate platelets in platelet-rich plasma and blood, anticoagulated with the thrombin inhibitor lepirudin. TRAP activation induced fluid-phase complement activation, as reflected by the generation of C3a and sC5b-9, which could be attenuated by the C3 inhibitor compstatin. Chondroitinase ABC treatment of supernatants from activated platelets totally inhibited the activation, indicating that platelet-derived CS had initiated the complement activation. Furthermore, addition of purified CS to plasma strongly triggered complement activation. C1q was identified as the recognition molecule, as it bound directly to CS, and CS-triggered complement activation could be restored in C1q-depleted serum by adding purified C1q. TRAP activation of whole blood increased the expression of CD11b on leukocytes and generation of leukocyte-platelet complexes. It was demonstrated that these leukocyte functions were dependent on C3 activation and signaling via C5a, as this expression could be inhibited by compstatin and by a C5aR antagonist. We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a.

CELL-DERIVED MICROPARTICLES AND COMPLEMENT ACTIVATION IN PREECLAMPSIA VERSUS NORMAL PREGNANCY

Background Inflammation plays a major role in the vascular dysfunction seen in preeclampsia, and several studies suggest involvement of the complement system. Objectives To investigate whether complement activation on the surface of microparticles is increased in plasma of preeclamptic patients versus healthy pregnant controls. Methods Microparticles from plasma of preeclamptic (n = 10), healthy pregnant (n = 10) and healthy nonpregnant (n = 10) women were analyzed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein [CRP], serum amyloid P component [SAP], immunoglobulin [Ig]M, IgG). Fluid phase complement activation products and activator molecules were also determined. Results Levels of microparticles with bound complement components showed no increase in complement activation on the microparticle surface in preeclamptic women, in line with levels of fluid phase complement activation products. In healthy nonpregnant and pregnant women, bound CRP was associated with classical pathway activation on the microparticle surface, and in healthy pregnant women IgM and IgG molecules also contributed. In preeclamptic women, microparticles with bound SAP and those with IgG seemed to contribute to C1q binding without a clear association to further classical pathway activation. Furthermore, significantly increased levels of microparticles with bound CRP were present in preeclamptic compared with healthy pregnant women (median 178 × 106/L versus 47 × 106/L, P < 0.01), but without concomitant increases in complement activation. Conclusions We found no evidence of increased complement activation on the microparticle surface in preeclamptic women. Microparticles with bound CRP were significantly increased, but in contrast to healthy pregnant and nonpregnant women, this was not associated with increased classical pathway activation on the surface of the microparticles.

Cell-Derived Microparticles and Complement Activation in Preeclampsia Versus Normal Pregnancy

Background Inflammation plays a major role in the vascular dysfunction seen in preeclampsia, and several studies suggest involvement of the complement system. Objectives To investigate whether complement activation on the surface of microparticles is increased in plasma of preeclamptic patients versus healthy pregnant controls. Methods Microparticles from plasma of preeclamptic ( n = 10), healthy pregnant ( n = 10) and healthy nonpregnant ( n = 10) women were analyzed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein [CRP], serum amyloid P component [SAP], immunoglobulin [Ig]M, IgG). Fluid phase complement activation products and activator molecules were also determined. Results Levels of microparticles with bound complement components showed no increase in complement activation on the microparticle surface in preeclamptic women, in line with levels of fluid phase complement activation products. In healthy nonpregnant and pregnant women, bound CRP was associated with classical pathway activation on the microparticle surface, and in healthy pregnant women IgM and IgG molecules also contributed. In preeclamptic women, microparticles with bound SAP and those with IgG seemed to contribute to C1q binding without a clear association to further classical pathway activation. Furthermore, significantly increased levels of microparticles with bound CRP were present in preeclamptic compared with healthy pregnant women (median 178 × 10 6/L versus 47 × 10 6/L, P < 0.01), but without concomitant increases in complement activation. Conclusions We found no evidence of increased complement activation on the microparticle surface in preeclamptic women. Microparticles with bound CRP were significantly increased, but in contrast to healthy pregnant and nonpregnant women, this was not associated with increased classical pathway activation on the surface of the microparticles.

Genetic and environmental factors influencing the human factor H plasma levels.

Factor H is a plasma protein that plays a critical role in the regulation of complement activation in fluid phase and on cellular surfaces. Over the years numerous reports have illustrated the association of factor H deficiencies with chronic renal and infectious diseases. Plasma levels of factor H show a five-fold range of variation in humans (116-562 microg/ml), which may also be relevant to disease susceptibility. To quantify the effects of the genetic and environmental factors responsible for the variation in the factor H plasma levels, we have applied variance-component methods to a family-based sample. Factor H plasma levels show an age-dependent increase ( P<0.0001) and are decreased in smokers ( P<0.0001). Interestingly, the heritability of the factor H trait is very high ( h(2)=0.62+/-0.07; P<0.0001), indicating that 62% of the factor H phenotypic variance is due to the additive effects of genes. On this premise, we conducted a genome-wide linkage screen in order to identify genes regulating the factor H trait. Three genomic regions (1q32, 2p21-24 and 15q22-24) provided suggestive evidence of linkage (LOD scores 2.03, 2.15 and 2.00, respectively) with the plasma levels of factor H.

