Erythrocyte Complement Receptor Research Articles

Antigen-based heteropolymers facilitate, via primate erythrocyte complement receptor type 1, rapid erythrocyte binding of an autoantibody and its clearance from the circulation in rhesus monkeys.

We investigated the feasibility of using the primate E complement receptor (CR1), in concert with Ag-based heteropolymers (AHP), as a potential therapy to remove autoantibodies from the circulation. AHP are prepared by cross-linking an anti-CR1 mAb with the acetylcholine receptor (AChR), the principal target Ag in myasthenia gravis. In vitro studies demonstrate that this methodology facilitates specific, rapid, and quantitative binding of an anti-AChR mAb to primate Es. In vivo experiments in rhesus monkeys indicate that AHP-mediated binding of an anti-AChR mAb to Es leads to the clearance of the mAb from the circulation. Once bound to the E via the AHP, the autoantibody is transported to the liver and spleen, where it is degraded without destruction of the E. It is therefore likely that the complexes of AHP and target mAb, when bound to Es, are recognized in vivo and processed by a mechanism quite similar to that which occurs when complement-opsonized immune complexes, bound to primate Es, are cleared from the circulation. It may be possible to extend and generalize this work to allow for the development of a simple, noninvasive therapy that can be made specific for the treatment of several different autoimmune diseases.

Another function of erythrocytes: transport of circulating immune complexes.

To review the main characteristics of the 'immune complex (IC) transport system of erythrocytes', and its possible relevance for transfusion medicine. Literature search, and results Of Studies performed in the laboratory of Dr J. Schifferli from 1986 to 1995. Peer review journals and work relevant to the topic. When antigen/antibody IC form in the presence of complement. C3b binds covalently to the complexes. Such opsonized complexes attach to cells bearing complement receptor 1 (CR1), which is mostly found on erythrocytes in the circulation. This allows IC to be transported through the circulation to the fixed macrophage system of the liver and spleen. In addition, C3b bound to the complexes is catabolized by factor 1 (with CR1 as a cofactor) so that the complement-activating properties of the complexes are reduced. Complement and erythrocyte CR1 contribute to the safe and effective elimination of IC in humans. This physiologic system prevents IC deposition in the vessel walls.

Erythrocyte complement receptor type 1 and interactions between immune complexes, neutrophils, and endothelium.

The deposition of immune complexes (IC) may play an important part in the pathogenesis of vasculitis. An increase in permeability of the vascular endothelial lining is a prerequisite for IC deposition. We used an in vitro model to examine the effects of interactions between IC, neutrophils, and endothelium on the integrity of endothelial cell monolayers. Human umbilical vein endothelial cells were grown to confluence on an FITC-labeled matrix, and monolayer integrity was assessed by the exclusion of an 125I-anti-FITC Ab. Alteration in endothelial monolayer permeability was associated with increased uptake of 125I-anti-FITC Ab, expressed as a percentage of the maximal uptake of Ab onto the FITC-matrix from which endothelial cells had been stripped. Neither resting nor cytokine-stimulated endothelial cells bound hepatitis B surface Ag (HBsAg/anti-HBsAg) IC. Immune complexes were shown to activate neutrophils to induce a 9.5% increase in the permeability of IL-1 beta-stimulated endothelium. This increase in endothelial permeability was abrogated by the addition of RBC bearing normal complement receptor type 1 (CR1) numbers (3.2%). This protective effect was shown to be related to the binding of IC to erythrocyte CR1 and was reduced by CR1 blockade using polyclonal rabbit anti-CR1 Abs. These observations demonstrate that IC are capable of directly activating neutrophils to induce increases in endothelial permeability, which may facilitate the deposition of circulating IC. The results support the hypothesis that the binding of IC to erythrocyte CR1 may inhibit damaging interactions between IC, neutrophils, and endothelium.

Cross-linked bispecific monoclonal antibody heteropolymers facilitate the clearance of human IgM from the circulation of squirrel monkeys.

