Regulation of complement functional efficiency by histidine-rich glycoprotein.

The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.     

Inhibitors of complement derived from the erythrocyte membrane in paroxysmal nocturnal hemoglobinuria

Extracts of the membranes of normal red cells and red cells from all subpopulations of paroxysmal nocturnal (PNH) red cells inhibited antibody-mediated complement activation. These extracts were shown to accelerate decay of the complement complex. C42, and the relative amount of inhibitory activity was similar in normal and PNH membranes. Inhibitors derived from normal red cells markedly decreased lysis of both PNH and normal cells when antibody was present in excess and complement was limiting. These same inhibitors decreased PNH cell lysis to a much lesser degree when complement was activated with cobra venom or acidified serum. The susceptibility of the PNH cell to complement lysis because of an increased fixation of C3 to its membrane is not due to a difference in membrane-associated accelerator of the decay of the C42 complex.     

Characterization of the complement sensitivity of calcium loaded human erythrocytes.

A deficiency of membrane proteins having a glycosylphosphatidylinositol (GPI) anchor is characteristic of the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) and is currently believed to be the basis for the enhanced susceptibility to lysis by activated complement observed in these cells. Our recent observation that GPI-anchored proteins are preferentially lost into membrane vesicles shed from normal erythrocytes after calcium loading led us to examine the hypothesis that the remnant erythrocytes might also have increased sensitivity to complement-mediated hemolysis. Indeed, red blood cells treated in such a manner became more sensitive to lysis by antibody and complement or to lysis initiated by activated cobra venom factor complexes (CoFBb). As a consequence of membrane vesiculation, the erythrocytes lost up to approximately 50% of their immunoreactive decay-accelerating factor and 25% to 30% of their immunoreactive membrane inhibitor of reactive lysis (MIRL). Closer examination of the defect responsible for the marked increase in sensitivity to CoFBb-initiated hemolysis seen in calcium-loaded erythrocytes showed that a complex combination of factors produced the defect. These included a decrease in both functional and immunoreactive MIRL and depletion of intracellular potassium and adenosine triphosphate (ATP). These results suggest the possibility that loss of DAF and MIRL via membrane vesiculation, as well as decreases in intracellular potassium and/or ATP, might contribute to the phenotype of PNH erythrocytes. Further, normal or pathologic red blood cells might develop a PNH-like defect after membrane vesiculation if sufficient decreases in potassium and ATP also occurred.     

The Lysis of Paroxysmal Nocturnal Haemoglobinuria Red Cells by Serum and Cobra Factor

S ummary . The complement-sensitive red cells of patients with paroxysmal nocturnal haemoglobinuria (PNH) are haemolysed in the presence of serum and a protein factor present in cobra venom (CoF). The reaction of CoF and a serum factor, C₃ proactivator, requires Mg²⁺. Once this interaction has taken place, the activation of C₃ and the terminal components of complement can occur in the presence of EDTA and result in the lysis of PNH red cells.
The concentration of reactants may be adjusted so that all of the complementsensitive red cells of PNH patients and the red cells of no other patients are lysed. A simple but specific and precise test for the presence of the complement-sensitive red cells of PNH was devised. Normal human red cells treated with trypsin and neuraminidase are not lysed, whereas these cells treated with glutathione or 2-aminoethylisothiouronium bromide (AET) are lysed.     

Potentiation of virulence by group B streptococcal polysaccharides

The ability of group B streptococcal (GBS) capsular polysaccharides to potentiate virulence was examined. Incubation of soluble type Ia or III polysaccharide in hypogammaglobulinemic human serum inhibited classical complement-dependent opsonophagocytic killing of type Ia strain 515. When functional complement components were measured, C1 activity increased in sera with added polysaccharide whereas C4 activity decreased 80%-90%. Incubation of purified C1 with type Ia polysaccharide inhibited lysis of EAC4 cells in a C1 transfer assay. In a mouse lethality model, tail-vein injection of 50 micrograms of type Ia or III polysaccharide decreased the 50% lethal dose (LD50) from 3.2 X 10(6) to 2.2 X 10(5). Total hemolytic complement levels in mice immediately after polysaccharide injection were increased over levels in control mice, but this difference was not seen 30 min later. The LD50 in mice depleted of C3 (with cobra venom factor) was 6 X 10(5) and simultaneous injection of polysaccharide did not further lower the LD50.     

Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement.

To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.     

Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum.

When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC.     

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab'')2 fragments

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug-cured mice challenged with a high inoculum of ring-infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary-infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont-infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc-dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody-mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcgammaR-mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.     

Activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement

Properdin is a positive regulator of alternative pathway (AP) complement. The current understanding of properdin function is that it facilitates AP complement activation by stabilizing the C3 convertase C3bBb. Properdin-deficient patients are susceptible to lethal meningococcal infection, but the mechanism of this selective predisposition is not fully understood. By gene targeting in the mouse, we show here that properdin is essential for AP complement activation induced by bacterial lipopolysacharride (LPS) and lipooligosacharride (LOS) and other, but not all, AP complement activators. LPS- and LOS-induced AP complement activation was abolished in properdin-/- mouse serum, and properdin-/- mice were unable to clear Crry-deficient erythrocytes, which are known to be susceptible to AP complement-mediated extravascular hemolysis. In contrast, zymosan- and cobra venom factor-induced AP complement activation, and classical pathway-triggered AP complement amplification were only partially or minimally affected in properdin-/- mice. We further show that the ability of human properdin to restore LPS-dependent AP complement activity in properdin-/- mouse serum correlated with the human properdin-binding affinity of the LPS. These results reveal a novel role of properdin in AP complement initiation and have implications for understanding the selective predisposition of properdin-deficient patients to meningococcal infection.     

Aberrant regulation of complement by the erythrocytes of hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS)

Susceptibility to hemolysis in acidified serum is a pathognomonic feature of hereditary erythroblastic multinuclearity with a positive acidified serum lysis test (HEMPAS, congenital dyserythropoietic anemia type II). The purpose of the studies reported herein was to determine if aberrant regulation of complement contributes to the susceptibility of HEMPAS erythrocytes to acidified serum lysis. The results of these experiments have demonstrated that regulation of both the C3 convertase of the alternative pathway and the membrane attack complex of complement by HEMPAS erythrocytes is aberrant. However, these abnormalities are not a consequence of quantitative or functional deficiencies of the erythrocyte complement-regulatory proteins, decay accelerating factor (DAF, CD55), or membrane inhibitor of reactive lysis (MIRL, CD59). Our recent studies have shown that glycophorin A (GPA), the major erythrocyte sialoglycoprotein is a complement regulatory protein. Analysis by radioimmunoprecipitation suggested that GPA on HEMPAS erythrocytes is abnormally glycosylated. Further analysis indicated that the abnormality involves the O-linked oligosaccharide moiety. Together, these studies show that complement regulation by HEMPAS erythrocytes is abnormal and that constituents other than DAF and MIRL participate in controlling complement activation on the erythrocyte membrane. Additionally, these studies suggest that the glycosylation defect that is characteristic of HEMPAS involves GPA.     

Defective regulation of complement by the sickle erythrocyte: evidence for a defect in control of membrane attack complex formation

A prominent clinical manifestation of sickle cell disease (SCD) is hemolytic anemia. Although complement activation can lead to intravascular hemolysis, its role in the hemolysis of SCD is not known. Because normal red blood cells induced to vesiculate by treatment with calcium and ionophore become sensitive to damage by activated complement and because sickle cells release microvesicles as they circulate, we postulated that sickle cells might also be unusually sensitive to complement-dependent hemolysis. Complement activation is tightly regulated on the membrane of the normal erythrocyte; therefore, defective complement regulation by the sickle cell would be necessary for complement-dependent hemolysis to occur. These studies show a defect in the regulation of membrane attack complex (C5b-9) formation in sickle erythrocytes, particularly in the most dense cells. The defect is characterized by increased binding of C5b-7 and of C9 to denser sickle cells and results in increased susceptibility of sickle cells to C5b-9-mediated (reactive) lysis initiated by either C5b6 or activated cobra venom factor. Among the densest sickle cells, irreversibly sickled cells are especially sensitive to reactive lysis. The similarity of this defect to that previously described in a patient with paroxysmal nocturnal hemoglobinuria suggests that complement- mediated hemolysis could play a role in the anemia of SCD.     

Acquired resistance to ticks: expression of resistance by C4-deficient guinea pigs.

Hartley and C4-deficient guinea pigs developed resistance to the ixodid tick. Dermacentor andersoni, after one infestation. Resistance was characterized by resistant animals of both groups allowing significantly fewer larvae (5--25%) to engorge during a second infestation than during an initial infestation (70--90%). Resistant animals in both groups developed cutaneous reactions at the site of tick attachment which were characterized by intraepidermal vesicles containing numerous basophils. In previous studies, tick-resistant Hartley guinea pigs depleted of complement by cobra venom factor were not able to express the resistance response and the skin reactions at the tick attachment sites were depleted of basophils. The use of cobra venom factor as an anti-complement probe could not distinguish the relative importance of the classical and/or alternate pathways of complement activation in the expression of tick resistance. The present study reports that C4-deficient guinea pigs, those with a total deficiency in the classical pathway of complement activation, but with an intact alternate pathway, can acquire and display tick resistance in a fashion similar to Hartley guinea pigs. This finding provides evidence that the alternate pathway of complement activation is important in the expression of tick resistance.     