Perspectives on complement in xenotransplantation

The crucial role of complement and naturally occurring anti-Gal antibodies in hyperacute rejection of pig transplants to Old World monkeys, apes and humans is well established. This rejection can be prevented by manipulating either system. Although cells, tissues or organs from pigs made transgenic for human complement regulatory proteins escape hyperacute rejection, there is an increasing evidence of a role for complement also in the subsequent acute vascular or delayed xenograft rejection. Furthermore, complement contributes in a general manner to ischemia-reperfusion injury (IRI), irrespective of the organ source. Early complement-mediated endothelial cell activation, although not sufficient to induce hyperacute rejection, may contribute to reduced long-time graft performance. Control of fluid-phase complement activation by soluble complement inhibitors might be an important adjuvant to transgenic organs from the time of organ harvesting through the first post-transplant period. Pharmacologic manipulation of complement is a field in expansion, though still in its infancy. Although an optimal transgenic and cloned pig may be close to reality, the role of complement in xenotransplantation needs to be fully elucidated and a treatment regimen for fluid-phase inhibition is warranted. The story of complement in xenotransplantation is not completed but under continuous revision.

Complement activation and expression of membrane regulators in the middle ear mucosa in otitis media with effusion.

The aetiopathogenesis of chronic otitis media with effusion (OME) in children is not yet fully understood. OME is characterized by metaplasia of the epithelium and accumulation of sticky, glue-like effusion in the middle ear containing different mediators of inflammation, including activation fragments of the complement system. Here we examined whether the fluid phase complement activation is reflected in the middle ear mucosa and how the mucosa is protected against the cytolytic activity of complement. Mucosal biopsies from 18 middle ears of children with a history of chronic OME were taken. The biopsies were analysed by immunofluorescence microscopy after staining for complement fragments iC3b/C3c, C3d and C9, and regulators membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and protectin (CD59). There was a strong staining for iC3b/C3c, and a weaker one for C3d and C9 on the surface of the middle ear epithelial cells of OME patients but not in controls without OME. MCP was expressed on the hyperplastic three to four outer cell layers of the epithelium, while CD59 was expressed throughout the middle ear mucosa. The results suggest a strong ongoing complement activation and consequent inflammation in the middle ear cavity. Unrestricted complement damage of the epithelial lining is prevented by the strong expression of MCP and CD59.

Modulation of fluid-phase complement activation inhibits hyperacute rejection in a porcine-to-human xenograft model

YPERACUTE rejection (HAR) of porcine organs is due to classical complement activation by naturally occurring human antibodies to the endothelium. Pigs that are transgenic for human regulators of complement (RCA) seem promising with respect to avoiding HAR, and their use in various studies has brought xenotransplantation closer to clinical reality. However, RCAs are usually expressed on the endothelial cells as clusters and the activation of complement may take place at some distance from RCA localisation. Thus, it should be emphasised that, although RCA pig xenografts may be protected against HAR, a certain degree of complement activation may still contribute to an inflammatory reaction with subsequent impaired graft function. Therefore, efforts should be taken to control the fluid-phase activation of complement, which was the main topic of our studies. We have established an ex vivo perfusion model 1 to investigate the effect of complement manipulation by intravenous immunoglobulin (IVIG), a C3-binding peptide (Compstatin) and C1-inhibitor (C1-INH) on HAR.

Xenotransplantation: how to overcome the complement obstacle?

The xenotransplantation research is driven by the shortage of allografts for transplantation of patients with end-stage organ failure and by lack of success in developing suitable artificial organs. Pigs are now generally accepted to be the donor species of choice, although a possible risk for transmission of xenozoonoses, particularly pig endogenous retroviruses, should be born in mind. A vascular xenograft from pig to human is usually rejected hyperacutely due to naturally occurring antibodies and complement. This hyperacute rejection can be prevented by manipulating with either of these systems. In this review we briefly describe the hyperacute rejection and how it can be prevented by complement modulation. Pigs made transgenic for human complement regulatory proteins seem very promising for future clinical experiments. Control of fluid-phase complement activation should be obtained as well. The latter is discussed based on our own recent studies in this field.

Inhibition of hyperacute pig-to-human xenograft rejection by modulating the fluid-phase complement activation: A.E. Fiane, V. Videm, J.D. Lambris*, O.R. Geiran, J.L. Svennevig and T.E. Mollnes. The National Hospital, Univ. of Oslo; Univ. of Trondheim; *Univ. of Pennsylvania, USA; Univ. of Tromsø, Norway

Inhibition of hyperacute pig-to-human xenograft rejection by modulating the fluid-phase complement activation: A.E. Fiane, V. Videm, J.D. Lambris*, O.R. Geiran, J.L. Svennevig and T.E. Mollnes. The National Hospital, Univ. of Oslo; Univ. of Trondheim; *Univ. of Pennsylvania, USA; Univ. of Tromsø, Norway

Immunoglobulin prolongs survival of pig kidneys perfused ex vivo with human blood.