We have previously demonstrated that cross-linked bispecific monoclonal antibodies (mAb) heteropolymers (HP), specific for primate erythrocyte (E) complement receptor type 1 (CR1) and target antigen (Ag), facilitate the binding of these target Ag to human and non-human primate E. Once bound in vitro to rhesus monkey E, upon re-infusion these HP/Ag complexes are recognized in vivo by cells of the reticuloendothelial system (RES) and removed from the circulation without loss of the E. We now show, in squirrel monkeys, that an HP specific for E CR1 and human IgM (anti-CR1 x anti-IgM) can be used to facilitate in vivo E binding and clearance from the circulation of a previously injected and circulating model protein pathogen, human IgM. Approximately 70-80% of 125I-labeled human IgM is cleared from the circulation of each of five squirrel monkeys via the HP system. We observe, in experiments analogous to previous studies on immune complex (IC) clearance, that subsequent to HP/Ag clearance there is a decrease in the number of CR1 epitopes per E which is manifested when we use both monoclonal and polyclonal anti-CR1 probes. Our results indicate that the primary organs responsible for uptake of the complexes are the liver and spleen. This work strongly suggests that the HP/Ag complexes, bound to E, function as IC prototypes and are recognized and processed as such in vivo. Thus, the HP-E system may eventually serve as a viable immunotherapy for the clearance of blood-borne pathogens from the circulation.

Effect of chronically increased erythrocyte complement receptors on immune complex nephritis

Experimental studies in humans and other primates have shown that the erythrocyte (E) complement receptor Type 1 (CR1), which is unique to the primate, plays an important role in clearing immune complexes (IC) from the circulation by binding C3b/C4b opsonized immune complexes and carrying the IC to liver and spleen for disposal. The results of these acute experiments suggest that increasing E-CR1 levels chronically should protect against IC-mediated glomerulonephritis (IC-GN) induced by chronic formation of IC in the circulation. In the present study this hypothesis was tested in the cynomolgus monkey (CYN). IC-GN was induced by daily bolus intravenous infusion of BGG into immunized CYN for 8, 10, or 14 weeks. Prior to and during the daily bolus infusions of BGG, sustained differences in E-CR1 levels were achieved between the experimental group (increased E-CR1 levels) and the control group (maintained or decreased E-CR1 levels), by one of two methods: (1) Twice weekly exchange transfusion. The CYN donors were blood type compatible with the recipients and had either constitutive high E-CR1 expression (3,000 to 5,000 CR1/E) or constitutive low E-CR1 expression (< 100 CR/E). The recipients of the exchange transfusions (N = 2) had constitutive mid-level E-CR1 expression. (2) Weekly phlebotomy (PL) or sham PL. CYN with mid-level E-CR1 expression were randomly assigned to receive weekly either PL (causing increased E-CR1 expression by stimulating erythropoiesis) (N = 8) or sham PL (which has no effect on E-CR1 expression (N = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens pre‐bound to the primate erythrocyte complement receptor via cross‐linked bispecific monoclonal antibody heteropolymers are rapidly cleared from the circulation

We have used cross-linked, bispecific monoclonal antibody heteropolymers, specific for primate erythrocyte (E) complement receptor (CR1) and target antigen (Ag), to attach 125I-labeled Ag to 51Cr-labeled monkey erythrocytes (E) in vitro. Injection of these sensitized E into monkeys leads to rapid clearance from the circulation of several different 125I-labeled Ag with little, if any sequestration, lysis, or clearance of the E. It should now be possible, in principle, to use the heteropolymer-E system to facilitate the clearance of blood-borne pathogens from the circulation.

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.

The binding of immune complexes by the erythrocyte complement receptor 1 (CR1).

Immune complexes, which have reacted with complement and bear C3b fragments, bind to the complement receptor 1 (CR1) on human erythrocytes. Indeed, CR1 on erythrocyte serves as a transport system for immune complexes in the circulation so as to prevent immune complex deposition outside the fixed macrophage system. A defect in this transport system has been described in several diseases, in which either complement levels or CR1 number on erythrocytes are diminished. Recent studies have shown that the binding of immune complexes to erythrocytes is favored by the multiple C3b binding sites per receptor and the clustered distribution of CR1 on the erythrocyte surface. Only a few immune complexes bind per erythrocyte but these complexes are tightly bound. The other main function of CR1 on erythrocytes is to enhance the inactivation of C3b by factor I present in plasma. This reaction allows the release of immune complexes from the erythrocyte surface and their transfer to fixed macrophages.

In vivo binding and clearance of circulating antigen by bispecific heteropolymer-mediated binding to primate erythrocyte complement receptor.

We have used the avidin/biotin system to construct soluble, cross-linked bispecific heteropolymers containing mAb to both the primate E C receptor and the DNP group. These heteropolymers facilitate in vitro binding of DNP-bovine gamma-globulin (DNP-BGG) to both human and squirrel monkey E. Intravenous injection in squirrel monkeys of DNP-BGG followed by heteropolymer leads to E binding and clearance from the circulation of a significant fraction of both heteropolymer and DNP-BGG, without lysis or clearance of the E. This methodology may potentially be used to treat a variety of infectious diseases and other syndromes associated with blood-borne pathogens.

