Erythrocyte Complement Receptor Research Articles

Mechanism of Regulation of Complement Receptor Type 1 Transcription by Cytosine Arabinoside in a Pre‐Erythroid Model

Binding to erythrocyte complement receptor type 1 (CR1) clears immune complexes from blood and tissues, preventing complement-mediated pathological inflammation in disease. Previous work has demonstrated that Ara-C, a cytosine analogue, induces an 11-fold increase in CR1 mRNA expression in K-562 erythroleukaemia cells. In this work we therefore investigated whether the Ara-C/K-562 system could be used as a model for studying the pre-erythroid regulation of CR1. We demonstrated that increased CR1 expression could be induced independently of increased haemoglobin expression. Increases in CR1 mRNA levels produced by Ara-C treatment were not a function of increased stability of the message. However, Ara-C induced a protein synthesis-dependent increase in transcription initiation rate as early as 12h after treatment. Further data suggest that the effect of Ara-C on transcription is not a result of its direct DNA-damaging or DNA polymerase-inhibition activities. Induction of receptor transcription was inhibited by tyrosine kinase (TK) and protein kinase C (PKC) inhibitors. These data suggest that TK, PKC and dCTP-adducted phospholipid signalling pathways may all play a role in the mechanism of Ara-C-induced CR1 transcription.

Escherichia coli Bound to the Primate Erythrocyte Complement Receptor via Bispecific Monoclonal Antibodies Are Transferred to and Phagocytosed by Human Monocytes in an In Vitro Model

Abstract We have prepared cross-linked, bispecific mAb complexes (heteropolymers) that facilitate rapid and quantitative binding of a prototype pathogen, Escherichia coli, to the complement receptor (CR1) on primate erythrocytes. Incubation of the erythrocyte-heteropolymer-E. coli complexes with freshly isolated human mononuclear cells leads to rapid removal of the E. coli from the erythrocytes, and phagocytosis and killing of the bacteria. The erythrocytes are not lysed or phagocytosed during this transfer reaction, but both heteropolymer and CR1 are removed from the erythrocytes along with the E. coli. These findings parallel observations made in previous in vivo experiments in which heteropolymers were used to facilitate clearance of innocuous prototype pathogens in a monkey model. It should now be possible to extend the heteropolymer paradigm to a live pathogen in a primate model.

Inflammatory Potential of C-Reactive Protein Complexes Compared to Immune Complexes

C-reactive protein (CRP) is an acute phase serum protein that binds to phosphocholine (PC) and to components of damaged tissue. CRP resembles antibody in that it binds to ligands and activates the classical complement pathway. To compare the processing of CRP complexes to that of IgG complexes, we have prepared complexes containing the same ligand, PC-conjugated BSA, and IgG antibody to either BSA or CRP. We previously demonstrated similar complement-mediated binding of these complexes to erythrocyte complement receptors. CRP and IgG also bind to receptors on neutrophils (PMN), providing another possible pathway for clearance of ligands. PMN binding of IgG complexes can lead to activation with damaging inflammatory consequences. In the present report we have used CRP and IgG complexes containing PC-BSA to compare binding to PMN and activation of PMN adherence to endothelial cells. The results indicate that CRP complexes do not activate PMN whereas IgG complexes do. Binding assays indicate that there is substantially greater binding of IgG than CRP complexes to PMN.

Clearance of blood-borne pathogens mediated through bispecific monoclonal antibodies bound to the primate erythrocyte complement receptor.

The primate erythrocyte complement receptor facilitates both the immune adherence reaction and the immune complex clearance properties of primate erythrocytes. These phenomena have been studied for more than 40 years. However, it has only recently become apparent that these characteristics of primate erythrocytes may be useful in the generation of a therapy based on bispecific monoclonal antibodies. Our approach uses bispecific monoclonal antibody constructs (heteropolymers) that promote binding of specific target pathogens to primate erythrocytes via the complement receptor. Once bound to the erythrocytes, the pathogen-heteropolymer complex should be cleared from the circulation, phagocytosed and destroyed in the liver. Results with several prototype target pathogens in monkey models indicate it may be possible to use this technology to develop a robust and general therapy for the treatment of diseases associated with blood-borne pathogens.

Bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model.

We used Anger camera imaging in a monkey model to investigate the organ localization of a prototype particulate pathogen, 131I-labeled bacteriophage phi X174, after it was bound to the primate erythrocyte complement receptor and then cleared from the circulation. This 131I-labeled phi X174 was infused into the circulation of an immunized monkey, and the nascently formed immune complexes showed rapid and quantitative binding to erythrocytes via the immune adherence reaction (complement-mediated binding). Alternatively, phi X174 was infused into the circulation of a naive animal, and then cross-linked bispecific mAb complexes (heteropolymers, anti-CR1 x anti-phi X174) were infused into the circulation. The infused heteropolymers also facilitated rapid and quantitative binding of phi X174 to erythrocytes. In both cases, after a short lag period, the erythrocyte-bound phi X174 was rapidly cleared from the circulation, and the vast majority of the radiolabel was cleared to the liver, with a small amount clearing to the spleen. Further liver imaging confirmed that within 24 h most of the bacteriophage previously cleared to the liver via the heteropolymer system was phagocytosed and destroyed. The findings in this model system provide additional evidence for the potential utility of heteropolymers to facilitate the safe and rapid clearance of blood-borne pathogens as a potential treatment for infectious diseases.

