Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice

Complement plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 Ags with C3 breakdown product C3dg, a CR2-specific ligand. We used a C3dg-opsonized IC model, anti-CR1/2 mAb 7G6, to investigate how such substrates are processed. We used RIA, whole body imaging, flow cytometry, and fluorescence immunohistochemistry to examine the disposition of 0.1- to 2-microg quantities of mAb 7G6 infused i.v. into BALB/c mice. The mAb is rapidly taken up by the spleen and binds preferentially to marginal zone (MZ) B cells; within 24 h, the MZ B cells relocate and transfer mAb 7G6 to follicular dendritic cells (FDC). Transfer occurs coincident with loss of the extracellular portion of MZ B cell CR2, suggesting that the process may be mediated by proteolysis of CR2. Intravenous infusion of an FDC-specific mAb does not induce comparable splenic localization or cellular reorganization, emphasizing the importance of MZ B cells in intrasplenic trafficking of bound substrates. We propose the following mechanism: binding of C3dg-opsonized IC to noncognate MZ B cells promotes migration of these cells to the white pulp, followed by CR2 proteolysis, which allows transfer of the opsonized IC to FDC, thus facilitating presentation of intact Ags to cognate B cells.     

Transfer of immune complexes from erythrocyte CR1 to mouse macrophages

We are developing a potential therapeutic approach for removing pathogens from the circulation of primates in which the pathogen is bound to the complement receptor (CR1) on E using a bispecific mAb complex, a heteropolymer (HP). We have used mAb this approach to demonstrate that cleared prototype pathogens are localized to, phagocytosed in, and destroyed in the liver. Extension of this work to a clinical setting will require a detailed understanding of the mechanism by which the E-bound immune complex substrates are transferred to fixed tissue macrophages in the liver, the transfer reaction. Therefore, we examined an in vitro system to study this process using bacteriophage phiX174 as a model pathogen. E containing phiX174 (bound via an anti-CR1/anti-phiX174 HP) were incubated with P388D1 murine macrophages, and the two cell types were separated by centrifugation through Ficoll. Both E and macrophages were then probed and analyzed by RIA or flow cytometry. The results indicate that all three components of the E-bound IC (phiX174, HP, and CR1) were removed from the E and internalized by the macrophages. We found that transfer requires the Fc portion of IgG, because little transfer of phiX174 occurs when it is bound to E CR1 using a HP containing only Fab fragments. These findings, taken in the context of other studies, suggest a general mechanism for the transfer reaction in which Fc receptors facilitate close juxtaposition of the macrophage to the E-bound IC which then allows a macrophage-associated protease to cleave CR1. The released IC are then internalized and processed by the macrophages.     

Hematin promotes complement alternative pathway-mediated deposition of C3 activation fragments on human erythrocytes: potential implications for the pathogenesis of anemia in malaria

Childhood malaria caused by Plasmodium falciparum is often characterized by severe anemia at low parasite burdens; the mechanism(s) responsible for this pathology remain to be defined. We have reported, based on clinical observations and in vitro models, that complement control proteins on erythrocytes such as CR1, the immune adherence receptor specific for C3b, may be reduced in childhood malaria, suggesting a possible role for complement in erythrocyte destruction. Intravascular lysis of iE by P. falciparum leads to release of erythrocyte breakdown products such as hemoglobin and hematin, which have inflammatory properties. In the present article, we demonstrate that in serum and in anticoagulated whole blood, moderate concentrations of hematin activate the alternative pathway of complement and promote deposition of C3 activation and breakdown products on erythrocytes. The degree of C3 fragment deposition is directly correlated with erythrocyte CR1 levels, and erythrocytes opsonized with large amounts of C3dg form rosettes with Raji cells, which express CR2, the C3dg receptor which is expressed on several types of B cells in the spleen. Thus, the reaction mediated by hematin promotes opsonization and possible clearance of the youngest (highest CR1) erythrocytes. A mAb specific for C3b, previously demonstrated to inhibit the alternative pathway of complement, completely blocks the C3 fragment deposition reaction. Use of this mAb in nonhuman primate models of malaria may provide insight into mechanisms of erythrocyte destruction and thus aid in the development of targeted therapies based on inhibiting the alternative pathway of complement.     

