CR1 and the cell membrane proteins that bind C3 and C4. A basic and clinical review.
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Complement C3↗
Biomedical subjects
D T Fearon
Publications and source records attributed to D T Fearon.
Human receptor for C3b/C4b: complement receptor type I.
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Humans↗
Hereditary angioedema: a decade of management with stanozolol.
Thirty-seven patients with hereditary angioedema, who, without therapy, had attacks of cutaneous angioedema, gastrointestinal colic, and/or upper respiratory symptoms at a frequency and severity sufficient to prompt treatment with an attenuated androgen, have been evaluated for the incidence of side effects and biochemical toxicity during various schedules leading to the minimal effective dose. Stanozolol was administered in a 2 mg daily dose, initially, and after the symptoms and signs were adequately controlled for 2 months at this dose or at 1 mg per day, the drug was administered every other day at 4 mg. Patients who responded adequately to this schedule were administered 2 or 1 mg every other day, and then the interval between doses was gradually increased to 1 week, after which the agent was stopped. Eighteen patients experienced adverse reactions to stanozolol while the minimal effective dose was attained. In each instance the side effect subsided with a reduction in dosage. The most common adverse reactions were biochemical evidence of hepatic dysfunction and, to a lesser extent, hirsutism and menstrual irregularities. Although 21 of 27 patients in an initial study of the minimal effective dose were maintained with daily therapy in 1980, by 1986 this group and 10 additional patients were distributed so that three patients were receiving daily maintenance, 18 were receiving alternate-day maintenance, and 16 patients were receiving no maintenance therapy [corrected]. Thus, stanozolol appears to be a safe and effective agent for management of hereditary angioedema when patients are continually monitored to define the minimal effective dose or the feasibility of stopping the drug.
Adolescent↗
Neutrophil activation after burn injury: contributions of the classic complement pathway and of endotoxin.
We attempt to elucidate the mechanisms of neutrophil (PMN) activation after burn injury. We previously reported prolonged elevations of PMN cell surface complement (C) opsonin receptor levels after burn trauma with a corresponding period of depressed PMN chemotaxis to C5a, which suggests that the C product, C5a, was responsible for PMN activation. However, a lack of direct correlation of C activation with C receptor levels soon after injury raised the possibility of a second PMN-activating substance. We therefore investigated the effect of endotoxin (LPS) on the expression of the C receptors (CR1 and CR3) by normal human PMNs. Concentrations from 0 to 50 ng/ml of LPS 026:B6 caused a dose response increase in the PMN surface expression of CR1 and CR3 as assessed by monoclonal antibody binding and indirect immunofluorescence. The relative CR1-dependent fluorescence rose from a mean of 50 to 385 and CR3 from 50 to 300. Chelation by ethylenediaminetetra acetic acid (EDTA) did not influence this dose response, thus ruling out the possibility of C activation by LPS--an inference supported by the lack of complement activation observed with these concentrations of LPS in normal serum. A similar dose response was obtained in the absence of other cell types or serum, which implies a direct effect that mimicked that of C5a. To determine the mechanism of the later, prolonged C activation after burn injury, we next examined C activation products in 22 patients with burn injuries. Elevations of plasma C3a desArg were present and persisted for 50 days. Elevations were at maximum levels on days 9 through 13 postburn (mean +/- standard error of mean [SEM], 496 +/- 47 ng/ml versus normal 113 +/- 32; p less than 0.01). These were accompanied by elevations of C4a desArg (917 +/- 154 ng/ml versus normal 424 +/- 50; p less than 0.01), which are indicative of classic pathway activation. Finally, we examined PMN function, phagocytosis and percentage killing of Staphylococcus aureus, and found PMN function to be unaltered in the 22 patients. Thus PMN activation after burn injury appears to be caused by LPS soon after injury and by C5a later after injury and affects only selected PMN functions.
Adolescent↗
Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils.
As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.
Actins↗
Analysis of multiple restriction fragment length polymorphisms of the gene for the human complement receptor type I. Duplication of genomic sequences occurs in association with a high molecular mass receptor allotype.