Stimulating erythropoiesis increases complement receptor expression on primate erythrocytes

Erythrocyte complement receptors (CR) are unique to primates and play a role in the clearance of immune complexes from the circulation. Immune complex-mediated diseases (e.g., systemic lupus erythematosus, SLE) cause decreased erythrocyte CR levels, resulting in decreased capacity of the erythrocytes to bind immune complexes that form in the circulation. Thus, raising erythrocyte CR levels might benefit patients with immune complex-mediated diseases. Because young erythrocytes express more CR than old erythrocytes, increasing erythropoiesis should increase the average number of CR expressed per erythrocyte. The present study was undertaken to test that hypothesis. Erythropoiesis was stimulated in nine cynomolgus monkeys (CYN) by weekly phlebotomy (30% of blood volume was removed, erythrocytes were discarded, and leukocytes were returned to the animal) for 8 weeks. Sham phlebotomy (30% of blood removed, then all components returned to the animal) was carried out weekly in four additional CYN for a period of 4 to 8 weeks. Sham phlebotomy did not change any of the parameters measured. However, phlebotomy resulted in a progressive increase in the mean number of CR expressed per erythrocyte (CR/erythrocyte): 2780 ± 700 to 4230 ± 820, P < 0.0005. The increase in erythrocyte CR was first detected at about 2 weeks after the start of phlebotomy and was sustained through the course of phlebotomy. The increase in CR/erythrocyte included an increase in the percentage of erythrocyte expressing CR in clusters (37.9 ± 6.4 vs 50.8 ± 8.7%, P < 0.01) as demonstrated by a flow cytometry study of the binding of fluorescent beads coated with anti-CR1 antibody. No significant change in leukocyte or platelet counts were observed. We conclude that stimulating erythropoiesis causes an increase in CR/erythrocyte. The magnitude of the increase suggests that it could be biologically significant.

Loss of erythrocyte complement receptors (CR1; CD35) in patients with acute episodes of septicaemia or bacterial meningitis.

14 patients with septicaemia or bacterial meningitis were examined for serum levels of erythrocyte complement receptors CR1 (C3b/C4b; CD35) by an enzyme-linked immunosorbent assay and levels of circulating immune complexes (IC) by a polyethylene glycol precipitation complement consumption method, and concentrations of complement C3d split products in plasma by intermediate gel rocket immunoelectrophoresis. The CR1 receptor levels were significantly lower on day 7-8 after admission than on day 1-2 (p = 0.01), and than the levels 3-4 months later (p = 0.002). Both the levels of IC in serum and the C3d concentrations were significantly higher on day 7-8 than 3-4 months later (p = 0.02 and p = 0.004). The mechanism behind a temporary decreased expression of CR1 in patients with acute episodes of septicaemia or purulent meningitis is not fully known, and further investigations are needed to clarify whether this reduction in erythrocyte CR1 causes an impaired disposal of IC in patients with infections.

A flow cytometric assay for measuring complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes

A flow cytometric assay (FCA) was developed to measure complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. It was possible to measure these parameters accurately with intra- and interassay coefficients of variation of 2.0% and 6.5% respectively. The method was able to discriminate between low and high levels of erythrocyte CR1, C3d and C4d. Comparison with a previously described RIA method gave excellent correlation coefficients with r 2 values of 0.94, 0.93 and 0.91 for CR1, C3d and C4d respectively. The flow cytometric assay was used to measure CR1, C3d and C4d on the erythrocytes of 98 healthy individuals and the 95% upper limits for C3d and C4d were established. There was a wide distribution of CR1 levels amongst these individuals but their C3d and C4d levels were low and often not above background. The possible application of this method in clinical medicine is discussed.

Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: a potential therapeutic treatment.

We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine gamma globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37 degrees C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

Elimination of soluble 123I‐labeled aggregates of IgG in patients with systemic lupus erythematosus. Effect of serum IgG and numbers of erythrocyte complement receptor type 1

Using soluble 123I-labeled aggregates of human IgG (123I-AHIgG) as a probe, we examined the function of the mononuclear phagocyte system in 22 patients with systemic lupus erythematosus (SLE) and 12 healthy controls. In SLE patients, a decreased number of erythrocyte complement receptor type 1 was associated with less binding of 123I-AHIgG to erythrocytes and a faster initial rate of elimination of 123I-AHIgG (mean +/- SEM half-maximal clearance time 5.23 +/- 0.2 minutes, versus 6.58 +/- 0.2 minutes in the controls), with possible spillover of the material outside the mononuclear phagocyte system of the liver and spleen. However, multiple regression analysis showed that serum concentrations of IgG were the most important factor predicting the rate of 123I-AHIgG elimination. IgG concentration may thus reflect immune complex clearance, which in turn, would influence the inflammatory reaction, in SLE.