Processing of C3b-opsonized immune complexes bound to non-complement receptor 1 (CR1) sites on red cells: phagocytosis, transfer, and associations with CR1

Severe anemia is a lethal complication of Plasmodium falciparum malaria, particularly in children. Recent studies in children with severe P. falciparum anemia have demonstrated elevated levels of E-bound Abs, reduced E-associated complement receptor 1 (CR1) and decay-accelerating factor (DAF), and pronounced splenic enlargement, suggesting a mechanism for E loss involving Abs, complement, and phagocytosis. Motivated by these reports, we have developed an in vitro model in which human E with Abs and complement bound to CR1, DAF, or glycophorin A are incubated with model human macrophages (the THP-1 cell line). Previous work has demonstrated that immune complex (IC) substrates bound to E CR1, either by an Ab or via C3b, are transferred to macrophages with loss of CR1. In this study, we report that IC bound to DAF or glycophorin A by an Ab linkage are also transferred to macrophages. DAF is lost from the E during the transfer of DAF-bound IC, but the transfer of CR1-bound IC does not lead to a significant loss of DAF. Using glycophorin A-bound IC, we observe competition between transfer of IC and phagocytosis of the E: a fraction (相似文献   

Binding of model immune complexes to erythrocyte CR1 facilitates immune complex uptake by U937 cells

The E C3b/C4b receptor (CR1) has been shown to rapidly bind large complement-fixing immune complexes (IC) both in vivo and in vitro. It has been proposed that E (RBC) CR1 act as a shuttle mechanism, binding circulating IC and transporting them to tissue macrophages, thereby preventing their deposition in target tissues. In this study we have established an in vitro model system with which to study the transfer of model IC from CR1 on the RBC surface to phagocytic cells. Aggregated IgG (AHG) was opsonized with C3b, bound to RBC CR1, and the binding of these RBC-bound IC by a human monocyte cell line (U937 cells) was examined. U937 binding of AHG from the RBC surface was complete within 2 min, whereas binding of the same AHG from solution required 30 to 60 min. Despite the difference in kinetics of binding, the total amount of IC bound by U937 cells at equilibrium was the same for RBC-bound AHG and for AHG in solution. The transfer of AHG from the RBC to the U937 cell did not require exogenous factor I and was not accompanied by binding of RBC to U937 cells or by erythrophagocytosis. Our data lend support to the hypothesis that binding of IC to RBC CR1 may facilitate the clearance of IC from the circulation by enhancing their uptake by phagocytic cells.     

Quantitative analyses of the binding of soluble complement-fixing antibody/dsDNA immune complexes to CR1 on human red blood cells

We have used direct binding isotherm analyses to measure the association constant (Ka) and number of binding sites for the binding of prepared complement-fixing antibody (Ab)/dsDNA immune complexes (IC) to human red blood cells (RBC). In order to generalize this study we have examined the binding reaction for a number of different anti-dsDNA Ab (from systemic lupus erythematosus plasmas), complement sources, RBC donors, and dsDNA sizes. The affinity of the IC for the RBC is quite high, and the Ka values fall within a narrow range (5 to 14 X 10(10) liter/mol). Similarly, the limiting stoichiometries for the number of IC bound per RBC were between 40 and 91. The very high affinity and limiting stoichiometries both suggest that the IC bind to the RBC via multiple contacts with clusters of complement receptor type 1 (CR1). Furthermore, we have used three specific monoclonal AB (mAb) to quantitate CR1 on human RBC in the presence and absence of bound IC. One of these Ab, mAb 1B4, is blocked from binding to the RBC if IC are previously bound, and we have used this observation to verify the multivalent nature of the interaction of complement-fixing IC with CR1 on human RBC.     

The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans. Complement- and erythrocyte complement receptor 1-dependent mechanisms