Human CR1 exhibits an unusual form of polymorphism in which allotypic variants differ in the molecular weight of their respective polypeptide chains. To address mechanisms involved in the generation of the CR1 allotypes, DNA from individuals having the F allotype (250,000 Mr), the S allotype (290,000 Mr), and the F' allotype (210,000 Mr) was digested by restriction enzymes, and Southern blots were hybridized with CR1 cDNA and genomic probes. With the use of Bam HI and Sac I, an additional restriction fragment was observed in 20 of 21 individuals having the S allotype with no associated loss of other restriction fragments. Southern blot analysis with a noncoding genomic probe derived from the S allotype-specific Bam HI fragment showed hybridization to this fragment and to two other fragments that were also present in FF individuals. Thus, an intervening sequence may be repeated twice in the F allele and three times in the S allele. A restriction fragment length polymorphism (RFLP) unique to two individuals expressing the F' allotype was seen with Eco RV, but the absence of persons homozygous for this rare allotype prevented further comparisons with the F and S allotypes. Analysis of the CR1 transcripts associated with the three CR1 allotypes indicated that these differed by 1.3-1.5 kb and had the same rank order as the corresponding allotypes. Taken together, these findings suggest that the S allele was generated from the F allele by the acquisition of additional sequences, the coding portion of which may correspond to a long homologous repeat of approximately 1.4 kb that has been identified in CR1 cDNA. We saw two other RFLPs with Hind III and Pvu II that were in linkage dysequilibrium with the Bam HI-Sac I RFLPs associated with the S allotype, and a third polymorphism was seen with Eco RI that was not in linkage dysequilibrium with the other polymorphisms. Thus, 10 commonly occurring CR1 alleles can be defined, making this locus a useful marker for the long arm of chromosome 1 to which the CR1 gene maps.
Alleles↗
Purification of the B lymphocyte receptor for the C3d fragment of complement and the Epstein-Barr virus by monoclonal antibody affinity chromatography, and assessment of its functional capacities.
The human C3d receptor (complement receptor type 2, CR2), that also serves as the B lymphocyte receptor for the Epstein-Barr virus, was purified from detergent lysates from the B lymphoblastoid cell lines, SB and Raji, by monoclonal antibody affinity chromatography using the anti-CR2 monoclonal antibody, HB-5. Relative to the concentration of cellular protein and receptor that was initially solubilized by detergent, the procedure provided a 37,000-fold purification with a 40-50% recovery of CR2. The purified receptor presented a single Coomassie blue-stained band when analyzed by SDS-PAGE, and it retained its function of binding to C3-Sepharose. The N-terminus of CR2 was blocked. The amino acid composition was significantly similar to that of the C3b/C4b receptor, factor H and C4 binding protein, suggesting that CR2 may be a member of this newly defined protein family. However, CR2 did not exhibit the regulatory functions of these proteins, namely, the decay dissociation of the classical or alternative pathway C3 convertases and serving as a cofactor for the cleavage of C3b.
Amino Acids↗
Identification of a restriction fragment length polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes.
A genetic basis for the regulation of the number of CR1 on E of different normal individuals was investigated by probing Southern blots of their genomic DNA with a 0.75-kb fragment of CR1 cDNA. Using Hind III, we observed a RFLP involving fragments of 7.4 kb and 6.9 kb that correlated with the number of CR1 on E. 32 individuals having only the 7.4-kb restriction fragment had a mean of 661 +/- 33 (SEM) CR1/E, 11 donors having both restriction fragments had a mean of 455 +/- 52 CR1/E, and 7 individuals having only the 6.9-kb fragment had a mean of 156 +/- 13 CR1/E, all means being significantly different (p less than 0.005). Cosegregation in a normal family of the Hind III restriction fragments with the S, F, and F' structural allotypes of CR1 confirmed that the regulatory element identified by these fragments is linked to the CR1 gene. Moreover, an analysis of the relative expression on E of these structural allotypes in association with either the 7.4-kb Hind III fragment or the 6.9-kb fragment showed that this regulatory element is cis-acting. In contrast, quantitation of CR1 of B lymphocytes and neutrophils revealed no differences in total CR1 expression between individuals homozygous for the 7.4-kb and 6.9-kb Hind III fragments. Thus, we have identified a genomic polymorphism that is linked to the CR1 gene and is associated with a cis-acting regulatory element for the expression of CR1 on E.
