Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD.     

p62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium

As a signaling hub, p62/sequestosome plays important roles in cell signaling and degradation of misfolded proteins. p62 has been implicated as an adaptor protein to mediate autophagic clearance of insoluble protein aggregates in age-related diseases, including age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE). Our previous studies have shown that cigarette smoke (CS) induces oxidative stress and inhibits the proteasome pathway in cultured human RPE cells, suggesting that p62-mediated autophagy may become the major route to remove impaired proteins under such circumstances. In the present studies, we found that all p62 mRNA variants are abundantly expressed and upregulated by CS induced stress in cultured human RPE cells, yet isoform1 is the major translated form. We also show that p62 silencing exacerbated the CS induced accumulation of damaged proteins, both by suppressing autophagy and by inhibiting the Nrf2 antioxidant response, which in turn, increased protein oxidation. These effects of CS and p62 reduction were further confirmed in mice exposed to CS. We found that over-expression of p62 isoform1, but not its S403A mutant, which lacks affinity for ubiquitinated proteins, reduced misfolded proteins, yet simultaneously promoted an Nrf2-mediated antioxidant response. Thus, p62 provides dual, reciprocal enhancing protection to RPE cells from environmental stress induced protein misfolding and aggregation, by facilitating autophagy and the Nrf2 mediated antioxidant response, which might be a potential therapeutic target against AMD.     

Complement Component C5a Primes Retinal Pigment Epithelial Cells for Inflammasome Activation by Lipofuscin-mediated Photooxidative Damage

Complement activation, oxidative damage, and activation of the NLRP3 inflammasome have been implicated in retinal pigment epithelium (RPE) pathology in age-related macular degeneration (AMD). Following priming of RPE cells, the NLRP3 inflammasome can be activated by various stimuli such as lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated whether products of complement activation are capable of providing the priming signal for inflammasome activation in RPE cells. We found that incubation of primary human RPE cells and ARPE-19 cells with complement-competent human serum resulted in up-regulation of C5a receptor, but not C3a receptor. Furthermore, human serum induced expression of pro-IL-1β and enabled IL-1β secretion in response to lipofuscin phototoxicity, thus indicating inflammasome priming. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum whereas recombinant C5a likewise induced priming. Conditioned medium of inflammasome-activated RPE cells provided an additional priming effect that was mediated by the IL-1 receptor. These results identify complement activation product C5a as a priming signal for RPE cells that allows for subsequent inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration and may provide novel therapeutic targets in this disease.     

Morin hydrate attenuates CSE-induced lipid accumulation,ER stress,and oxidative stress in RPE cells: implications for age-related macular degeneration

Oxidative stress has a key role in the pathogenesis of age-related macular degeneration (AMD). Cigarette smoking is known to the one of the main risk factors of AMD through oxidative stress-mediated endoplasmic reticulum (ER) stress and lipid accumulation in human retinal pigment epithelium (RPE) cells. A number of studies have investigated the benefits of antioxidants in the AMD. However, previous studies have not shown that efficacy of antioxidant in the treatment of AMD. Recent studies demonstrated that morin hydrate (MH) has antioxidant properties, anti-inflammatory, and antiapoptosis effects, however, the protective effects of MH against cigarette smoke extract (CSE)-induced AMD have not been studied in detail. We tested the potential effect of MH against the CSE-induced lipid accumulation in RPE cells and mice RPE layer. Herein, we observed that expose of RPE cells to CSE reduced cell viability, increased the lipid accumulation, ER stress, and oxidative stress. Concomitantly, CSE treatment to mice induced AMD associated histopathological changes, lipid accumulation, ER stress and oxidative stress in RPE layer. MH significantly attenuated cytotoxicity, lipid accumulation, ER stress, and oxidative stress via activated AMPK-Nrf2 signaling pathway in RPE cells and mice RPE layer. In addition, AMPK inhibition reversed MH-induced RPE cell protection against CSE. Thus, we conclude that MH protects RPE cells from CSE through reduced oxidative stress, ER stress, and lipid accumulation via activated AMPK-Nrf2-HO-1 signaling pathway. These findings suggest that MH treatment may be exploited in effective strategy against CSE-induced AMD.     

Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.     

Altered function of factor I caused by amyloid beta: implication for pathogenesis of age-related macular degeneration from Drusen

The results of recent studies have implicated local inflammation and complement activation as the processes involved in the pathogenesis of age-related macular degeneration (AMD). We have demonstrated that amyloid beta (Abeta), which is deposited in drusen, causes an imbalance in the angiogenesis-related factors in retinal pigment epithelial cells. We have also shown that neprilysin gene-disrupted mice accumulate Abeta, and develop several features of AMD. The purpose of this study was to investigate the mechanisms involved in the development of AMD that are triggered by Abeta. Our results showed that Abeta binds to complement factor I which inhibits the ability of factor I to cleave C3b to inactivated iC3b. Factor H and factor I are soluble complement-activation inhibitors, and preincubation of factor I with Abeta in the presence of factor H abolished the ability of Abeta to cleave C3b, and also abolished the ability of factor I to cleave FGR-AMC. In contrast, Abeta did not affect the function of factor H even after binding. The production of iC3b was significantly decreased when C3b and factor H were incubated with the eyes from neprilysin gene-disrupted mice as compared with when C3b and factor H were incubated with eyes from age-matched wild-type mice. These results suggest that Abeta activates the complement system within drusen by blocking the function of factor I leading to a low-grade, chronic inflammation in subretinal tissues. These findings link four factors that have been suggested to be associated with AMD: inflammation, complement activation, Abeta deposition, and drusen.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.     

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.     

A non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration

Age related macular degeneration (AMD) is the most common cause of blindness amongst the elderly. Approximately 10% of AMD patients suffer from an advanced form of AMD characterized by choroidal neovascularization (CNV). Recent evidence implicates a significant role for complement in the pathogenesis of AMD. Activation of complement terminates in the incorporation of the membrane attack complex (MAC) in biological membranes and subsequent cell lysis. Elevated levels of MAC have been documented on choroidal blood vessels and retinal pigment epithelium (RPE) of AMD patients. CD59 is a naturally occurring membrane bound inhibitor of MAC formation. Previously we have shown that membrane bound human CD59 delivered to the RPE cells of mice via an adenovirus vector can protect those cells from human complement mediated lysis ex vivo. However, application of those observations to choroidal blood vessels are limited because protection from MAC- mediated lysis was restricted only to the cells originally transduced by the vector. Here we demonstrate that subretinal delivery of an adenovirus vector expressing a transgene for a soluble non-membrane binding form of human CD59 can attenuate the formation of laser-induced choroidal neovascularization and murine MAC formation in mice even when the region of vector delivery is distal to the site of laser induced CNV. Furthermore, this same recombinant transgene delivered to the intravitreal space of mice by an adeno-associated virus vector (AAV) can also attenuate laser-induced CNV. To our knowledge, this is the first demonstration of a non-membrane targeting CD59 having biological potency in any animal model of disease in vivo. We propose that the above approaches warrant further exploration as potential approaches for alleviating complement mediated damage to ocular tissues in AMD.     

Immunohistochemical localization of complement regulatory proteins in the human retina

