C1q and immune complexes in liver cirrhosis sera

The highest mean value of the serum C1q concentrations among chronic liver diseases was obtained in patients with liver cirrhosis (LC). C1q serum levels seemed to increase along the progression of liver damage. Serum levels of C1q and CH50 in patients with LC and systemic lupus erythematosus (SLE) were simultaneously estimated. No correlation between C1q and CH50 levels was observed in LC sera, while a significant correlation was demonstrated in SLE sera. It could be suggested that the mechanism for the low CH50 in LC sera seemed different from that in SLE sera, and that the activation of classical complement pathway was not the predominant cause in LC sera. Correlations of serum levels between immune complexes and C1q were examined in patients with LC and SLE. It could be deduced from the results of a positive correlation in LC sera and the reverse tendency in SLE sera that the significances of C1q and immune complexes in LC sera differed from those in SLE sera.     

Kupffer cell complement receptor clearance function after surgical injury and phagocytosis of immune complexes: effect of changes in plasma fibronectin

The present study evaluated the effect of changes in plasma fibronectin levels on the degree of depression of in vivo clearance function of Kupffer cell complement receptors after injury and the phagocytosis of immune complexes. In vitro studies have suggested that fibronectin may act as an opsonin for the clearance of immune complexes from the blood by binding to C1q and may influence the expression and activation of immune receptors on macrophages. Complement receptor clearance function was assessed in rats from the hepatic uptake of rat erythrocytes coated with antierythrocyte IgM. Increasing plasma fibronectin by the injection of purified fibronectin or decreasing fibronectin by the injection of gelatin had no effect on complement receptor function in otherwise normal animals. Surgical injury depressed both plasma fibronectin levels and complement receptor function. Injection of fibronectin at the time of injury prevented the depression of receptor function; however, when fibronectin was given 1 hour after injury, receptor function remained depressed even though fibronectin levels were increased. The phagocytosis of immune complexes (IgG-coated rat erythrocytes [EIgGs]) depressed complement receptor function but did not depress plasma fibronectin levels. The depression of receptor function caused by the phagocytosis of EIgGs was prevented by administering fibronectin and was potentiated by administering gelatin, which decreased plasma fibronectin levels. Therefore, plasma fibronectin concentrations can influence in vivo Kupffer cell complement receptor function under certain conditions that lead to the depression of complement receptor function.     

Effect of complement binding on a solid-phase immunometric TSH assay

The binding of serum thyrotropin (TSH) to plastic beads coated with a monoclonal antibody to human TSH was inhibited unless EDTA was present during the incubation. The inhibitory factor in serum was heat labile, and its effect could be abrogated by the addition of human albumin-anti-albumin immune complexes. Subsequently it was shown that the antibody-coated beads were able to bind the first component of complement, C1q, and that this binding was inhibited by addition of albumin-anti-albumin complexes. The results show that a surface coated with a monoclonal murine antibody is able to bind complement, and that binding of complement may interfere in solid-phase immunometric assays.     

Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

A sensitive and reproducible procedure for the detection of soluble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled C1q is reacted with sera containing immune complexes. Separation of free from complex bound [(125)I]C1q is achieved by selective precipitation with polyethylene glycol (PEG). The method is based on both the large molecular size and the C1q-binding property characterizing immune complexes. The minimal amount of aggregated immunoglobulins thus detected is about 10 mug and that of soluble human IgG-anti-IgG complexes is about 3 mug of complexed antibody. Some immune complexes formed in large antigen excess (Ag(2)Ab) can still be detected by this radiolabeled C1q binding assay. The specificity of the radiolabeled C1q binding test was documented by the inability of antigen-F(ab')(2) antibody complexes to lead to a precipitation of [(125)I]C1q in PEG.In a second step, this radiolabeled C1q binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes.Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the hepatitis B antigen (HB-Ag). Significantly increased [(125)I]-C1q binding values were observed in 52 sera from SLE patients when compared to values obtained with healthy blood donors (P<0.001). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients.No increased [(125)I]C1q binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [(125)I]C1q percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [(125)I]C1q. The results were also used for a correlative study of [(125)I]C1q binding to IgG levels in the sera but increased [(125)I]C1q binding could not be attributed to high serum IgG levels which are likely to account for gammaglobulin aggregates.These examples suggest the utility of the radiolabeled C1q binding assay for the evaluation of immune complex diseases in human pathology.     