The role of complement and its receptor on erythrocytes (CR1) in the physiologic elimination of large immune complexes from the circulation of humans was assessed. Large radiolabeled soluble tetanus toxoid- anti-tetanus toxoid complexes were injected i.v. into three normal individuals and three patients with SLE. These complexes were prepared in antibody excess and were 45S in size, fixed C and bound to E CR1 in vitro. The percentage of complexes bound in vitro was directly proportional to CR1 number/E in four normal subjects and three SLE patients. After i.v. injection into normal subjects, complexes were cleared rapidly, with a monoexponential rate constant (10.3 to 11% complexes cleared/min). In the SLE patients, clearance was best explained by two phases: the first occurred within the first minute indicating immediate trapping of a fraction of the complexes (19.5 to 25.3% of injected complexes trapped), the second was monoexponential and was similar to the normal range. A large fraction of complexes bound within the first minute to E in vivo; the percentage of binding was variable, ranging from 16.3% to 71.5% and was related to E CR1 number. In a second study complexes were injected that had been attached to autologous E by opsonization with C in vitro. Their elimination was similarly monoexponential, except in one SLE patient in whom there was significant initial trapping (30.9%). A fraction of these complexes were released from E within the first minute, the percentage release being greatest in the patient with the lowest CR1 number (81.4%). E bearing immune complexes remained in the circulation and were not transiently sequestered in the liver or spleen. This is the first study of the clearance of soluble immune complexes in vivo in humans and shows that C and CR1 on E participate in immune complex clearance reactions, and that abnormal clearance can be detected in the form of rapid removal of immune complexes from the circulation.     

Expression, molecular association, and functions of C3 complement receptors CR1 (CD35) and CR2 (CD21) on the human T cell line HPB-ALL.

We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.     

Human erythrocytes inhibit complement-mediated solubilization of immune complexes

Incubation of precipitable immune complexes (IC) with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed human E were added to human serum or guinea pig serum, binding of IC to the E occurred and IC solubilization was significantly inhibited. By contrast, SRBC did not bind IC nor inhibit IC solubilization. Because IC binding to human E is mediated by CR type 1 (CR1) we evaluated whether CR1 was responsible for the inhibition of IC solubilization. Human E were treated with trypsin or anti-CR1 mAb. Both treatments abrogated IC binding to human E but did not affect the ability of the human E to inhibit IC solubilization. Human E inhibited C activation by IC. Thus, incubation of IC in human serum caused significant activation of C3 and C5, but not C4. However, when IC were incubated in whole blood or with isolated human E and serum, C3 activation by IC was inhibited significantly. In addition, we demonstrated that the C3b generated during C activation by IC deposited on both IC and human E. Thus, human E may compete for nascent C3 generated during C activation by IC. In conclusion, human E inhibit both complement-mediated solubilization of IC and C activation by IC.     

Release of arachidonic acid by stimulation of opsonic receptors in human monocytes: the FcgammaR and the complement receptor 3 pathways

The role of the opsonic receptors FcgammaR and CR3 on the release of arachidonic acid (AA) by human monocytes was studied using IgG-ovalbumin (OVA) equivalence immune complexes (IC), anti-OVA IgG bound to OVA-coupled latex beads, and C3bi-bound IC. Release of AA was produced by IC and latex-OVA beads bound to IgG, whereas binding of C3bi to IC inhibited the ability of IC to release AA. In contrast, coating of zymosan particles with C3bi enhanced AA release as compared with that produced by non-coated particles. Masking of C3bi on C3bi-bound IC by incubation with anti-C3 IgG resulted in the recovery of their ability to release AA, thereby suggesting that binding of C3b by IC reduces their flogogenic effects, whereas opsonization of microbial walls by complement may enhance their proinflammatory potential. The binding/uptake of opsonized zymosan particles was inhibited by anti-CR3 Ab and C3bi-bound IC, but not by beta-glucan, mannan, and anti-Toll-like receptor 2 Ab. These findings show that cooperative engagement of CR3 on both the lectin-like site involved in beta-glucan binding and the I-domain involved in C3bi binding, as it can be observed in the innate immune response, produces AA release, whereas the unique interaction of C3bi-bound IC with the I-domain of CR3, as it may occur in the adaptive immune response, diverts the IC lattice from a productive interaction with FcgammaR linked to AA release.     

Defective immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with mixed essential cryoglobulinemia type II

Mixed essential cryoglobulinemia type II (monoclonal Ig/polyclonal IgG) is characterized by systemic vasculitis caused by the deposition of circulating immune reactants that include the monoclonal component. Such reactants may include immune complexes (IC) formed from exogenous Ag. IC binding to E C receptor type 1 appears to play a role in transport and buffering of such IC (immune adherence: IA). To define the mechanisms responsible for immune deposition, 7 patients with cryoglobulinemia type II (IgM kappa/polyclonal IgG) and 14 normal volunteers were injected i.v. with hepatitis B surface Ag/antibody complexes. Two minutes after injection, only 19.4% (mean) of the circulating complexes were bound to E in patients as compared with 63.1% in normal subjects. This IA correlated directly with C4 and inversely with the IgM rheumatoid factor (RF) titer. Disappearance of IC was faster in patients (mean elimination rate: 15.7%/min) than in normal subjects (9.3%). In vitro experiments demonstrated that C depletion, interference with IC opsonization by monoclonal IgM RF, and decreased binding of opsonized IC in the presence of monoclonal RF are each associated with decreased IA. These observations suggest that, in patients with cryoglobulinemia type II, monoclonal IgM RF and low C contribute to reducing IA of circulating IC that might be rapidly trapped in tissues, resulting in injury.     