DNA↗
Neutrophil activation in thermal injury as assessed by increased expression of complement receptors.
We studied neutrophil activation in patients with burns by serial immunofluorescent measurement of neutrophil expression of the complement opsonin receptors CR1 and CR3. CR1-dependent fluorescence was initially (days 0 through 5 after the burn) elevated (mean +/- SEM, 294 +/- 42 vs. 63 +/- 6 in the controls; P less than 0.001) and gradually returned to normal (days 6 through 8, 270 +/- 62, P less than 0.001; days 9 through 13, 185 +/- 38, P less than 0.001; days 14 through 19, 143 +/- 27, P less than 0.001; and days 20 through 50, 93 +/- 5, P less than 0.04). CR3-dependent fluorescence paralleled that of CR1. Neutrophil chemotaxis in response to zymosan-activated serum, a source of C5a, was depressed (days 0 through 5, 77 +/- 4 percent of control, P less than 0.001; days 6 through 8, 70 +/- 4 percent, P less than 0.001; days 9 through 13, 74 +/- 3 percent, P less than 0.001; days 14 through 19, 90 +/- 4 percent, P less than 0.01; and days 20 through 50, 97 +/- 3 percent, P not significant) and inversely correlated with CR1- and CR3-dependent fluorescence (r = -0.559, P less than 0.001; and r = -0.709, P less than 0.001, respectively). Plasma C3a desArg levels were above normal (100 +/- 5 ng per milliliter) throughout (days 0 through 5, 305 +/- 42; days 6 through 8, 546 +/- 69; days 9 through 13, 490 +/- 72; days 14 through 19, 409 +/- 54; and days 20 through 50, 260 +/- 36; all P less than 0.005). Thus, neutrophils in burned patients were activated as indicated by increased expression of complement receptors. The correlation between this increase and the depression of chemotaxis in response to zymosan-activated serum suggests that C5a is responsible for systemic neutrophil activation, which may contribute to the increased susceptibility to infection of patients with burns.
Adult↗
Tissue-specific phosphorylation of complement receptors CR1 and CR2.
CR1 of neutrophils and monocytes may exist in a resting state, in which it only binds ligand-coated particles, or an activated state, in which it mediates phagocytosis. Because the activated state of CR1 can be induced by the stimulation of protein kinase C with PMA, CR1 was assessed for phosphorylation. Purified human neutrophils, monocytes, eosinophils, tonsilar lymphocytes, SB cells, and erythrocytes were labeled with 32PO4 and incubated with buffer or 100 ng/ml PMA. Membrane proteins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. CR1, unlike HLA class I heavy chain, was not constitutively phosphorylated by any cell type. PMA induced phosphorylation of CR1 in three phagocytic cell types, but did not induce the phosphorylation of CR3 or FcR. FMLP also induced the phosphorylation of CR1 in neutrophils. In contrast, PMA did not induce phosphorylation of CR1 in tonsilar B lymphocytes, SB cells, or erythrocytes, indicating restriction of this reaction to phagocytic cell types. This may be due to differences in the structure or presentation of the cytoplasmic domain of CR1 in phagocytic vs. nonphagocytic cells. Phosphorylation of CR2, however, did occur in PMA-treated B lymphocytes and SB cells, suggesting that this receptor, rather than CR1, may be involved in regulation of B lymphocyte function.
B-Lymphocytes↗
Decreased expression of the C3b/C4b receptor (CR1) and the C3d receptor (CR2) on B lymphocytes and of CR1 on neutrophils of patients with systemic lupus erythematosus.