The Raji cell radioimmune assay for detecting immune complexes in human sera.

A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.     

Cryoglobulins as indicators of upregulated immune response in schizophrenia

OBJECTIVE: In the present work the concentration of abnormal immune complexes, cryoglobulins (Cgs), in the blood of schizophrenic patients was determined, and immunochemical composition of these complexes was studied. PATIENTS AND METHODS: Eighty multiple-episode schizophrenia-affected subjects (55 medicated, 25 drug-free) and 40 healthy controls were involved in the study. Cgs were isolated by exposure of blood serum samples to precipitation at low temperature followed by extensive washings of Cg-enriched pellets. The immunochemical composition of Cgs was analyzed using different electrophoretic and immunoblotting systems. RESULTS: Significantly increased blood serum levels of type III Cgs were detected in all schizophrenia-affected subjects, as compared to controls. We also revealed the presence of C1q and C3 complement proteins and their activation products in Cgs isolated from the blood of schizophrenic patients. CONCLUSIONS: The results of the present study suggest that Cgs are involved in schizophrenia-associated upregulated immune response by binding the complement proteins, activating the complement cascade and triggering aberrant apoptosis.     

Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE

Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1–nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1β, IL-6, IL-10, and tumor necrosis factor (TNF) α and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2–dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1–nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.     

The development of a C1q anti-C1q immunoadsorbent for removal of immune complexes from plasma

An immunoadsorbent based on immobilized C1q has been developed to remove possibly pathogenic immune complexes from plasma deriving from patients suffering from systemic lupus erythematosus or rheumatoid arthritis. Traditional immobilization procedures based on, e.g., cyanogen bromide activation could not be used to produce an efficient adsorbent. However, by using antibodies directed towards C1q as handles for the immobilization of C1q it was possible to make an adsorbent that efficiently bound immune complexes in plasma. The capacity of the C1q anti-C1q adsorbent to bind artificial immune complexes such as aggregated human globulins or immune complexes from various plasma samples was evaluated. Both batch and column experiments were conducted. The typical capacity in batch was about 1 mg immune complexes/ml gel when incubated with patient plasma samples with high titers of immune complexes. Special attention has to be paid to leakage of undesirable components from the adsorbent. It was found that leakage of C1q occurred but it was not more than after covalent immobilization procedures such as cyanogen bromide.     

Complement depletion accelerates the clearance of immune complexes from the circulation of primates.

Binding of immune complexes (IC) to erythrocytes in vitro is the result of interaction between C3b sites on the IC, and complement receptors type I (CRI) expressed on primate erythrocytes. Recent evidence indicates that primate erythrocytes can also rapidly bind large, preformed IC in vivo. This study was undertaken to determine if the binding of IC to baboon erythrocytes in vivo is complement dependent and to examine the effect of complement depletion on IC clearance from the circulation. The results indicate that complement depletion in vivo reduced the binding of IC to erythrocytes. There was relatively little binding of IC to leukocytes in both the complement-depleted and complement-repleted condition. Thus, the majority of IC not bound to erythrocytes remained free in the plasma and, consequently, IC infusion during the complement-depleted state resulted in increased plasma IC concentrations. This was associated with a rapid disappearance of IC from the circulation. By contrast, in the normal or complement-repleted state, a large fraction of the IC became bound to erythrocytes during IC infusion, which resulted in lower plasma IC concentrations. Under these conditions, a more gradual rate of disappearance of IC from the circulation was observed. The relatively abrupt clearance of IC from the circulation in the complement-depleted state could not be accounted for by increased hepatic or splenic uptake. These data indicate that, in contrast to previous studies in nonprimates, complement depletion in primates results in accelerated removal of IC from the circulation. This suggests that factors such as hypocomplementemia and deficient expression of erythrocyte CRI, which are known to occur in certain IC-mediated diseases, may promote IC uptake by organs vulnerable to IC-mediated injury.     

A kinetic study of factor I-mediated modification and release of RBC-bound DNA-anti-DNA immune complexes.

