Release of Immune Complexes bound to Erythrocyte Complement Receptor (CR1), with Particular Reference to the Role of Factor I

The release of 125I-bovine serum albumin (BSA)-anti-BSA immune complexes (IC) bound to human erythrocyte complement receptors (E-CR1) was studied. IC were complement-solubilized in normal human serum (NHS), and reacted with human erythrocytes at conditions optimal for binding of the IC to E-CR1. E-CR1-bound IC could be released by the addition of NHS or purified factor I. Factor I-deficient or I-depleted serum mediated no release, and addition of purified factor I restored the release. Factor H was not required for the release of IC. The kinetics of IC release was influenced by the NHS concentration, the presence of EDTA, and the time of prior storage of the erythrocytes at 4 degrees C. NHS (1:5 to 1:10) in the presence of EDTA caused nearly maximal release within 10-20 min at 37 degrees C. In the absence of EDTA the NHS-induced IC release was markedly slower. IC released within the first 30 min showed significant rebinding to new E. The release of IC was not associated with loss of the IC binding activity of E-CR1. The NHS-mediated release of IC could be inhibited by rabbit anti-CR1 and by a mixture of protease inhibitors. Release induced by purified factor I was also inhibited by protease inhibitors. The affinity of IC binding to E-CR1 was reduced after cleavage of CR1-bound C3b-IC to iC3b-IC by factor I.     

Erythrocyte CR1 Determination Using Monoclonal Antibody in a Microtiter Plate ELISA; Receptors Are Not Masked by Immune Complexes

The microtiter plate ELISA using monoclonal antibody is a specific, sensitive and quantitative technique for measuring CR1 on human erythrocytes. The present investigations established that receptor occupancy by immune complexes did not affect the measurements. The monoclonal anti-CR1 antibody To5 bound unimpeded to receptors that had reacted with an excess of complement-opsonized tetanus toxoid anti-tetanus toxoid complexes prepared at antigen:antibody ratios between 32:1 and 1:8. The CR1 levels on erythrocytes from 11 patients with systemic lupus erythematosus (SLE) were not increased (P greater than 0.30) after release of CR1-bound immune complexes by incubation with factor I. Neither did the serum from these patients contain blocking anti-CR1 activity (P greater than 0.10). Additionally, the number of antigenic CR1 sites in 10 normals and in the 11 patients with SLE was well correlated with the number of functional receptor sites as assessed by binding of soluble complexes (P less than 0.001). These data establish that the true CR1 levels are determined using the microtiter plate ELISA for quantitation of CR1 in patients with diseases involving immune complexes and/or autoantibodies.     

Interaction of complement-solubilized immune complexes (IC) with CR1 receptors on human erythrocytes. Polysulfated compounds inhibit IC binding and induce IC-release from CR1

The effect of the polysulfated compounds heparin, dextran sulfate, chondroitin sulfate and suramin, and non-sulfated poly-, oligo-, and monosaccharides on binding and release of complement-solubilized 125I BSA-anti BSA immune complexes (IC) reacting with complement C3b receptors (CR1) on human erythrocytes (E) was investigated. Following presolubilization of IC in normal autologous human serum (NHS) a clear dose-dependent inhibition of IC-binding to E-CR1 was obtained by addition of polysulfated compounds. The inhibitory effect was dependent on the sulfate content of the reagents used but independent of their anticoagulant activity as heparin preparations with high and low affinity for antithrombin III inhibited IC binding to E-CR1 to approximately the same extent. Dextran sulfate caused a stronger inhibition than heparin while chondroitin sulfate was inhibitory only at high concentrations. The inhibitory effect was exerted at the IC-C3b level as normal IC-binding occurred following preincubation of E with the polysulfated compounds. Non-sulfated saccharides showed no inhibition of IC binding to E-CR1. All polysulfated compounds, apart from chondroitin sulfate, induced a dose-dependent release of E-CR1 bound IC in the absence of NHS. No release was obtained by use of non-sulfated saccharides. Heparin induced IC-release was rapid (40-45% after 3 min) and incubation beyond 30 min caused only an insignificant further release of IC from E-CR1. Following release of IC the E-CR1 retained full binding capacity for freshly added IC-C3b.     