Decreased numbers of complement receptor type 1 (CR1) have been observed on erythrocytes of patients with systemic lupus erythematosus (SLE) and on glomerular podocytes of patients having proliferative nephritis of SLE. In the present study, the analysis of the cellular expression of CR1 has been extended to include leukocytes. In addition, expression by B lymphocytes of the C3d receptor (CR2), which also serves as the receptor for the Epstein-Barr virus, was assessed. Receptor expression was measured by 2-color fluorescent flow cytometry of peripheral blood B cells, identified by the presence of the B1 antigen, that had also been stained with anti-CR1 or anti-CR2. B cells from 17 patients with SLE exhibited a mean relative fluorescence for CR1 that was 61% of that found in 17 normal individuals (P less than 0.001). The expression of CR2 by the patients' B cells (n = 14) was 62% of that of the B cells from normal subjects (n = 17) (P less than 0.001). The expression of CR1 correlated with that of CR2 among patients (r = 0.63; P less than 0.01) but not with the expression of CR2 among normal individuals (r = 0.36; P greater than 0.1). The mean CR1 content of the patients' neutrophils was only 59% of the normal mean (P less than 0.001). Thus, abnormalities of complement receptor expression occur on the leukocytes of patients with SLE. These deficiencies may be secondary to interaction of the cells with the products of complement activation, or, in some individuals, the deficiencies may be familial.
Antibodies, Antinuclear↗
The role of receptors for complement in the induction of polyclonal B-cell proliferation and differentiation.
A panel of monoclonal antibodies and ligands that bind to the CR1 or CR2 complement receptors of B cells has been used to investigate the role of these membrane molecules in regulating B-cell proliferation and differentiation. When CR2 was modulated from the surface of B cells by treatment with the HB-5 antibody and a secondary goat anti-mouse immunoglobulin antibody, Epstein-Barr virus-induced polyclonal B-cell proliferation and immunoglobulin production were inhibited by 83 and 90%, respectively. In contrast, modulation of other cell surface molecules, HB-2, B1, and the C3b receptor (CR1), or pretreatment of B cells with C3d,g (a CR2 ligand) or HB-5 antibody, alone minimally inhibited these responses. Neither the HB-5 antibody C3d,g, nor a monoclonal antibody (YZ-1) reactive with CR1 induced resting B cells to proliferate, nor did they alter anti-mu antibody-induced proliferation. Similarly, treatment with C3d,g or with the HB-5 or YZ-1 antibodies did not induce B cells to secrete immunoglobulin or affect pokeweed mitogen-induced plasma-cell formation. Whereas CR2 appears to be the functionally relevant receptor for Epstein-Barr virus on B cells, the effects of ligand interactions with CR1 and CR2 on normal B-cell proliferation or differentiation remain unidentified.
Adult↗
Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement.
Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low-stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross-hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2.
Amino Acid Sequence↗
Decreased expression of C3b receptor (CR1) on erythrocytes of patients with systemic lupus erythematosus contrasts with its normal expression in other systemic diseases and does not correlate with the occurrence or severity of SLE nephritis.
Expression of the C3b/C4b receptor (CR1) on erythrocytes is decreased in patients with systemic lupus erythematosus (SLE) compared to normal individuals, and the CR1 antigen is absent from podocytes in severe diffuse proliferate nephritis of SLE. In the present study, we examined the relationship between the number of CR1 on erythrocytes and the occurrence and severity of SLE nephritis, and assessed the expression of CR1 on erythrocytes and the occurrence and severity of SLE nephritis, and assessed the expression of CR1 on erythrocytes in non-SLE nephritis and other systemic inflammatory diseases by measuring the binding of 125I-labeled rabbit F(ab')2 and murine monoclonal IgG anti-CR1 antibodies to erythrocytes of normal individuals and patients in a French population. The number of binding sites for monoclonal anti-CR1 antibody on erythrocytes of 116 normal individuals was 743 +/- 22 (mean +/- SEM) with a range of 169-1,333, and the frequency distribution of this number in the population was bimodal. In 112 patients with SLE, the mean number of CR1 sites on erythrocytes was decreased to 62% of the mean for normal individuals (p less than 0.001). No correlation was found between CR1 expression on erythrocytes and the presence or immunohistopathological type of glomerulonephritis in biopsy specimens from these patients. The mean number of CR1 on erythrocytes of 29 patients with non-SLE glomerulonephritis was slightly decreased to 89% of the normal mean (p greater than 0.05), which could not be attributed to glomerular immune complex disease or vasculitis.(ABSTRACT TRUNCATED AT 250 WORDS)
Arthritis, Rheumatoid↗
The identification of N-linked oligosaccharides on the human CR2/Epstein-Barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding.