Complement alterations in inflammatory bowel disease.

A prospective evaluation of the activity of the complement system was undertaken in 32 patients at the time of diagnosis of inflammatory bowel disease, before the onset of therapy. Serum classical pathway components and function were normal, while significant abnormalities of the alternative pathway were found. Depressions of serum properdin and properdin convertase were noted in association with diminished consumption of C3--C9 after reaction with cobra venom. These abnormalities of alternative pathway integrity were most significant in regional enteritis and in ulcerative colitis with extraintestinal complications. Sequential studies extending into clinical remission revealed resolution of all significant abnormalities.     

Complement activation by isolated myelin: activation of the classical pathway in the absence of myelin-specific antibodies.

Many pathological conditions of the central nervous system involve damage to and removal of myelin membrane. Very little is known about initiation of this membrane damage and the mechanisms of disposal of the damaged tissue. We are interested in the interaction between complement (the components of complement are designated C1, C2, C3, etc.) and myelin membranes and the possible role of complement in amplifying myelin damage and in the disposal of damaged myelin in vivo, because activation of complement generates both membrane-attack complexes and opsonin(s). In this study, we found that isolated rat or human myelin consumes complement in the absence of specific antibodies. Activation of complement was demonstrated by showing C3 cleavage in fresh serum incubated with myelin. Incubation of central nervous system myelin with C2-deficient serum produced no C3 consumption and only minor factor B conversion, thus excluding the alternative pathway of activation. Involvement of the classical pathway was shown directly by the C1 fixation and transfer assay. Myelin incubated with C2-deficient serum or with purified C1 and then washed contained C1 activity that could lyse sheep erythrocytes sensitized with anti-Forssman IgM antibody and carrying C4, together with C2 and C3-C9. Membranes in brain tissues other than myelin (heavy membrane fraction obtained on sucrose density gradient centrifugation) were unable to activate C1.     

Complement activity in normal rabbit bronchoalveolar fluid. Description of an inhibitor of C3 activation

Hemolytically active C4, C3, C5, and C6, and trace amounts of C1, were present in bronchoalveolar lavage fluid from healthy adult rabbits. Both the alternative and classical pathways were functionally intact through C5. Complement depletion by intravascularly administered cobra venom factor caused a parallel decrease in lavageable C5 from the rabbit's lungs. Whereas BALF C5 had specific activity comparable to serum C5, that of BALF C3 was lower than predicted. This was found to be due to the action of a substance that selectively blocked C3 activation. The inhibitor did not cause irreversible inactivation of C3, and it had no effect on C5 activity. It blocked the formation of EAC423 from EAC42 + C3, but not the lysis of preformed EAC423 + C5-9 or EAC423 rosetting with rabbit neutrophils. Hydrophobic chromatography of BALF separated the activity of the inhibitor from that of C3. Further chromatography showed that it had a net negative charge at physiologic pH, and molecular weight between 14 and 30 kilodaltons. The presence of an inhibitor of C3 activation in the airways would provide control of complement activation in an environment that is constantly exposed to bacteria, dust, and other potential inflammatory stimuli.     

Acute pancreatitis induced by intrapancreatic complement activation in the rat.

Acute necrotizing pancreatitis was introduced in rats by intrapancreatic complement activation. As an immunopathogenetic model for complement activation an intrapancreatic Arthus reaction was produced by intraductal injection of rabbit IgG into sensitized animals. Antibody independent complement activation was accomplished by intraductal injection of particulated inulin and cobra venom factor resp. As shwon by histological and immunohistological examination the development of acinar cell necroses and the consecutive inflammatory reaction followed an identical course in all three models. The generation of cytolytically active complement is suggested to be the common link in the formal pathogenesis of the initial membrane demage of the acinar cells which enables its autodigestion by pancreatic enzymes. The region of the initial complement activation within the pancreas but not the mode of its activation (by immunological or non-immunological processes) determine the topography of acinar cell necroses and connective tissue proliferation.     