Zinc Ions Inhibit Factor I-Mediated Release of CR1-Bound Immune Complexes and Degradation of Cell-Bound Complement Factors C3b and C4b

ZnCl2 exerted a dose-dependent inhibition of citrate-phosphate-dextrose (CPD) plasma-induced release of 125I-labelled BSA-anti-BSA immune complexes (IC) bound to complement receptor type 1 (CR1, CD35) in human whole blood. Maximal inhibition was observed at 10 mM of ZnCl2. Furthermore, the release of IC bound to erythrocyte (E)-CR1 by purified factor I, factor I-deficient serum plus purified factor I, or normal human serum was reduced by approximately 90%, 64%, and 52%, respectively, in the presence of 10 mM ZnCl2. The effect of ZnCl2 on factor I-mediated degradation of cell-bound C3b/C4b was also investigated employing CPD blood or E from a factor I-deficient donor. These cells expressed covalently bound C3b and C4b as demonstrated by a simple agglutination technique. Upon incubation of CPD whole blood with purified factor I, or of E with purified factor I or normal CPD plasma, the C-fragments were cleaved and the cells were no longer agglutinated by antibodies to C3c and C4c. The presence of ZnCl2 prevented this factor I-mediated degradation of C3b and C4b, as evidenced by the unaffected agglutination of the cells by the antibodies. We conclude that ZnCl2 inhibited factor I activity since: (1) release of complement-preopsonized IC from E-CR1 by purified factor I was markedly inhibited (90%) in the presence of ZnCl2, (2) preincubation of the cells with ZnCl2 caused only a moderate inhibition (32-38%) of the IC release, and (3) degradation by purified factor I of covalently cell-bound C3b and C4b was abrogated in the presence of 10 mM ZnCl2.     

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.     

C3 nephritic factor protects bound C3bBb from cleavage by factor I and human erythrocytes

C3 nephritic factor is an autoantibody to the alternative-pathway C3 convertase (C3bBb) which increases the half-life of the convertase both in the presence and absence of serum regulatory proteins. Human erythrocytes contain membrane proteins which also can regulate C3bBb. One of these proteins, the C3b/C4b receptor (CR1), plays an important role in the processing of soluble immune complexes. C3b which is fixed to immune complexes binds to CR1 and is cleaved by factor I to C3c and C3dg. We have tested the effectiveness of the nephritic factor in protecting bound C3b from cleavage by factor I and human erythrocytes. Sheep erythrocyte intermediates EAC1423b were prepared using 125I-labeled C3 and incubated with factors B and D in the presence and absence of nephritic factor. Breakdown of C3b was measured by release of 125I-C3c following incubation with human erythrocytes and factor I. Purified IgG from two patients with nephritic factor prevented C3c release in a dose-dependent manner. Normal human IgG was ineffective as was nephritic factor in the absence of factor B. Factor P also inhibited the release of C3c in the presence of factor B with equivalent activity at approx. 20-fold higher concns than nephritic factor. These results indicate that nephritic factor can impair human erythrocyte dependent degradation of C3b in alternative-pathway-activating immune complexes.     

The role of hypocomplementaemia and low erythrocyte complement receptor type 1 numbers in determining abnormal immune complex clearance in humans.

Defective clearance of immune complexes (IC) may contribute to the pathogenesis of diseases such as SLE. We studied the effect of hypocomplementaemia and the influence of erythrocyte complement receptor type 1 (CR1, CD35) number on the clearance of radiolabelled tetanus toxoid (TT)-anti-TT IC from the circulation. These were injected intravenously into 9 normal subjects and 15 patients with diseases characterized by IC formation and/or hypocomplementemia, including 2 with hereditary complement deficiency. IC were found to bind to erythrocyte CR1 in a complement-dependent manner and their degree of uptake was directly correlated with CR1 numbers. Two phases of IC clearance were identified. The first was rapid, occurring within 1 min. Since this phase might represent inappropriate deposition of IC in target organs we called it trapping. It was seen predominantly in subjects with low CR1, low complement, and low binding of complexes to red cells. The second phase was monoexponential with a mean elimination rate of 14.1%/min; it was inversely correlated with CR1 numbers and binding of complexes to red cells. In a second study each individual was injected with IC bound to autologous erythrocytes in vitro using normal serum so that the effects of complement deficiency were eliminated. Up to 81.4% of these bound IC were released in vivo from erythrocytes in 1 min, and the proportion was inversely correlated with CR1 numbers. Only five patients showed trapping, and these had low CR1 numbers and high percentage release of IC. The second phase of elimination was inversely correlated with CR1 numbers and the proportion of IC remaining bound to red cells at 1 min. The two complement-deficient patients had normal CR1: when IC were injected, trapping and very fast clearance rates were observed; however complexes that had been opsonized and bound to erythrocytes were cleared at a slower rate without evidence for trapping. These studies show that complement and erythrocyte CR1 may determine the physiological clearance of certain types of IC and suggest that this system may function abnormally when CR1 number or complement function are reduced.     