A tritiated DNA-anti-DNA complement--containing immune complex probe was used to define the mechanism of factor I-mediated modification and release of erythrocyte CR1-bound immune complex in 10 normal control subjects. Experiments were performed with preformed immune complex, autologous erythrocytes, and autologous serum. Control experiments were performed by using a single donor source of erythrocytes and commercial factor I. The data demonstrate that the modification-release rate was first order with respect to factor I concentration, the mean half-life of red blood cell-bound immune complex was 1.7 +/- 0.7 minutes, and the mean percent of total immune complex present as unbound complexes was 19 +/- 11. These data are consistent with a clearance model in which factor I-mediated modification and release of red blood cell-bound immune complex plays a significant role in immune complex handling.     

Binding of complement component C5 to model immune complexes and the use of anti-C5 antibodies for determination of C5-containing circulating immune complexes

When normal human or mouse serum is added to micro ELISA plates coated with monomeric or aggregated IgG, complement component C5 binds to IgG. C5 binding was demonstrated with a specific chicken anti-C5 antibody. Hydrazine treatment of the serum or addition of EDTA to the serum abolished the binding of C5. C5-deficient mouse serum was negative for C5 binding, whereas the same serum supplemented with human C5 restored the binding of C5. Chicken anti-C5-coated plates were used for determination of C5-containing circulating immune complexes (CIC). Increased concentrations of CIC were found in sera from patients with rheumatoid arthritis and Bell's palsy.     

Evaluation of the C1q solid-phase binding assay for immune complexes. A clinical and laboratory study.

The solid-phase C1q binding assay for circulating immune complexes has been evaluated. The assay provides a rapid, sensitive (detecting as little as 1 microgram of aggregated IgG) and reproducible procedure for the detection of immune complexes in biological fluids. Using artificially prepared immune complexes, the assay detects complexes at four-times antigen-excess. Gel filtration over Sepharose 6B showed that these complexes were distributed over a range of molecular weights from greater than 4 x 10(6) to 300,000 daltons. Using radiolabelled anti-BSA, antigen (BSA) could be detected in these complexes. Screening of gel-filtered SLE showed that the assay detects complexes of both high and low molecular weight, but does not detect all complexes in the SLE sera. Clinical studies showed that immune complexes are frequently found in the sera of patients with SLE and measurement of the concentrations of complexes provides a more sensitive index of disease activity than either serum C3 or C4 concentrations or DNA binding capacity. In patients with RA concentrations of immune complexes were generally higher in synovial fluid than serum, although a patient with systemic rheumatoid disease with hypocomplementaemia had an extremely high level of circulating immune complexes. The assay only infrequently detects circulating immune complexes in glomerulonephritis and in renal transplant recipients. It is concluded that the assay provides a useful clinical tool, but detects only a limited species of immune complexes. It can be used in the detection of antigens in complexes.     

Altered erythrocyte CR1 binding kinetics compensate for decreased binding capacity in rheumatoid arthritis

Patients with rheumatoid arthritis have decreased numbers of CR1 per erythrocyte and decreased binding of immune complexes to erythrocytes. Overall erythrocyte immune complex binding activity depends on both the number and the binding kinetics of CR1. We measured kinetic parameters for the interaction between a complement-containing dsDNA:anti-dsDNA probe and erythrocytes in patients with rheumatoid arthritis and normal controls. The results indicate that: 1) the maximum quantity of immune complexes bound per erythrocyte was significantly decreased in rheumatoid arthritis compared with normal controls (p less than or equal to 0.009); 2) the steady state binding constant, Kss, and the association rate constant for binding of immune complexes to erythrocytes, ka, were significantly increased in rheumatoid arthritis versus normal controls (p less than or equal to 0.0001 and 0.002 respectively); 3) the dissociation rate constant for the release of bound immune complexes from erythrocytes, kd, was slightly smaller in rheumatoid arthritis but this difference was not statistically significant; and 4) the energies of activation for the association and dissociation reactions, Eaa, and Ead, did not differ between the two groups. These data confirm that while the maximum quantity of immune complexes bound per erythrocyte is decreased in rheumatoid arthritis, the association rate constants are larger and dissociation rate constants slightly smaller than those of normal controls. Changes in these kinetic parameters compensate for the decrease in the maximum quantity of immune complexes bound per erythrocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

Summary.  Background:  Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets. Objective:  Here we test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase. Methods and results:  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. Conclusions:  We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a.     

