Peripheral catabolism of CR1 (the C3b receptor, CD35) on erythrocytes from healthy individuals and patients with systemic lupus erythematosus (SLE).

The present study investigated the rate of catabolism of CR1 (the C3b receptor, CD35) on erythrocytes (E) in vivo, in relationship with the expressed number of CR1/E, the CR1.1 HindIII quantitative CR1 polymorphism, and cell age. The relationship between the number of CR1/E and cell age was analysed by measuring G6PDH activity in E that had been sorted according to high or low expression of CR1 (CD35), by assessing the expression of CR1 (CD35) on E separated according to cell density, and by comparing the number of CR1 (CD35) antigenic sites on reticulocytes and on E. A physiological catabolism of CR1 (CD35) manifested by a reduction in the number of CR1 (CD35) antigenic sites/E with cell ageing was consistently observed in healthy individuals. The number of CR1/E decreased with ageing of E according to a complex pattern that associated an exponential decay and an offset. Calculated half-lives of CR1 (CD35) ranged between 11 and 32 days in healthy individuals. A more rapid loss of CR1 (CD35) with cell ageing occurred on cells from individuals expressing high numbers of CR1/E. In patients with systemic lupus erythematosus (SLE), half-lives of CR1 (CD35) on E were in the same range as those of healthy individuals with a similar quantitative CR1 genotype; the number of CR1 (CD35) on reticulocytes was reduced and linearly related to the number of CR1/E, independently of the patients' quantitative CR1 genotype. Transfusion experiments with E bearing high or low amounts of CR1/E indicated the lack of preferential removal of E bearing high numbers of CR1 (CD35) in patients with SLE. These results indicate that the rate of loss of CR1 (CD35) from E with cell ageing is directly related to the quantitative CR1 phenotype and suggest that enhanced peripheral catabolism is not the sole mechanism of the acquired loss of CR1 (CD35) on E in patients with SLE.     

The C3b/C4b receptor (CR1, CD35) on erythrocytes: methods for study of the polymorphisms

This report is devoted to methodologies used in analyzing the C3b/C4b receptor (CR1, CD35) on erythrocytes (E), its soluble form, the CRI structural or allotype polymorphism, and CR1 density polymorphism. In primates E CR1 serves as the main system for processing and clearance of complement opsonized immune complexes (IC). CR1 copy numbers decrease with aging of E in normal individuals. Erythrocyte CR1 is also decreased in pathological conditions such as systemic lupus erythematosus (SLE), HIV infection, certain hemolytic anemias, and many other conditions featuring immune complexes. Consequently, CRI on E has an important physiological role in immune complex handling and has interesting alterations in disease.     

CR1 on erythrocytes of Brazilian systemic lupus erythematosus patients: the influence of disease activity on expression and ability of this receptor to bind immune complexes opsonized with complement from normal human serum

Hypocomplementaemia and low expression of CR1 on erythrocytes (E) of patients with systemic lupus erythematosus (SLE) are associated with defective clearance of circulating immune complexes (IC) and so they may have pathogenic significance. Here, we investigated whether the reduced CR1/E in SLE patients per se might affect the binding of IC to CR1/E. First, we analysed the expression of CR1 on E of active (n=30) and inactive (n=34) SLE patients using a FITC-conjugated mouse anti-CR1 monoclonal antibody E11 and flow cytometry. Both groups of patients had a significantly reduced CR1/E expression compared with healthy controls (n=40). It was also observed that the number of E bearing CR1 was reduced in both groups of SLE patients studied. Second, we determined the functional activity of CR1/E by measuring the binding to E of FITC-bovine serum albumin (BSA)/rabbit anti-BSA complexes, formed at equivalence, which were opsonized with complement from normal human serum (NHS). On the other hand, we did not find differences between the patient and control groups in the ability of E to bind IC/NHS. There was also a positive correlation between the CR1/E expression and the number of E bearing CR1 in control and inactive SLE groups, which was not observed in the group of active SLE patients. Considering the involvement of low levels of complement and CR1/E expression on complex processing, in this in vitro model the results show that an effective coating of the complexes with complement is sufficient to bind them preferentially to CR1 over normal levels of receptor expression.     