Age-related macular degeneration (AMD) is a complex disease. Genetic studies have found strong associations between AMD and variants of several complement pathway-associated genes. The regulation of the complement cascade seems to be critical in the pathogenesis of AMD. In 45 human donor eyes immunohistochemistry was performed using antibodies directed against major regulators of the complement system: complement factor H (CFH), decay accelerating factor (DAF/CD55), complement receptor 1 (CR1/CD35), and membrane cofactor protein (MCP/CD46). All eyes were classified in AMD and controls. 11 eyes were graded as early AMD. 34 eyes were controls. In all eyes staining was found in intercapillary pillars of choroid adjacent to Bruch's membrane for CFH, at the basal surface of RPE cells for MCP, and at the apical side of the retinal pigment epithelium for CR1. DAF immunoreactivity was increased along the inner segments of rod and cone photoreceptor cells at the level of the external limiting membrane Labeling of soft drusen was found for CFH and CR1. In addition, DAF and CR1 showed staining of ganglion cells in all eyes. CFH and particularly MCP showed decreased or absent staining in eyes with early AMD adjacent to Bruch's membrane. The overlapping expression of regulators at the level of Bruch's membrane and the retinal pigment epithelium shows the importance of this site for control of the complement system. Decreased and therefore unbalanced expression of regulators, as shown in this study for CFH and MCP, may ultimately lead to AMD.     

Oxidative stress induces mitochondrial dysfunction and a protective unfolded protein response in RPE cells

How cells degenerate from oxidative stress in aging-related disease is incompletely understood. This study’s intent was to identify key cytoprotective pathways activated by oxidative stress and determine the extent of their protection. Using an unbiased strategy with microarray analysis, we found that retinal pigmented epithelial (RPE) cells treated with cigarette smoke extract (CSE) had overrepresented genes involved in the antioxidant and unfolded protein response (UPR). Differentially expressed antioxidant genes were predominantly located in the cytoplasm, with no induction of genes that neutralize superoxide and H₂O₂ in the mitochondria, resulting in accumulation of superoxide and decreased ATP production. Simultaneously, CSE induced the UPR sensors IRE1α, p-PERK, and ATP6, including CHOP, which was cytoprotective because CHOP knockdown decreased cell viability. In mice given intravitreal CSE, the RPE had increased IRE1α and decreased ATP and developed epithelial–mesenchymal transition, as suggested by decreased LRAT abundance, altered ZO-1 immunolabeling, and dysmorphic cell shape. Mildly degenerated RPE from early age-related macular degeneration (AMD) samples had prominent IRE1α, but minimal mitochondrial TOM20 immunolabeling. Although oxidative stress is thought to induce an antioxidant response with cooperation between the mitochondria and the ER, herein we show that mitochondria become impaired sufficiently to induce epithelial–mesenchymal transition despite a protective UPR. With similar responses in early AMD samples, these results suggest that mitochondria are vulnerable to oxidative stress despite a protective UPR during the early phases of aging-related disease.     

Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.     

CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H₂O₂) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Release of iC3b from apoptotic tumor cells induces tolerance by binding to immature dendritic cells in vitro and in vivo

Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Apoptotic cells are known to activate homologous complement and to be opsonized with iC3b. Since maturation of dendritic cells (DC) can be inhibited by binding of iC3b to the complement receptor 3 (CR3, CD11b/CD18) and because immature DC induce tolerance, we investigated the induction of tolerance after pulsing DC with apoptotic cells in the presence or absence of native serum. Apoptosis in pancreatic carcinoma cells was induced either by heat-stress, chemotherapy or anti-Her2 antibody. Monocyte-derived DC were pulsed with apoptotic cells with or without native serum. DC were analyzed for the maturation state by flow cytometry and the cytotoxic activity was determined. Tolerance was prevented by addition of substances such as anti CD11b or N–acetyl-D-Glucosamine (NADG) which block iC3b binding to CR3. Furthermore, binding of iC3b from apoptotic cells to DC was blocked in a syngeneic pancreatic carcinoma mouse model. All of the former strategies for apoptosis induction resulted in iC3b release. Pulsing DC with apoptotic cells in the presence of serum prevents maturation of DC and induces finally tolerance. This tolerance could be prevented almost completely by blocking the interaction of iC3b with the CR3 receptor. This could be shown as well in an immunocompetent mouse model. Chemo- as well as immunotherapeutical approaches induce apoptosis in tumor cells. Release of iC3b from apoptotic tumor cells prevents fully maturation of DC and immature DC induce antigen-specific silencing or tolerance. Blocking of iC3b-binding could mostly prevent this effect.