Immune complex processing in patients with systemic lupus erythematosus. In vivo imaging and clearance studies.

Abnormal processing of immune complexes (IC) may be important in the pathogenesis of systemic lupus erythematosus (SLE). The clearance of large soluble IC (comprising hepatitis B surface antigen (HBsAg)/anti-HBsAg) radiolabeled with 123I was examined in 12 normal subjects and 10 patients with SLE. IC localization was analyzed by static and dynamic gamma-scintigraphy. Initial IC clearance from blood was more rapid in patients (median t1/2 = 2.15 min) than normals (median t1/2 = 5.15 min) due to more rapid uptake in the liver. However, in the SLE group, up to 12% of complexes were released from the liver after 30-50 min. Splenic uptake of immune complexes was reduced in the patients and there was reduced ability to retain IC in this organ. Plasma complement levels and erythrocyte complement receptor type 1 numbers were reduced in the patients, resulting in defective opsonization of IC and reduced red cell binding in vivo. These observations support the hypothesis that IC handling is abnormal in SLE.     

Human platelet-initiated formation and uptake of the C5-9 complex of human complement.

We have studied the interaction of radiolabeled complement components with normal human platelets, platelets from a patient with paroxysmal nocturnal hemoglobinuria, and rabbit platelets in the absence of known complement activators or in the presence of cobra venom factor (CVF). When unwashed platelets in platelet-rich plasma, or washed platelets suspended in serum or autologous plasma, were incubated for 30 min, C3 and terminal components (C5, C8, and C9) were found to bind to them. The terminal components were shown to be bound as the C5-9 complex, rather than as individual proteins, by eluting them from the platelet membrane and examining their behavior on ultracentrifugation. They cosedimented at a rate characteristic of the stable C5-9 complex (22S). As many as 370-1,380 C5-9 complexes/platelet were calculated to have been bound during the incubation period. The complex so formed did not differ by ultracentrifugational criteria from that binding to rabbit platelets after CVF activation of complement. Though C3 was not included in the complex, it did not appear to be bound by nonspecific absorption. It could not be removed by washing but rather was eluted by the freeze-thaw technique used to elute the C5-9 complex. Incubation of radiolabeled components in platelet-free plasma did not result in C5-9 complex formation, indicating an initiating role for platelets in this reaction. In contrast to platelets, erythrocytes incubated in analogous plasma did not induce detectable C5-9 formation. Neither EDTA, phenylmethylsulfonylfluoride, nor epsilon-amino-N-caproic acid prevented platelet-initiated formation of C5-9, suggesting that the reaction may involve mechanisms of complement activation not previously described.     

Increased efficiency of binding of nascent C3b to the erythrocytes of chronic cold agglutinin disease.

The pathogenesis of chronic cold agglutinin disease (CCAD) has been enigmatic. To determine if abnormal erythrocyte membrane constituents might provide the stimulus for antibody production, we compared the electrophoretic pattern of radiolabeled membrane glycoproteins of four patients with CCAD to that of normal control erythrocytes. For the CCAD erythrocytes, fluorographs revealed the appearance of an abnormal band whose molecular weight was estimated at 126,000 D. Using two-dimensional gel analysis and immunoblotting techniques, it was determined that the 126,000 D glycoprotein consisted predominately of polymeric glycophorin-alpha. Previous investigations had suggested that abnormalities in glycophorin-alpha influence the functional activity of the complement system. When purified complement (C)3 was activated in the fluid-phase by cobra venom factor complexes, CCAD erythrocytes bound nascent C3b 7-27 times more efficiently than normal erythrocytes. Normal erythrocytes could be induced to manifest the appearance of the 126,000 D band, and the increased efficiency of binding of nascent C3b by incubation with CCAD serum or with the purified cold agglutinin antibody plus autologous serum, but not with the purified antibody alone or the purified antibody plus EDTA-chelated autologous serum. These studies demonstrate that the interactions of IgM cold-reacting antibody and complement with glycophorin induce changes in the biophysical properties of the erythrocyte membrane which modify subsequent interactions with complement.     