The binding of immune complexes to human red cells: complement requirements and fate of the RBC-bound IC after interaction with human phagocytic cells.

Soluble immune complexes (IC) are known to bind to human red blood cells (HRBC). Most authors have attributed this binding to the interaction between IC-bound C3b and a red cell CR1 receptor, but contradictory data has been published concerning the ability of IC to bind to HRBC in the absence of complement. Using soluble tetanus toxoid-rabbit anti-tetanus toxoid (TT-ATT) IC, we have shown that binding through the CR1 receptor takes place when IC are formed at antibody excess, while IC formed at antigen excess do not require complement for erythrocyte binding. Once absorbed to HRBC, IC are recognized by CR1 and/or Fc receptors on phagocytic cells. This interaction is not associated with red cell engulfment, but using radiolabelled S. aureus protein A as a probe, we have demonstrated the transfer of IC from HRBC to phagocytic cells. Such transfer without red blood cell (RBC) damage agrees with the postulated role of RBC in the elimination of soluble IC from circulation. However, we have also demonstrated that the interaction between HRBC-IC and phagocytic cells is associated with the release of mediators of inflammation. It is, therefore, not absolutely clear whether the interaction of RBC-adsorbed IC and phagocytic cells will always have beneficial consequences.     

Visualization of the transfer reaction: tracking immune complexes from erythrocyte complement receptor 1 to macrophages

Immune complexes (IC) bound to human erythrocytes (E) via complement receptor 1 (CR1) are transferred to phagocytes in the liver and spleen. In an in vitro model system using bispecific mAb reagents (antigen-based heteropolymers) to link IC to E, we have made time-lapse movies in which fluorescently labeled IC cross the E-human macrophage interface and remain associated with the macrophage. Both these movies and fixed-time experiments reveal transfer intermediates in which IC hinge E to macrophages. Examination of model macrophages after transfer indicates that the majority of IC are on the surface at short times (2 min) but are internalized at long times (1-4 h). More than half of the surface IC colocalize with CR1 at 2 min. This evidence supports a model in which CR1-bound IC provide a secure linkage between E and macrophages, allowing rearrangements of the macrophage surface necessary for release of CR1, and IC, from the E.     

Interaction of Complement-Solubilized Immune Complexes with CR1 Receptors on Human Erythrocytes. The Binding Reaction

The binding of complement (C)-solubilized ¹²⁵I bovine serum albumin (BSA) anti-BSA immune complexes (IC) to CR1 receptors on human red blond cells (RBC-CR1) was studied. The binding of IC to CRI was strongly dependent on the molar antigen lo antibody ratio, and IC formed in moderate antigen excess showed no binding. IC solubilized, in 50% human serum in the presence of autologous RBC bound rapidly lo RBC-CRI, with maximal binding within less than 1 min at 37°C. Release of CRI-hound IC under these conditions occurred slowly, requiring more than.30 min. Only binding of 'partially' solubilized, e.g., anti C3c (C4c) and conglutinin-reactive 1C occurred, whereas fully solubilized complexes (IC-C3dg. C4d) showed virtually no binding. Solubilization of IC in the presence of Mg-EGTA or in C2-deficient serum resulted in a markedly delayed binding of IC ti RBC, indicating the importance of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solobilized IC could be absorbed to solid-phase conglutinin or antibody to C3c abd C4c, and tgese kugabds were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect on the binding of presolubilized IC to RBC-CR1, whereas the binding was unaffected by the addition of monosaccharides or by the concentration of Ca²⁻ or Mg²⁻ ions.     

Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes.

Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Non-complement-dependent adsorption of soluble immune complexes to human red cells

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.     

A technique for the purification of immune complexes using rheumatoid factor.

A method for the purification of immune complexes (IC) from human serum is described, using as a model complexes of tetanus toxoid with human antitoxoid antibodies (TAT). The technique is based on the ability of IC to bind to tubes coated with rheumatoid factor (RF). Small amounts of TAT IC were added to human serum and the mixtures were rotated overnight in tubes coated with RF. The tubes were then washed and the bound material was eluted with sodium dodecyl sulphate and iodinated with 125I. Analysis of the labeled preparation by electrophoresis in polyacrylamide gels revealed the presence of the toxoid component. The technique should be useful in isolating small amounts of IC for analytical purposes.     