Human complement receptor type 2 (CR2) was biosynthetically labeled by pulsing SB B lymphoblastoid cells for 25 min with [35S]methionine followed by chase in the presence of excess unlabeled methionine. An Mr 134,000 polypeptide represented the major form of the receptor at the end of the pulse period, and within 1 h of chase this disappeared coincident with the appearance of the Mr 145,000 mature form of CR2. Precursor, but not mature, CR2 was sensitive to endoglycosidase H, indicating that maturation of CR2 represented processing of N-linked high mannose oligosaccharides to the complex type. The processing of precursor CR2 was impaired by monensin. In the presence of tunicamycin an Mr 111,000 form of CR2 was synthesized by SB cells, and this did not chase into either precursor or mature CR2. This Mr 111,000 form of CR2 did not incorporate [3H]glucosamine, indicating that it lacked both N- and O-linked oligosaccharide. The half-lives of mature CR2 and nonglycosylated CR2 pulse-labeled in the presence of tunicamycin were 13.8 and 2.8 h, respectively; the turnover rate of B1, a membrane protein normally lacking carbohydrate, was unaffected by the presence of the antibiotic. The percentage of pulse-labeled, nonglycosylated CR2 that was expressed at the cell surface after 1 h of chase in the presence of tunicamycin was 30%, identical to that of mature CR2 in cells chased in the absence of the antibiotic. However, after 6 h of chase there was no additional net accumulation of nonglycosylated CR2 at the plasma membrane, while the proportion of pulse-labeled mature CR2 at this site had risen to 81%. Therefore, N-linked oligosaccharides are essential for the stability of CR2 and have some role in its plasma membrane expression. In contrast, the observation that all three forms of CR2 bound to Sepharose C3 indicates that oligosaccharides are not necessary for the interaction between CR2 and its complement ligand.
Cell Membrane↗
Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography.
The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.
Amino Acid Sequence↗
Acquired deficiency of the inhibitor of the first component of complement: report of five additional cases with commentary on the syndrome.
The association of late onset recurrent angioedema with a deficiency of the inhibitor of the first component of complement (C1INH) and of the binding subunit of the first component, Clq, defines the syndrome of acquired C1INH deficiency. The description of five new cases, along with the original two and the 18 others in the literature, brings the total reported cases to 25 and highlights the associated B cell abnormalities that are present in 23 and are of a malignant nature in 19 cases. In three of the five newly reported cases, the occurrence of angioedema, which prompted recognition of the acquired deficiency of C1INH, C1q, and C4, preceded the delineation of the underlying B cell malignancy by 2 to 3 yr despite efforts to recognize neoplastic disease in two of these patients throughout the interval. Because the acquired C1INH deficiency reflects increased catabolism rather than impaired biosynthesis, only high-dose attenuated androgens elicit a measurable increment in serum C1INH. The occurrence of the syndrome with multiple myeloma is noted for the first time.
Aged↗
Identification of a partial cDNA clone for the human receptor for complement fragments C3b/C4b.
Redundant oligonucleotides were synthesized based on amino acid sequences of tryptic peptides from the purified receptor for human complement fragments C3b/C4b (CR1). These probes were used to screen a size-selected human tonsilar cDNA library. A single positive clone was identified that hybridized to three oligonucleotide probes. The cDNA insert was 1.5 kilobases in length and contained sequences homologous to those of the oligonucleotide probes as well as nucleotide sequences corresponding to another independent CR1 tryptic peptide. Blot-hybridization analysis using fragments of the cDNA insert as probes revealed two distinct species of the CR1 message of 9 and 11 kilobases in human tonsil mRNA. The two EcoRI fragments of the CR1 cDNA insert hybridized to each other, suggesting the presence of homologous sequences. When used as probes in Southern blot analysis of human DNA, each fragment identified similar but not identical patterns of multiple restriction fragments, indicating either a series of homologous domains in a single CR1 gene or the presence of multiple CR1 genes. Furthermore, an additional BamHI fragment was found to segregate with the expression of the S allotype of the CR1 protein in a family. Thus, the molecular weight difference in the polymorphic variants of the CR1 protein is based on differences in nucleotide sequences.
Base Sequence↗