Possible role of glucocorticoids in a complement-activated state induced by cobra venom factor in rats

Activation of the complement system of adrenalectomized rats with an injection of cobra venom factor (CVF) caused death of the rats within 2.5 h. Morphologically, this activation provoked distinct congestion of the gastric glandular mucosa and pulmonary leukostasis. Pretreatment of the animals with dexamethasone abolished the undesirable responses completely. Injection of CVF to intact rats produced only slight responses, but caused a marked increase in the serum levels of corticosterone. Dexamethasone was found to be replaced by promethazine (H1-antihistamine) or dimethylsulphoxide (scavenger of hydroxyl radicals) but not by indomethacin, ibuprofen (cyclo-oxygenase inhibitors), deferoxamine mesylate (iron chelator) or imidazole (thromboxane synthetase inhibitor). These results suggest that glucocorticoids protect the animals from the adverse effects of excessive complement activation and that they act as an inhibitor of the production or action of histamine and toxic oxygen products induced by complement activation.     

Reduction of total hemolytic complement activity with Naja haje cobra venom factor does not prevent endotoxin-induced lung injury in sheep

We studied the effects of reducing total hemolytic complement activity with Naja haje cobra venom factor on the lung injury caused by intravenously infused endotoxin in 5 unanesthetized sheep with lung lymph fistulas. In normal sheep, infusions of lipopolysaccharide W from Escherichia coli (1.0 micrograms/kg) intravenously over 30 min caused increases in protein-rich lung lymph flow as well as the appearance in plasma and lung lymph of complement (C5)-derived chemotactic activity for polymorphonuclear leukocytes. Reduction of total hemolytic complement activity by treatment with Naja haje cobra venom factor (12 to 17 U/kg intraperitoneally) did not prevent the lung injury caused by endotoxin and also did not prevent the appearance in plasma and lung lymph of chemotactic activity. We conclude that although complement appears to be activated following intravenously infused endotoxin in sheep, a completely intact complement system is not necessary for endotoxin-induced lung injury.     

Structural and functional differences between decay-accelerating factor and red cell acetylcholinesterase

The abnormal erythrocytes in paroxysmal nocturnal hemoglobinuria, both PNH II (the moderately abnormal cells) and PNH III (the markedly abnormal cells), lack both acetylcholinesterase (AChE) activity and decay-accelerating factor (DAF) activity. Both of these activities are found on glycoprotein molecules with a molecular weight of about 70 Kd. To demonstrate that these two activities are in fact on different proteins, we have shown that binding to normal red cells of antibody to DAF does not inhibit the subsequent binding of monoclonal antibody to AChE nor AChE activity. Inhibition of DAF activity by polyclonal antibody increases the susceptibility of normal erythrocytes to lysis by complement but inhibition of AChE activity by antibody does not. The rate of decay of the C3 convertase complex of the classical pathway of complement activation was inhibited by DAF added in the fluid phase but not by AChE. When DAF was exhaustively immunoprecipitated from a solution of the erythrocyte membrane proteins, AChE remained and vice versa. These studies indicate that acetylcholinesterase and decay-accelerating factor are two different proteins, both of which are lacking on PNH II and PNH III erythrocytes.     

Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack

Decay-accelerating factor (DAF) and CD59 are 2 glycosylphosphatidylinositol-anchored membrane proteins that inhibit complement activation at the C3 and C5b-9 step, respectively. CD59 is considered critical for protecting erythrocytes from spontaneous complement attack, as deficiency of CD59 or CD59/DAF, but not of DAF alone, on human erythrocytes renders them sensitive to complement lysis in paroxysmal nocturnal hemoglobinuria syndrome. To evaluate the relative roles of CD59 and DAF in vivo, we have generated and studied a CD59 knockout and a CD59/DAF double-knockout mouse. CD59-deficient and CD59/DAF-double-deficient mouse erythrocytes were highly sensitive to antibody-induced complement lysis in vitro, yet neither CD59 knockout nor CD59/DAF double-knockout mouse developed spontaneous hemolytic anemia. Consistent with the latter observation, erythrocytes from the 2 strains of mutant mice were shown to have a normal lifespan in vivo. In contrast, mouse erythrocytes deficient in complement receptor 1 (CR1)-related gene y (Crry), a membrane C3 inhibitor with DAF and membrane cofactor protein activities, were rapidly eliminated from the circulation by a complement-dependent mechanism. Compared with DAF-deficient erythrocytes, Crry-deficient erythrocytes incurred higher levels of spontaneous C3 deposition in vivo. These findings demonstrate that CD59 and DAF are not indispensable on murine erythrocytes. Rather, effective C3 regulation on the cell surface, provided by Crry rather than DAF, is necessary for mouse erythrocytes to resist spontaneous complement attack. Our results raise the possibility that proper control of C3 activation may also be critical on human erythrocytes, where CR1 but not DAF could be the principal regulator of spontaneous C3 activation.