Interaction between immune complexes and C3b receptors on erythrocytes

We studied the interaction between immune complexes (IC) and C3b receptors (CR1) on erythrocytes (E) and showed that activation of the classical complement pathway is essential for the binding of IC to CR1, that C3b inactivator (I) and beta 1H (H) are essential for the release of IC from CR1, that CR1 retain the capacity to bind IC after repeated binding and release of IC, and that on the other hand, IC lose the capacity to bind to CR1 after repeated binding and release. These results suggest a dynamic in vivo interaction between IC and CR1 on E which are supposed to transport IC to the reticuloendothelial system. CR1 on E, with a help of I and H, might make IC less harmful to the tissues in the process of releasing IC from E.     

Influence of TPA (12-O-tetradodecanoyl-phorbol-13-acetate) on human B lymphocyte function

A deficiency of C3b receptors (CR1) on erythrocytes from patients with systemic lupus erythematosus (SLE) has already been reported and assumed to be one of the causes of the impaired immune complex clearing function found in these patients. In the present study, we developed a functional assay to quantify the amount of CR1 on human erythrocytes. Sample erythrocytes were reacted with tetanus toxoid-anti-tetanus toxoid immune complexes (IC) in the presence of complement. The amount of CR1 was expressed as the amount of IC bound to sample erythrocytes. Determination of CR1 showed a decrease in erythrocytes from patients with SLE, rheumatoid arthritis and other connective tissue diseases. The activity of CR1 in erythrocytes from patients with SLE changed in parallel with complement activity and also reflected the clinical status of two of three patients. These results imply that the reduction of CR1 found in SLE patients might be cause not only by hereditary factors but by unknown factors that influence the amount or function of CR1.     

Influence of serum complement and rheumatoid factor on detection of immune complexes by the C1q and monoclonal rheumatoid factor solid-phase assay

Soluble purified monoclonal and polyclonal rheumatoid factor, total serum complement, and soluble C1q all inhibit the detection of model tetanus toxoid/anti-toxoid immune complexes in the solid-phase C1q assay. The binding of these immune complexes to solid-phase monoclonal rheumatoid factor is less inhibited by soluble C1q and by total serum complement, but clearly decreased by soluble monoclonal or polyclonal rheumatoid factor. Serum complement does not reduce the size of these model complexes. We recommend the use of low ionic strength EDTA (10 mM) to partly neutralize the complement-mediated inhibition. This procedure is shown to be superior to currently used higher EDTA concentrations and to the use of IgG-Sepharose.     

Functional characterization of non-human primate erythrocyte immune adherence receptors: implications for the uptake of immune complexes by the cells of the mononuclear phagocytic system.

Erythrocytes from primates express an immune adherence (IA) receptor that binds complement-opsonized immune complexes (IC) both in vivo and in vitro. We have analyzed the immunochemical and functional properties of the IA receptor from erythrocytes from species that have been used for in vivo IC clearance studies and have compared these properties to the human IA receptor (which is called complement receptor type 1, CR1). Erythrocytes from all species (chimpanzee, baboon, rhesus and cynomolgus monkey) bind antibody/double-stranded DNA IC when opsonized with autologous complement. However, IC which are bound to chimpanzee erythrocytes are not released upon addition of chimpanzee serum (which contains factor I activity), while IC bound to baboon erythrocytes and human erythrocytes are released upon addition of autologous serum. Anti-human CR1 monoclonal antibodies (mAb) E11 and HB8592 bind to erythrocytes from all species examined and the number of mAb epitopes per erythrocyte correlated with the number of IC that could bind to the erythrocyte under saturating conditions. However, a number of interesting differences between the species are observed with other mAb. The anti-CR1 mAb 1B4 and 3D9, which block recognition of ligand by CR1, did not bind to chimpanzee erythrocytes and bound partially to rhesus and cynomolgus monkey erythrocytes. In addition, the ability of autologous serum to induce release of erythrocyte-bound IC correlates with the presence of these epitopes. These findings, taken in context with previous clearance studies, suggest that serum-mediated release may not be required for the rapid transfer of the IC from the erythrocyte to the mononuclear phagocytic system.     