Complement receptor 1 (CR1) on erythrocytes of patients with systemic lupus erythematosus

Ninety-five (85%) of the 112 Japanese patients with systemic lupus erythematosus (SLE) were negative for the complement receptor 1 (CR1) activities on erythrocytes, while 770 (91%) of the 847 normal subjects were positive for CR1, as determined by immune-adherence hemagglutination. Pedigree analyses of the normal population suggested that the phenotype of negative CR1 was determined by a autosomal recessive gene. Among 112 SLE patients, 73 (65%) showed persistently negative CR1 during remission for over 26 months of follow-up, although the CR1 levels did vary with the disease activity in 22 SLE patients. These results show that the relative risk for developing SLE in persons with negative CR1 is 19. CR1 activity appears to be an important genetic factor related the development of SLE.     

Decreased C3b receptors (CR1) on erythrocytes from patients with systemic lupus erythematosus.

Using 125I-F(ab')2 anti-CR1 we have measured C3b receptors (CR1) on the erythrocytes of 56 normal individuals 26 patients with systemic lupus erythematosus (SLE) and 24 with rheumatoid arthritis (RA). The mean number of CR1 sites in SLE (1150/cell) but not RA (1460/cell) was significantly lower (P less than 0.01) than normal (2200/cell). Although the cumulative frequency curve for normals showed minor inflections at frequencies of 18% and 64%, these were not sufficiently marked to permit us to conclude that they distinguished subpopulations of different CR1 phenotypes. Measurement of CR1 numbers of two normal families and four families of SLE patients indicated that low CR1 numbers aggregated in families as did high CR1 numbers, a finding which suggests that CR1 numbers are under genetic control. However, certain observations in SLE patients indicated that low CR1 numbers could be an acquired abnormality. These included, (a) absent CR1 phenotype in a patient whose family had moderate and high CR1 numbers, (b) increasing CR1 numbers as SLE patients went into remission, (c) CR1 numbers were lower in patients with active compared with inactive disease and (d) CR1 numbers were different in each of two sets of identical twins (Fig. 4A). Our conclusions are that, (a) genetic factors probably influence CR1 numbers in normal individuals, (b) that our findings were not inconsistent with the two codominant allele models (Wilson et al., 1982), and (c) the low CR1 phenotype of SLE patients may be secondary to the disease process.     

A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b

The erythrocyte type one complement receptor (E-CR1) mediates erythrocyte binding of complement-opsonized immune complexes (IC), and helps protect against random deposition of circulating IC. Two linked CR1 polymorphisms occur in binding domains, at I643T and Q981H. In Caucasians, the variant alleles (643T, 981H) are associated with low constitutive E-CR1 expression levels. This study was conducted to determine if these polymorphisms affect ligand binding, and if so, represent risk factors for the autoimmune IC disease, systemic lupus erythematosus (SLE). In an ELISA comparing relative ligand binding differences, E-CR1 from individuals homozygous for the variant residues (643TT/981HH) exhibited greater binding to C4b, but not C3b, than homozygous wild-type E-CR1. Analysis of single-binding domain CR1 constructs demonstrated that the 981H residue imparted this enhanced C4b binding. No differences were observed in the 981H allele frequency between Caucasian controls (0.170, n = 100) and SLE patients (0.130, n = 150, P = 0.133), or between African American controls (0.169, n = 71) and SLE patients (0.157, n = 67). In a subset of individuals assessed for CR1 size, excluding from this analysis those expressing at least one B allele revealed a trend for over-representation of the 981H allele in Caucasian controls (0.231 frequency, n = 26) versus SLE patients (0.139, n = 83, P = 0.089), but again no difference between African American controls (0.188, n = 24) and SLE patients (0.191, n = 34). These data suggest that the 981H residue compensates for low constitutive expression of E-CR1 in Caucasians by enhancing C4b binding. This may contribute protection against SLE.     

Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS

The number of complement receptor type 1 (CR1; CD35) on human erythrocytes (E) decreases during normal in vivo aging. Patients with acquired immunodefiziency syndrome (AIDS) have an acquired deficiency of CR1 on E. The possible mechanisms responsible for the loss of CR1 from E include the release of small vesicles from the E membrane and proteolytic cleavage of CR1. When compared to E of normal donors and of asymptomatic human immunodeficiency virus HIV⁺ subjects, E of patients with AIDS had fewer CR1/E (p < 0.001), but had the same number of two glycosylphosphatidyl-inositol-anchored proteins, decay-accelerating-factor (DAF) and CD59. When compared to young E, old E separated by density gradients on Percoll had fewer CR1 [six normal subjects, mean loss: 50.4 ± 4.9 (SEM) %], DAF (34.4 ± 1.2%) and CD59 (34.5 ± 2.7%). The loss of CR1 was significantly higher than the loss of DAF and CD59 (p < 0.02). In vitro, ATP depletion of E is responsible for the release of vesicles from the E surface, a reaction that has been called in vitro aging. CR1, DAF and CD59 were lost on ATP-depleted E; however, the loss of CR1 and DAF were identical (six experiments, mean loss for CR1: 28.7 ± 2.7%, DAF: 26.3 ± 4.6% and CD59: 20.5 ± 4%). Thus, the release of vesicles from E cannot explain the specific loss of CR1 in patients with AIDS and would explain only incompletely the loss of CR1 during in vivo aging. In vitro experiments indicated that CR1 was more sensitive to trypsin and papain cleavage than DAF and CD59. Enhanced chemiluminescence Western blotting, using a monoclonal antibody (E11) recognizing fragments of CR1 down to 43 kDa on E exposed to trypsin or papain, indicated that normal E bear fragments of CR1, which are not found on polymorphonulcear leukocytes or on CR1-bearing vesicles in urine. The relative amount of these fragments was increased in patients with AIDS. Taken together these data suggest that the specific loss of CR1 on E in AIDS is due to proteolytic cleavage. The loss of CR1 during in vivo aging also involves proteolytic cleavage, although part of the loss might be explained by other mechanisms including the release of vesicles by E.     

Immune complex binding to erythrocyte-CR1 (CD 35), CR1 expression and levels of erythrocyte-fixed C3 fragments in SLE outpatients

Erythrocytes (E) from a cross-sectional group of 22 outpatients with systemic lupus erythematosus (SLE) and/or mixed connective tissue disease (MCTD), the majority without active disease (n = 14), were analyzed for CR1 antigen expression and capacity to bind complement opsonized, radiolabelled immune complexes (IC). Furthermore, E-bound C3 fragments and the plasma C3d concentration were determined. E-bound C3b/iC3b fragments were not elevated in patients with SLE, whereas E from 11 out of 22 SLE patients had increased C3d levels which correlated with the plasma C3d concentration (Rs 0.73, p less than 0.001). E-fixed C3d fragments did not affect the binding of Mab or preopsonized IC to E-CR1 and were not correlated with disease activity or medical treatment. Antigen expression of E-CR1 measured by ELISA or agglutination showed positive correlation with the IC binding capacity of E-CR1 (Rs 0.92 and 0.72 respectively, p less than 001). The IC binding capacity of E-CR1 from SLE patients was significantly reduced (p less than 0.005), whereas the antigen expression of CR1 (ELISA) on E from the patients did not differ from that of E from healthy donors (p greater than 0.1). E-CR1 antigen was measured by Mab reacting with an epitope outside the IC-binding site of E-CR1. E-CR1 antigen expression or IC binding showed no correlation either with disease activity or prednisolone treatment. However, 4 og 5 patients with MCTD and 4 of 5 patients receiving Imurel were found to have low E-CR1 expression and capacity to bind IC. Thus, measurement of antigenic E-CR1 in a cross-sectional group of SLE outpatients by use of Mab reacting with an epitope outside the ligand-binding region of CR1 did not reveal a significantly reduced CR1 expression. However, an assay for CR1-mediated IC binding showed a clearly reduced E-CR1 function.     