Pneumococcal immune adherence to human erythrocytes

BACKGROUND: Human red blood cells bind various C3b-coated microorganisms via their C3b/CR1 receptor, a phenomenon referred to as immune adherence. The aim of the present study was to measure pneumococcal adherence to human red blood cells by flow cytometry and to study kinetic aspects of this binding. MATERIAL AND METHODS: We quantified pneumococcal adherence to human erythrocytes by FACS analysis and tested the involvement of antibodies and complement activation in this process. RESULTS: Pneumococci are able to bind to human red blood cells in the presence of human serum. Coating with C3b/C4b appeared obligatory for pneumococcal adherence to red blood cells. The ligand on erythrocytes was confirmed to be complement receptor 1. Kinetic studies showed that innate (mannose-binding lectin) and specific immune factors (IgG antibodies) contributed to the binding of C3b-coated pneumococci to human erythrocytes. After initial binding, serum-derived factor I was found to induce bacterial detachment from the erythrocyte. CONCLUSIONS: Pneumococci are able to adhere to red blood cells. Both the classical and lectin complement pathways are important for optimal C3b-coating of pneumococci for immune adherence. Bound pneumococci are detached from red blood cells by factor I. These findings are in line with the hypothesis of immune adherence in which human erythrocytes are able to bind pneumococci and target the bacteria to the reticulo-endothelial system in the spleen.     

Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men in varying risk groups for acquired immune deficiency syndrome.

We studied levels of erythrocyte C3b receptors (E-CR1) and correlated them to the level of circulating immune complexes (CIC) and complement activation in patients with or at risk for acquired immunodeficiency syndrome (AIDS). A significant reduction was found in patients with AIDS (185 +/- 93 CR1/cell), AIDS-related complex, and generalized lymphadenopathy, whereas healthy male homosexuals or normal controls had 434 +/- 193 and 509 +/- 140 CR1/cell, respectively (P less than 0.001). Family studies indicate that this defect is acquired. Reduction in E-CR1 was associated with increased levels of CIC when assayed by binding to Raji cells, but not when tested by C1q binding. Complement activation was assessed by levels of C3bi/C3d-g in plasma, measured with a monoclonal antibody specific for a neoantigen in C3d. AIDS patients had increased C3 activation (2.68 +/- 1.67%) when compared with normal controls (0.9 +/- 0.22%) (P less than 0.01). The decreased E-CR1, the presence of CIC, and C3 activation suggest that complement activation by immune complexes may play a role in the clinical expression of the disease.     

The role of complement in the clearance of cold agglutinin-sensitized erythrocytes in man.

To define the pathophysiologic mechanisms of cold agglutinin disease, we investigated a human model of this syndrome in normal volunteers and in patients with diminished levels of serum complement. Subjects received intravenous injections of autologous, chromated (51Cr) erythrocytes which had been exposed in vitro to purified cold agglutinin preparations and to fresh autologous serum (as a source of complement). In vitro tests confirmed that such cells were coated with activated complement components (C3b), but not with immunoglobulin. Studies of erythrocyte clearance and simultaneous organ scanning showed that erythrocytes sensitized with low levels of cold agglutinin primarily undergo reticuloendothelial sequestration by the liver rather than intravascular hemolysis. After the initial sequestration of C3b-coated erythrocytes, a fraction of the cells are released back into the circulation and survive normally thereafter. Both phenomena are dose dependent and closely follow the sequestration and release pattern observed with IgM isoagglutinin sensitization. Experiments that used heated autologous serum as a source of B3 inactivator demonstrated that functionally intact C3b is required for hepatic sequestration. Erythrocytes coated with C3d were not cleared from the circulation. In vitro assays that sued human macrophage monolayers suggested that the intrahepatic conversion of C3b to C3d is responsible for the release of sensitized erythrocytes back into the circulation. The clearance of cold agglutinin-sensititzed erythrocytes was compared to the clearance mediated by IgM isoagglutinin. We found that the rate of complement fixation by an IgM antibody proceeds rapidly in vivo that the time for complement activation is not a factor in limiting the rate of hepatic sequestration. The major limiting factor appears to be the rate of liver blood flow. Maximal in vitro coating of erythrocytes with C3d conferred protection from further cold agglutinin sensitization but not from IgM isoagglutinin-mediated clearance. This suggests a mechanism for the resistance to lysis observed in cells obtained from patients with the cold agglutinin syndrome and confirms the marked dependence of the site of C3 attachment on the site of membrane localization of the sensitizing antibody.