Experimental immune complex-mediated glomerulonephritis in the nonhuman primate

This study was undertaken to develop a model of immune complex (IC)-mediated glomerulonephritis (GN) in the nonhuman primate that could be used in subsequent studies to examine critically the role of the erythrocyte complement receptor (E-CR) in the pathogenesis of IC-mediated disease. Cynomolgus monkeys were chosen for study because they constitutively express E-CR levels that are either less than, equal to, or greater than that seen in normal man. After immunization with bovine gamma globulin (BGG), the GN induction protocol was begun in 10 cynomolgus by initiating daily i.v. administration of BGG in amounts sufficient to achieve or exceed antigen/antibody equivalence (assessed by the quantitative precipitin assay) for precipitating antibody present in the plasma volume. We found that within eight weeks of daily BGG administration of all the cynomolgus developed IC-mediated GN, irrespective of the initial E-CR level of the animals. However, the high E-CR cynomolgus tended to receive the higher BGG doses because of higher initial antibody levels to BGG. When the total number of glomerular deposits (determined by morphometric studies) per total BGG dose for each animal was plotted against the initial CR/E of that animal, there was a tendency for the animals with higher CR/E levels to have a lower number of glomerular deposits/BGG dose (r = 0.62, P = 0.06). Also, the total number of glomerular deposits correlated with the severity of the GN. During the early weeks of the GN induction protocol, the IC that formed in vivo (assessed by infusion of 125I-BGG) bound in large amounts to the circulating erythrocytes of the cynomolgus with medium or high E-CR levels. However, when tested after the onset of heavy proteinuria, which occurred between weeks 5 and 8 of daily BGG administration, the IC that formed in the circulation bound only poorly to circulating erythrocytes. By this time the E-CR levels had declined to 43 +/- 9% of initial values (P less than 0.01). This study demonstrates that: 1) A workable model of IC-mediated GN has been developed in the nonhuman primate. 2) During the induction of GN, CR/E and the ability of the erythrocyte to bind IC in vivo are decreased significantly. This suggests that an intact E-CR system could play a role in the protection against IC-mediated disease. However, further study will be needed to test that hypothesis critically. The present model should be useful in such studies.

Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates.

Abstract Patients with immune complex-(IC) mediated diseases frequently have low levels of CR1 on E. The present study was undertaken to determine the role of circulating IC in causing low E-CR1 levels. E-CR1 were enumerated by measuring the binding of anti-CR1 mAb (E11) and rabbit anti-CR1 antibodies (RbaCR1) to E. In addition, the distribution of CR1 among E was assessed by flow cytometry of E stained with E11 and RbaCR1 and by evaluating the binding of E11-coated fluorescent beads (E11-beads) to E. E11-beads bind to clusters of CR1 on E. Five cynomolgus monkeys (CYN) were preimmunized to bovine gamma-globulin (BGG). E-CR1 changes in these animals were assessed: 1) acutely, during the first 60 min after an infusion of BGG and 2) chronically, during daily administration of BGG infusions over 2 wk. Acutely, there was a decrease in the number of E-CR1 as measured by E11 binding to E (E11/CR1). This decrease was not attributable to occupancy of CR1 by IC because the decrease in E11/CR1 number persisted after the IC had been cleared from E. By comparing the E11/CR1 levels in arterial blood to hepatic vein blood (n = 5), or in pulmonary artery blood (n = 1), we determined that the acute decrease in E11/CR1 number did not occur whereas E circulated through liver, spleen, or lung. The decrease in E11/CR1 number required the binding of IC to E because it did not occur after BGG was infused into nonimmunized CYN (n = 2) or into a preimmunized complement-depleted CYN. The decrease in E11/CR1 number was not due to loss of CR1 from E because E11/CR1 number recovered 24 h after infusion of BGG and in addition, enumeration of E-CR1 with RbaCR1 and E11-beads did not reflect a decrease in E-CR1 number. After several daily BGG infusions there was a persistent decrease in E-CR1 levels and that decrease appeared to be mainly the result of loss of CR1 from E because the decrease was confirmed with all methods of E-CR1 measurement and because E-CR1 levels recovered only slowly after BGG infusions were discontinued. Both in vitro and in vivo IC bound preferentially to subpopulations of E, identified by their ability to bind multiple E11-beads and by their high intensity staining with the anti-CR1 antibodies E11 and RbaCR1.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity of Type 1 Erythrocyte Complement Receptors in Uremia and after Renal Transplantation