A simple method for accurate quantification of complement receptor 1 on erythrocytes preserved by fixing or freezing

The mean number of complement receptor 1 (CR1) molecules on erythrocytes differs between normal individuals within the range of 100-1000 molecules per cell. In some disease states such as systemic lupus erythematosus (SLE), acquired immune deficiency syndrome (AIDS), insulin-dependent diabetes mellitus and malaria, erythrocyte CR1 levels are reduced and CR1 function may be impaired. Current methods for determining erythrocyte CR1 levels by flow cytometry require the use of freshly drawn blood samples because CR1 is lost from erythrocytes during storage. In order to facilitate field studies of associations between erythrocyte CR1 levels and disease, we have developed and validated an assay to quantify CR1 on both healthy and diseased erythrocytes that have been fixed in 5% formaldehyde or frozen in glycerol. These methods enable blood samples to be collected in areas lacking the facilities for flow cytometry and stored for later accurate quantification of CR1. Such procedures will be of particular benefit for future investigations of erythrocyte CR1 expression level and malaria susceptibility.     

Physiological up-regulation of inhibitory receptors Fc gamma RII and CR1 on memory B cells is lacking in SLE patients

Under physiological conditions immune complexes (IC) are efficiently cleared from the circulation and meanwhile provide important feedback signals for the immune system via Fc gamma Rs and complement receptors. Dysregulation of these mechanisms have been implicated in conditions where IC concentrations reach pathological levels and inflict diseases, like systemic lupus erythematosus (SLE). Our aim was to compare distinct sub-populations of CD19(+) B cells of healthy individuals and SLE patients with regard to their expression of Fc gamma R type II (Fc gamma RII, CD32), complement receptor type 1 (CR1, CD35) and complement receptor type 2 (CR2, CD21) and sIgG/IgM. The following four groups of peripheral CD19(+) B cells were investigated: IgM(+)/CD27(-) naive, IgM(+)/CD27(+) and IgM(-)/CD27(+) memory cells and CD27(high) plasmablasts. We demonstrate that the expression of the inhibitory receptors Fc gamma RII and CR1 is up-regulated on peripheral memory B cells of healthy controls, whereas this up-regulation is considerably impaired on the memory B cells of SLE patients. This reduction affects both the IgM(+) and switched memory B cells. We found a striking difference between the expression of complement receptors CD21 and CD35; namely, no up-regulation of CD21 occurred on the memory B cells of healthy donors, and its decreased expression in SLE patients was characteristic for both the CD27(-) naive and the CD27(+) memory B-cell populations. Our results clearly demonstrate that the previously reported reduced expression of IC-binding receptors is mainly due to the disturbed memory compartment; however, the higher frequency of CD19(+)/CD27(high)/sIg(low) plasmablasts expressing minimal levels of these receptors also contributes to this diminution.     

Enumeration of CR1 complement receptors on erythrocytes using a new method for detecting low density cell surface antigens by flow cytometry

A sensitive enhancing system was developed for detecting low density cell surface antigen by flow cytometry. The system termed the 'super avidin-biotin system' (SABS) uses biotinylated antibody, phycoerythrin-streptavidin (StreptA-PE), biotinylated goat anti-streptavidin antibody, and StreptA-PE. CR1 complement receptor antigenic sites were quantified on erythrocytes from healthy individuals and patients with antibodies against human immunodeficiency virus (HIV) using SABS and a conventional radioimmunoassay (RIA) with monoclonal anti-CR1 antibody. As little as 50 sites/cell were detected using either SABS or RIA. Accurate quantification of CR1 antigenic sites was achieved within the range of 100-1300 sites/cell. Similar results were obtained using either of the two methods. Intra-assay and day-to-day reproducibilities using SABS were 2% and 12% respectively, comparable to those of conventional RIA measurements. In addition to enumeration of CR1 on erythrocytes for clinical purposes, the use of SABS may probably be extended to a wide number of situations where a sensitive detection of low density cell surface antigen is needed.     