Differences in interaction between immune complexes and complement receptors in serum and cerebrospinal fluid

Antigen—antibody complexes (Ag—Ab), prepared from ¹²⁵I-radiolabelled bovine serum albumin (BSA) and guinea-pig antibody, were (1) pre-incubated at 37°C for 30 min with serum or cerebrospinal fluid (CSF) in different proportions and then reacted with cells, (2) incubated at 37°C directly with cells suspended in serum or CSF for different time periods, or (3) bound to cells (following incubation with serum in optimal proportions) and the cell-bound immune complexes (IC) incubated with serum or CSF at 37°C for different time periods. When Ag—Ab were pre-incubated with serum or CSF and reacted with unfractionated blood cells or mononuclear cells, binding decreased as serum to Ag—Ab proportion was increased above 1:16, but increased as CSF to Ag—Ab proportion was increased. When serum diluted 200-fold (to approximate the protein concentration of CSF) was used in place of undiluted serum, serum-mediated binding paralleled CSF-mediated binding. Inactivation of serum, CSF, and 1:200 serum in different ways and substitution of human red blood cells (RBC) (known to possess C3b receptors) or sheep RBC (known not to possess C3b receptors) demonstrated that binding was to C3b receptors. Addition of CSF to serum did not alter serum-mediated binding. When Ag—Ab were incubated directly with unfractionated blood cells suspended in serum or CSF, binding increased rapidly in serum, reaching a maximum within 2—4 min, and IC then rapidly dissociated, whereas binding increased gradually in CSF and IC remained associated with cells. When serum diluted approximately 100-fold was used in place of undiluted serum, kinetics of serum-mediated interaction approached that of CSF-mediated interaction. When IC were bound to Raji cells or human RBC and the cell-bound IC incubated in serum or CSF, > 85% of IC dissociated in serum after 30 min, but no dissociation occurred in CSF. Dilution of serum > 1:16 and > 1:64 abolished dissociation from the two cell types, respectively. These results indicate that CSF mediates binding of IC to complement receptors on cells but lacks the activities of serum which convert IC into a non-binding state.     

Reduced Erythrocyte CR1 (CD 35) Receptor Function and Complement Opsonization in Factor I-Deficient Patients is Restored by Plasma Infusion

Erythrocytes (E) from three factor I-deficient patients were investigated for surface-bound complement factors and CR1 (CD 35) expression and function. The E were coated with C4b, C3b, and factor H. Following plasma infusion or in vitro incubation of the patients' E with normal human serum (NHS) or purified factor I, cell-bound C4b and C3b could no longer be detected. The E now expressed C3d, and factor H was unaffected, indicating that factor H was bound to the C3d part of the C3b molecules, providing the co-factor for effective cleavage of E-bound C3b when purified factor I was added. The binding of monoclonal anti-CR1 antibodies (M710) to the patients' E was markedly reduced compared with control E, and was not normalized by treatment with NHS, probably because covalently bound C3d/factor H interfered with the binding of M710. By contrast, the reduced ability of the patients' E-CR1 to bind complement-opsonized immune complexes (IC) was normalized after plasma infusion. This shows that the impaired CR1 function was acquired and emphasizes the importance of performing functional CR1 assays. Complement opsonization of IC for binding to normal E was severely compromised in the patients' sera due to consumption of factor B and C3. After plasma infusion the opsonization capacity of the patients' sera was restored. Thus, two mechanisms of importance for normal clearance of IC were compromised in factor I-deficient patients: (1) the opsonization of IC for binding to E-CR1, and (2) the capacity of E-CR1 to bind opsonized complexes. Both dysfunctions were temporarily corrected by plasma infusion.     

The modulation of immune complex aggregation by classical pathway-mediated reactions

Classical pathway (CP)-triggered reactions of complement-modulated immune complex (IC) aggregation (tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) were investigated turbidimetrically during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) directed against certain complement components were used to block the complement function in normal human serum (NHS). Additionally, parts of the reactions were studied using purified complement components. C1q in serum generated by the addition of EDTA as well as purified C1q were found to increase the IC aggregation. In contrast to C1q, macromolecular C1 is able to inhibit IC aggregation, whereas additional participation of C-1 INH reversed this process. The cooperation of the remaining CP proteins (C4, C2, C4bp, and I) reconstituted the inhibition capacity of the complement. Whereas C3 supported significantly inhibition, a significant influence of other effector pathway (EP) components (C5-C9) was not detectable turbidimetrically.