Elevated NK sensitivity of Raji cells carrying acceptor-bound C3 fragments

The majority of cell lines derived from Burkitt lymphomas carry CR2 on their plasma membrane cell lines of haematopoietic origin can activate C3 present in human serum through the alternative pathway. However, only the lines that carry CR2 were shown to bind C3 fragments. This bond can be either fixation to acceptor sites or attachment to the CR. Our studies with Raji cells showed that when the possibility for the covalent acceptor bond was eliminated by using methylamine (MA)- or zymosan-treated serum, considerably lower amounts of C3 were bound. In the zymosan-treated serum C3 fragments are present that can bind to receptors but their capacity for acceptor bond is absent. These results indicate that when Raji cell are incubated in human serum some of the generated C3 fragments are bound to acceptors and a lower proportion through the specific interaction with complement receptors. Pretreatment of the CR2 carrying cell lines with human serum elevated their sensitivity to the lytic effect of human blood lymphocytes. We showed in this work that MA-treated serum did not induce this elevation. Zymosan-treated serum under conditions that excluded activation of the residual native C3 molecules, i.e., in the presence of EDTA, did not have the enhancing effect either. These results suggest that the increased lytic efficiency imposed by human serum was due to cleavage of C3 molecules by Raji and fixation of the C3 fragments by acceptor sites. Natural killer cells carry CR3; therefore it is likely that the attached C3 fragments bind also to the effector cells. The C3 molecules could elevate thereby the avidity between the target and the lytic lymphocytes. The observation that C3 fragments are not bound to the surface of CR2 negative lines in spite of their capacity to activate C3 suggests that the receptor molecule is either involved in the activation and/or serves also as an acceptor.     

Abnormal immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with AIDS.

C3b-coated immune complexes (IC) adhere to complement receptor 1 (CR1) on human E in the circulation. E from AIDS patients have an acquired low CR1 number. To study immune adherence and IC elimination in AIDS, radiolabeled hepatitis B surface Ag/antibody complexes were injected i.v. in six AIDS patients and in 14 healthy controls. The binding of IC to E was reduced in AIDS patients (mean binding 2 min after injection: 24.9 +/- 13.3%) compared with healthy individuals (63 +/- 3.7%) (p = 0.0005). The low binding correlated directly with the number of CR1/E and to the capacity of these E to bind IC in vitro. During the first 15 min disappearance of IC was faster in AIDS patients than in normal subjects and correlated with CR1 number. Thereafter, elimination was very slow in AIDS patients, which suggested that a fraction of IC might be released back into the circulation similarly to what has been observed for C3b-coated E. When the data were analyzed with a mathematical model allowing for such release to occur, five of six AIDS patients had a high release rate compared with little or no release in normal individuals (p less than 0.001). Thus, low CR1 on E is responsible for defective immune adherence, and might determine abnormal disappearance of IC from the circulation as well.     

C3 synthetic peptides support growth of human CR2-positive lymphoblastoid B cells

The nature of CR type 2 (CR2)-ligand interactions which leads to the activation of human B cells was analyzed by using synthetic peptides and CR2-positive cell lines. The third component of C (C3) supported the growth of human lymphoblastoid B cells in serum-free medium containing human transferrin. This effect was inhibited by an antibody to C3d (mAb 130) which specifically inhibits C3d binding to CR2, but not by other anti-C3 mAb. Synthetic peptides corresponding to the CR2-binding site on C3d, P28 (residues 1187-1214) or multivalent P13 [1202-1214)4-template), supported the proliferative response of CR2-positive human lymphoblastoid lines in a similar way as C3 and this response could be inhibited by the anti-CR2 mAb OKB7. The proliferative response to C3 or peptides was dose dependent and a 60-fold higher concentration of P28 peptide was required to induce the same level of proliferation as C3. This stimulation of growth was observed only on CR2 expressing cell lines Raji and Daudi, and not on the CR2-negative Burkitt lymphoma cell line Rael and the monocytic cell line U937. In contrast to the stimulatory effect of P28 and P13-template, monomeric P14 (1201-1214) was not able to support the growth of these cell lines. This peptide, however, inhibited the proliferative response of the CR2-positive lines to C3, P28, and multivalent-P13, thus indicating that cross-linking of the CR2 receptor is necessary for B cell proliferation. Another peptide, E12 (from glycoprotein (GP)350, the major EBV outer membrane GP) which shows a high degree of similarity with P14, also inhibited the proliferative response of Raji cells, suggesting that this segment on GP350 is involved in the interaction of EBV with CR2. The possibility of using the above peptides as well as other peptides with "tailor-made" structure in studying the multifunctional role of C3 is discussed.     