The effect of uremia on the activity of the erythrocyte complement receptors type 1 (CR1), and the changes occurring after renal transplantation, were studied. The complement receptor activity was measured by immune adherence utilizing a rosette technique. Patients with terminal kidney failure on the hemodialysis program exhibited significantly lower values of the erythrocyte CR1 activity in comparison with healthy controls. The circulating immune complexes did not affect erythrocyte CR1 activity. After successful renal transplantation, irrespective of the immunosuppressive program used, a significant increase in erythrocyte CR1 activity appeared, similar to control group values. However, the activity of erythrocyte CR1, in the graft recipients under cyclosporin A treatment, was significantly higher than in the patients receiving azathioprine with prednisone. Therefore, it is possible that cyclosporin A, transported in the erythrocytes, modifies the complement receptor function.

Role of IgA in IgA nephropathy

IgA nephropathy (Berger disease) is defined by the dominant or codominant deposition of IgA in the renal mesangium. There is much evidence in vitro to suggest up-regulation of the IgA immune response in patients. Data from tonsillar and bone marrow-derived lymphocytes and from in vivo immunization studies indicate that the primary defect is an up-regulated systemic one, rather than mucosal IgA production. Several lines of evidence suggest that increased IgA production alone is inadequate to explain the pathogenesis of Berger disease. Murine models of IgA nephropathy indicate that local complement activation mediated by deposited IgG is essential for mesangial cell proliferation and subsequent renal injury. Circulating immune complexes from patients with Berger disease contain IgA and IgG within the same lattice. In vitro studies of model immune aggregates containing various mixtures of IgA and IgG indicate that the IgG is the site of complement activation and fixation. The IgA in the aggregate actually inhibits both complement activation and binding to erythrocyte complement receptor CR1. This effect of IgA may prevent effective immune complex clearance. In future studies, more emphasis should be placed on the roles of IgG and complement in the pathogenesis of IgA nephropathy.

Erythrocyte complement receptor type 1 in non-SLE rheumatic diseases

An investigation was conducted to measure the levels of erythrocyte complement receptor type 1 (CR1) in patients with various rheumatic diseases other than systemic lupus erythematosus, and to evaluate the clinical significance of this receptor in patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA). Erythrocyte CR1 was measured in 37 normal controls and 106 patients with various rheumatic diseases. The levels of erythrocyte CR1 decreased significantly in patients with AS, juvenile rheumatoid arthritis, Sjögren's syndrome, and RA, while there was no statistical difference in levels of erythrocyte CR1 between normal controls and patients with gouty arthritis. This suggests that erythrocyte CR1 deficiency may occur in immune-mediated rheumatic diseases such as crystal-induced arthritis. In this study, we noted that the levels of erythrocyte CR1 were not related to the disease activity and severity of AS. The levels of erythrocyte CR1 were also not correlated with the clinical and laboratory parameters of disease activity in RA patients. However, there was a negative association between the levels of erythrocyte CR1 and titer of rheumatoid factor in RA patients. Further study is needed to determine whether or not the level of erythrocyte CR1 is related to prognosis in patients with RA.

The Activity of the Erythrocyte Complement Receptors Type 1 (CR1) in Patients with Glomerulonephritis

Journal Article The Activity of the Erythrocyte Complement Receptors Type 1 (CR1) in Patients with Glomerulonephritis Get access M. Klinger, M. Klinger Department of NephrologyTraugutta 57 st. Wroclaw Poland Search for other works by this author on: Oxford Academic PubMed Google Scholar E. Slowik, E. Slowik Department of NephrologyTraugutta 57 st. Wroclaw Poland Search for other works by this author on: Oxford Academic PubMed Google Scholar Z. Szewczyk, Z. Szewczyk Department of NephrologyTraugutta 57 st. Wroclaw Poland Search for other works by this author on: Oxford Academic PubMed Google Scholar W. Kopec W. Kopec Department of NephrologyTraugutta 57 st. Wroclaw Poland Search for other works by this author on: Oxford Academic PubMed Google Scholar Nephrology Dialysis Transplantation, Volume 5, Issue 9, 1990, Pages 834–835, https://doi.org/10.1093/ndt/5.9.834-a Published: 01 January 1990