CR1/CR2 deficiency alters IgG3 autoantibody production and IgA glomerular deposition in the MRL/lpr model of SLE

CR1 and CR2 expression is decreased by approximately 50% on B cells of patients with systemic lupus erythematosus (SLE). Expression is also decreased in the MRL/lpr murine model of SLE prior to the development of clinical disease, suggesting that this alteration may play a role in pathogenesis. To determine whether the decrease in receptor levels affects the development of SLE, we analyzed MRL/lpr mice in which CR1/CR2 expression was altered by gene targeting. Mice from each cohort (Cr2+/+, Cr2+/-, and Cr2-/-) were analyzed biweekly for the development of proteinuria and autoantibodies. Kidneys were examined at 12 and 16 weeks for evidence of immune complex deposition and renal disease. Deficiency of CR1/CR2 did not affect survival or development of renal disease as measured by proteinuria. Mice deficient in CR1/CR2 had significantly lower levels of IgG3 rheumatoid factor (RF) and total serum IgG3, suggesting a specific defect in production of IgG3 in response to endogenous autoantigens. Since IgG3 RF has been associated with the development of vasculitis in this model, we examined the mice for alterations in development of this clinical manifestation. Although there was no difference in the development of ear necrosis among the three groups, renal arteritis was not identified in any of the Cr2+/- mice, whereas it was present in 20% of the Cr2+/- and 40% of the Cr2+/+ mice. Finally, significantly higher levels of IgA were seen in the glomeruli of Cr2+/- mice compared to Cr2+/- or Cr2+/+ mice, suggesting that CR1/CR2 are involved in either the regulation of IgA production or the clearance of IgA immune complexes. Together these data support the concept that alterations in CR1/CR2 expression or function affect the regulation of autoantibody production and/or clearance and may have clinical consequences.     

C3b receptor (CR1) expression on the polymorphonuclear leukocytes from patients with systemic lupus erythematosus.

Polymorphonuclear leukocytes (PMN) C3b receptor (CR1) numbers have been measured in 14 normal individuals and 15 patients with SLE. The results in the normals showed that PMN possess three distinct pools of CR1. CR1 expression was lowest at 0 degrees C (mean 86,000 +/- s.e.m. 7,000), but increased when the cells were incubated at 37 degrees C (125,000 +/- 16,000) or when the cells were exposed to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 10(-5) mol 1) at 37 degrees C (207,000 +/- 21,000). The increased expression at 37 degrees C was not dependent upon protein synthesis, an intact cytoskeleton or energy. Although the response to FMLP did not require de novo protein synthesis, increased CR1 expression was dependent upon an intact cytoskeleton and energy. All three PMN CR1 pools were reduced in patients with active SLE, but were normal in those in whom the disease was inactive. Serial studies performed on three SLE patients showed that PMN CR1 numbers were low during periods of disease activity and increased during remission. These data suggest that low PMN CR1 numbers in SLE are a consequence of the disease.     

The polymorphism of the C3b/C4b receptor in the normal population and in patients with systemic lupus erythematosus.

One hundred and sixty-two unrelated, healthy normals and 118 unrelated SLE patients were subdivided by sex, race, and disease, and each group was in Hardy-Weinberg equilibrium for autosomal codominant expression of the four CR1 alleles. There were no significant phenotypic differences between males and females (P greater than 0.3) or between normals and SLE patients (P greater than 0.2). However, gene frequencies among whites (A:0.87; B:0.12; C:0; D:0.01), blacks (A:0.74; B:0.22; C:0.04; D:0) and orientals (A:0.98; B:0.02; C:0; D:0) were significantly different (P less than 0.05). We had previously reported that SLE patients of phenotype AC had higher relative expression of the C allele than normals and this was confirmed: 61% (SLE, n = 5) vs 22% (normals, n = 3; P = 0.014). Total CR1/E in the AC group (193 in SLE vs 393 in normals 0.05 less than P less than 0.10), was suggestive of the decreased CR1 number seen in larger SLE populations regardless of phenotype. In one large three-generation family with SLE and the C allele, an association between SLE and the C allele is suggested by the presence of the C allele in all three females with SLE versus 3 of 13 healthy females. In informative families in which receptor phenotype and CR1 number/E were determined, it was possible to assign a receptor number to an allele. These data provide evidence for a cis-acting regulatory element that is inherited in association with the CR1 structural gene.     

Normal C3b receptor (CR1) genomic polymorphism in patients with insulin-dependent diabetes mellitus (IDDM): is the low erythrocyte CR1 expression an acquired phenomenon?