Quantitative analyses of C3b capture and immune adherence of IgM antibody/dsDNA immune complexes

We isolated the IgM fraction from the plasma of an SLE patient with high titer anti-dsDNA antibodies and prepared soluble IgM/dsDNA immune complexes (IC) that fixed C and captured sufficient C3b to bind to human E via their C3b/C4b receptor, CR1 (immune adherence, IA). We used specific 125I-labeled mAb to IgM, C3b, and IgG to measure the stoichiometries of these C-opsonized IC. They contained 10 to 60 C3b and 10 to 30 IgM per PM2 dsDNA, had no detectable IgG, and the vast majority of the C3b was bound to the IgM, and not to the dsDNA. These stoichiometries are in contrast to those we observed for comparable E-bound IC prepared with IgG anti-dsDNA antibodies (100 to 200 C3b, and 200 to 500 IgG). Our results help explain the greater lability of the IgM IC with respect to IA as evidenced by their plasma-mediated release from human E (presumably due to factor I), and confirm previous predictions of a lower density of "packing" of IgM on dsDNA, compared to IgG. The detailed stoichiometry of C3b capture by the IgM IC (typically 1.5 to 3 C3b per IgM) suggests that individual IgM molecules with multiple C3b facilitate IC binding to clusters of CR1. Finally, comparison of the IgM/dsDNA IC with other IgM IC which have been investigated with respect to C activation, and review of the proposed mechanism by which IgM activates C, suggests that the nature of the Ag plays a fundamental role in determining whether or not an IgM IC can activate C and participate in IA.     

Characterization of the baboon erythrocyte C3b-binding protein

E from primates demonstrate type 1 CR (CR1) with binding specificities for C3b and C4b. In the present study we characterized the E C3b-binding protein of baboons. We showed that three out of four mouse mAb and one polyclonal antiserum, raised against human E CR1, cross-reacted with baboon E. In addition, one anti-human CR1 mAb (1B4) and a polyclonal anti-human CR1 inhibited the binding of C3b opsonized immune complexes to baboon E. Finally, a mAb to human CR1 (E11) recognized epitopes on E of a variety of nonhuman primates, including baboons. SDS-PAGE analysis of biochemically purified baboon E membrane fractions reactive with E11 demonstrated a 65-kDa protein as a major component. Affinity absorption and elution experiments verified this protein to be E11 reactive as well as a C3b binding protein. E surface radiolabeling, followed by C3i affinity purification, confirmed that this 65-kDa protein is the only C3b-binding protein present on the baboon E membrane. We postulate that the baboon E 65-kDa protein is the equivalent of the human E CR1. In addition, there appear to be antigenic similarities between the baboon E 65-kDa protein and the human E CR1.     