Intravenous immunoglobulin (IVIG) (Octagam), was used to determine the effect on hyperacute rejection in an ex vivo xenograft model. Six pig kidneys were perfused with IVIG and fresh human AB blood, and six control pig kidneys were simultaneously perfused with albumin and blood from the same donation. The survival of the IVIG-perfused xenografts (median, 6.5 h) was significantly (P = 0.03) longer than the albumin-perfused xenografts (median, 3.5 h). Complement was activated in both groups. The administration of IVIG to the perfused blood resulted in immediate and significantly higher complement activation in the fluid phase as compared with the albumin group. At rejection the fluid phase complement activation was higher in the IVIG group than in the albumin group for C1rs/C1inh complexes, C4bc, Bb and TCC. At the time of rejection both the albumin and the IVIG group demonstrated interstitial tubular haemorrhage, vasculitis or necrosis of glomerular capillaries and glomerular microthrombi. IgM, C1q, C3c, C4 and fibrin were located in arteries and glomeruli and IgG in the interstitium in both groups at rejection. The fluid phase findings are consistent with a modulatory effect of IVIG on complement activation by deviating the classical pathway activation towards the fluid phase. The prolonged survival of the IVIG-perfused kidneys suggests that IVIG may be useful to dampen hyperacute rejection.

Two modes of homologous C3 deposition on Ramos Burkitt's lymphoma cell substrains co-expressing DAF (CD55), CD59, and CR2 (CD21), and on cells lacking them.

We established decay-accelerating factor (DAF)/CD59-positive and -negative substrains of a human B cell line, Ramos, R(DAF+/CD59+) and R(DAF-/CD59-) respectively. Unexpectedly, treatment of R(DAF+/CD59+) cells with Mg2(+)-EGTA-serum resulted in efficient C3 deposition, while treatment of R(DAF-/CD59-) cells did not. All six substrains of R(DAF-/CD59-) cells were CR2-negative, and treatment of the cells with M177 [a membrane cofactor protein (MCP) cofactor-blocking antibody] and/or acidic buffer only minimally affected the extent of C3 deposition. However, when R(DAF-/CD59-) cells were pretreated with M177 followed by incubation with low conductivity (3 mS) Mg(2+)-EGTA-serum, C3 deposition leading to effective cytolysis was provoked. On the other hand, all seven R(DAF+/CD59+) substrains were CR2-positive and could potentially induce C3 autoactivation without cytolysis under physiological conditions. Both M177 and pH again minimally affected the extent of C3 deposition. However, conductivity altered the sensitivity to C3: at under 3.0 mS, R(DAF+/CD59+) cells became almost insensitive to alternative pathway-mediated C3 deposition. Anti-CR2 partially inhibited C3 deposition on R(DAF+/CD59+) cells and C3 deposition was abrogated on the CR2-lacking R(DAF-/CD59-) cells, suggesting that CR2 was associated with the deposition of C3. These results, together with the finding that fluid phase activation of complement did not enhance C3 deposition, suggest that there are two distinct modes of spontaneous homologous C3 deposition on human lymphoma cells. In one case, CR2 or its related molecules participates in C3 deposition overcoming the protective function of DAF/MCP and this type of C3 deposition is maximized under physiological conditions. In the other case, C3 deposition is induced by another homologous C3 activator that becomes functional under low conductivity conditions and the absence of DAF/MCP. These two modes of homologous alternative pathway activation would explain the reported instances of spontaneous C3 deposition on human B lymphoid cell lines (under physiological conditions in the presence of DAF/MCP) and on paroxysmal nocturnal hemoglobinuria erythrocytes (under low conductivity conditions in the absence of DAF/MCP).

Lysis of complement-sensitive Entamoeba histolytica by activated terminal complement components. Initiation of complement activation by an extracellular neutral cysteine proteinase.

Activation of complement by Entamoeba histolytica may be initiated by the extracellular 56-kD neutral cysteine proteinase which cleaves the alpha chain of C3. To determine the relationship between the fluid-phase activation of complement and our observation that only strains isolated from patients with invasive disease are resistant to complement-mediated lysis, we investigated the fate of C3 with recent amebic isolates. When 125I-C3 was incubated with trophozoites in serum, C3 in the fluid phase was cleaved to C3b or C3bi, but the alpha chain of the C3 molecules on the cell surface appeared intact. Since the lysis of nonpathogenic strains takes place in the absence of bound C3b, we demonstrated that this reaction occurs by reactive lysis initiated in the fluid phase: (a) the killing of nonpathogenic strains was enhanced when alternative pathway activation was accelerated by the addition of cobra venom factor; (b) non-pathogenic strains were lysed by purified terminal components; and (c) sera incubated with pathogenic E. histolytica produced passive lysis of chicken erythrocytes. These results demonstrate for the first time that complement-sensitive E. histolytica are lysed by activation of the terminal complement components in the fluid phase where the 56-kD neutral cysteine proteinase cleaves C3, and not by the surface deposition of activated C3.