Expression of the erythrocyte complement receptor (C3bR = CR1 = CD35) and its genomic polymorphism (HindIII RFLP) was studied in a group of 80 patients with IDDM, 31 healthy siblings and 101 healthy blood donors. Defective CR1 expression was found in 26% of the patients with IDDM compared with 9% of the controls (P less than 0.05) and 0% of the siblings. The CR1 gene polymorphism of the IDDM patients did not significantly differ from that of the controls. The presence of a 6.9 kb (L) CR1 gene fragment was associated with a low CR1 expression in the patients (P less than 0.05) and especially in the controls (P less than 0.001). No significant association was found between the presence or absence of the HLA risk antigens for IDDM and CR1 expression. The results confirm that erythrocyte CR1 expression is genetically determined, but the CR1 deficiency associated with IDDM seems to be an acquired rather than a genetic phenomenon.     

Erythrocyte complement receptor type 1 (CR1) expression and circulating immune complex (CIC) levels in hydralazine-induced SLE.

Family studies were carried out to look at CR1 expression in 24 hydralazine-induced SLE patients (Hz Reactors), who had been off the drug for at least 1 year and were clinically well at the time of the study. Mean expression of CR1 was reduced by 27% in the group of hypertensives who had developed Hz-induced SLE compared with a group of 35 normal individuals. CR1 expression was also slightly reduced in the relatives of the Hz Reactors compared to the normal group. Using a solid-phase Clq binding assay, CIC levels were found to be elevated in the plasma of the Hz reactors and an inverse relationship was found between CR1 levels and CIC levels in this patient group. Both CR1 levels and CIC levels in Hz Reactors and normal individuals were constant over the 36 weeks studied. This study suggests that there is an association between an inability to deal efficiently with CIC and susceptibility to developing Hz-induced SLE.     

Anti-C3b-receptor (CR1) antibodies in patients with systemic lupus erythematosus

Using an enzyme-linked immunoadsorbent assay, IgG in the plasma and purified IgG from 2 patients with systemic lupus erythematosus (SLE) were found to strongly react with purified C3b receptor (CR1) insolubilized on microtiter plates. The amount of IgG that bound to CR1 in 201 plasma samples from 179 other patients with SLE did not significantly differ from that which bound in 72 control samples from normal individuals. Purified IgG from the patients with anti-CR1 reactivity did not inhibit CR1 function in vitro. The number of CR1 antigenic sites expressed on erythrocytes from both patients was much lower than that observed in a normal population and in the lowest range of the decreased numbers found in patients with SLE. The occurrence of anti-CR1 antibodies in patients with SLE could provide an acquired mechanism for decreased expression of CR1 through antigenic modulation of the receptor on precursor cells and/or alter the function of cells of the immune system bearing C3b receptors.     

Effect of plasmapheresis on ligand binding capacity and expression of erythrocyte complement receptor type 1 (CR1) of patients with systemic lupus erythematosus (SLE)

The functional activity and the expression of CR1 on the erythrocytes (E) of patients with SLE were, respectively, determined by measuring the binding to E of either complement-opsonized bovine serum albumin (BSA)-anti-BSA immune complexes (ICC) or specific anti-ECR1 MoAbs. We found that both the functional activity and levels of ECR1 in SLE patients homozygous for ECR1 high density allele were significantly lowered compared with healthy controls having the same allele. Soon after plasmapheresis there was a significant increase in E ICC binding activity, and this increased functional activity was stable. Moreover, plasmapheresis reduced the level of immune complexes demonstrable in the circulation of the patients. The expression of ECR1 determined with several different anti-CR1 MoAbs was also elevated as a consequence of plasmapheresis. This elevation was observed for both MoAb 1B4, which competes for the ICC binding site of ECR1, and for MoAb HB8592, which does not, but the time course for the increase in binding of the two MoAbs was different, in that the epitope recognized by MoAb 1B4 increased more rapidly. The present results, considered in the context of previous findings, suggest that more than one mechanism may be operative with respect to the effects of the plasmapheresis in increasing ECR1 levels defined by different epitopes on the molecule.