A study of in vivo immune complex formation and clearance in man

C and CR1 have been shown to participate in the clearance of injected, preformed, immune complexes in humans and in non-human primates. Their role in the physiologic disposal of immune complexes formed in vivo in humans was investigated in three patients receiving radioimmunotherapy for ovarian carcinoma. On day 0 each patient received, by intraperitoneal injection, 10 mg of 131I-mouse anti-tumor mAb (10 mCi/mg). On days 1 and 2, 18 mg of trace-labeled, 125I-human anti-mouse IgG was administered by i.v. infusion over 15 min, to accelerate the clearance of the 131I-anti-tumor antibody from the circulation and reduce the radiation dose to the marrow. Sequential blood samples were obtained after the injection of the second (anti-mouse) antibody, to monitor clearance. Immune complexes (shown by sucrose gradient centrifugation to be 19 to 40 S in size) formed within 5 min, and were cleared with a half-life of 11 +/- 1.7 min in the liver. Complexes were measured by 4% polyethylene glycol precipitation, and by solid phase C3d- and C1q-binding assays. Between 8 and 11% of the total available complexed material bound to CR1 on E. Peak binding of immune complexes to red cells occurred 10 min after the maximal complex load was detected by precipitation with polyethylene glycol. At that time, immune complexes bound to E constituted one-fifth of the total circulating pool of complexes. Coincident with immune complex formation and clearance, a 47% fall in serum C4, C3, and CH50 was measured, with the deposition of up to 1230 molecules of C4, and 2590 molecules of C3 on the surface of red cells. During 20 min after immune complex formation there was a mean loss of 32% of erythrocyte CR1. The changes in complement and CR1 on E and in serum observed in these patients resembled those seen in patients with SLE: i.e., a reduction in CR1 and an increase in C3 and C4 on E, and reduced serum C.     

Direct evidence for the clustered nature of complement receptors type 1 on the erythrocyte membrane

C receptors 1 (CR1) of human E are involved in the transport of C3b-coated immune complexes (IC) in the circulation. Many studies have suggested that the binding of IC to E is multivalent. This would require CR1 to be clustered on the cell membrane, but no direct evidence for such clustering is available. We studied the distribution of CR1 on human E by immunofluorescence and shadow-casting immuno-electron microscopy techniques with the use of a monoclonal anti-CR1 antibody followed by FITC- or gold-conjugated second antibodies, respectively. By immunofluorescence, CR1 appeared as small dots (clusters) on fixed and unfixed E prepared either at 4 degrees C or at 37 degrees C. In the same donor, the number of clusters varied extensively from cell to cell (e.g., 1 to 43 clusters/E for a donor with 520 CR1/cell), but the mean number of clusters per cell correlated significantly with the mean number of CR1/cell. These images contrasted with those obtained for Rhesus D (RhD) Ag used as controls (RhD Ag are known to be evenly distributed): only a faint uniform fluorescence was seen despite the presence of 10,000 antigenic sites. As determined by immunocytochemical method, more than 65% of the total gold particles were organized in clusters (2 to 15 gold particles/cluster) whether cells were prefixed or not. Quantitative determinations suggested that each gold particle corresponded to one CR1. The fraction of gold particles grouped into clusters of three or more receptors, the mean size of the clusters, and the maximal size of clusters correlated with the mean number of CR1 per cell. By contrast, RhD Ag were distributed homogeneously (less than 2% gold particles in clusters). These data are the first to demonstrate the preclustered nature of CR1 on E. Such distribution could explain the high binding efficiency of C3b-coated IC to E despite the low number of CR1 per cell.     

Quantitative analyses of the relationship between C3 consumption, C3b capture, and immune adherence of complement-fixing antibody/DNA immune complexes

We have studied the turnover of the third component of C (C3) and capture of the major cleavage fragment of C3 produced during C activation (C3b) that occurs when soluble antibody/DNA immune complexes (IC) active C. We used the Amersham RIA kit for the minor cleavage fragment of C3 produced during C activation (C3a), and a new assay utilizing mAb to C3b to measure the fraction of active C3 in a C source after the IC activate C. These mAb, along with a mAb to human IgG, allowed us to measure IC stoichiometries. The efficiency of C3 turnover by the IC is quite high, and under conditions of Ab excess, the maximum number of IgG bound per dsDNA corresponds to 1 IgG/20 to 30 base pairs. The maximum number of C3b found in the IC corresponds to less than 1 C3b/IgG, and the vast majority of the captured C3b is bound to the IgG, and not to the DNA. We identified several IC that consumed large amounts of C3, and captured large amounts of C3b, but did not bind to human E via C3b receptors (C receptor type 1). This finding suggests that the ability of IC to bind to human E depends upon the number and distribution of captured C3b molecules and the conformation and size of the DNA Ag, which reflects the need for multivalent binding between several properly arrayed C3b and a "cluster" of C receptor type 1 on the human E membrane. IC that activate C3 but do not bind to E would presumably "escape" the E IC clearance mechanism, but could deposit in susceptible organs and tissues and play a role in the pathogenesis of SLE because of their potential to generate the inflammatory products of C activation.     

Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing effector cells than direct internalization by the B cells

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of ～40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.