Do plasma anti phospholipid antibodies in patients with autoimmune haemolytic anaemia correlate with reduced levels of erythrocyte complement receptor 1?

Do plasma anti phospholipid antibodies in patients with autoimmune haemolytic anaemia correlate with reduced levels of erythrocyte complement receptor 1?

The Primate Erythrocyte Complement Receptor (CR1) as a Privileged Site: Binding of Immunoglobulin G to Erythrocyte CR1 Does Not Target Erythrocytes for Phagocytosis

AbstractThe primate erythrocyte (E) complement receptor, CR1, is a transmembrane glycoprotein located in clusters on the surface of E. In vivo studies have demonstrated that during processing and clearance of complement-opsonized immune complexes, large amounts of immunoglobulin G (IgG) can be bound to primate E via CR1 with no E loss or lysis. However, when comparable amounts of IgG are bound to other sites on E, in many cases the E are cleared from the circulation by the mononuclear phagocytic system. Therefore, due to its role in immune complex processing, CR1 may represent a privileged site on the primate E. To delineate further this property of E CR1, we performed in vitro phagocytosis assays in the absence of complement and examined the ingestion of E, opsonized at various sites with IgG, by peripheral blood monocytes. When either human or rhesus monkey E were opsonized at sites other than CR1, with between 1,000 and 15,000 IgG per E, substantial phagocytosis of E was evident. However, when comparable amounts of IgG were bound exclusively via CR1, little, if any, phagocytosis was observed. The key to the low phagocytic level of E opsonized via CR1 may be related to the requirements of a “zipper mechanism” for phagocytosis first annunciated by Griffin et al. Based on their findings, we suggest that due to the presence of preexisting clusters of CR1 on the E membrane, large amounts of IgG can be bound to E under conditions that preclude circumferential engagement (and phagocytosis) of the entire E by Fc receptors on the monocyte.

Bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitate virus clearance in a monkey model.

We investigated the feasibility of using bispecific mAb complexes to redirect and improve the efficiency of the primate E complement receptor 1-based clearance reaction to remove a virus from the circulation. As an initial approach, we used bacteriophage phiX174 as an immunologic model for mammalian viruses. Bispecific complexes were prepared by chemically cross-linking a mAb specific for complement receptor 1 with a mAb specific for the bacteriophage phiX174. In a monkey model these complexes facilitate rapid and quantitative binding of the target bacteriophage to E in vitro and in vivo. Moreover, after in vivo binding to E, the complexes containing mAb and prototype virus are rapidly cleared from the circulation of rhesus and cynomolgus monkeys without loss of E. Our findings suggest that bispecific mAb complexes, in concert with primate E complement receptor 1, may have therapeutic utility in the treatment of diseases associated with blood-borne pathogens.

Serum Complement Activation of SLE Patients During Plasmapheresis

The erythrocyte complement receptor 1 (ECR1)-immune complex binding assay is a sensitive method for the determination of complement fragments which can be activated by bovine serum albumin (BSA)-anti-BSA in vitro. When the C3b/C4b containing bovine serum albumin (BSA)-anti-BSA was formed in the presence of the serum of patients with systemic lupus erythematosus (SLE) its binding to ECR1 was found to be lower than that formed in sera of normal volunteers. The plasmapheresis of SLE patients homozygous for the CR1/E high density allele displays a beneficial effect on the formation of C3b/C4b containing BSA-anti-BSA and its binding to ECR1. There was no significant correlation between the serum C3/C4 level and the percentage of C3b/C4b containing BSA-anti-BSA binding to the ECR1 of SLE patients during plasmapheresis. At the same time, there was an inverse correlation between the serum immune complex level and the ECR1 binding, which was significant in 3 of 5 cases. These data suggest that, besides the determination of different components of complement activation, the functional assay of complement activation might be useful in monitoring the effect of plasmapheresis in SLE.

Immune Complexes Bound to the Primate Erythrocyte Complement Receptor (CR1) via Anti-CR1 Mabs Are Cleared Simultaneously with Loss of CR1 in a Concerted Reaction in a Rhesus Monkey Model

In the circulation of primates, C3b-opsonized immune complexes (IC) bound to erythrocyte (E) CR1 are taken to the liver and spleen where IC are removed and destroyed without lysis or sequestration of E. Individuals with diseases associated with IC processing often have decreased E CR1 levels, and in previous primate animal models of IC disease, E CR1 was shown to be reduced, but the relationship between IC processing and CR1 loss remained to be clarified. We have developed a simple model to study this question. In naive (nonimmunized) rhesus monkeys, E-bound mouse anti-CR1 mAbs (1500 IgG/E) are not rapidly cleared from the circulation. Infusion of monkey anti-mouse IgG leads to rapid indirect binding of this second antibody to E CR1. Subsequently, in what appears to be a concerted reaction, CR1-bound nascent IC are rapidly cleared from the circulation and CR1 is removed from E at the same rate. Clearance of bound IC and loss of CR1 were both independently followed by RIA. Imaging studies localized the cleared anti-CR1 mAbs to the liver. Western blots indicated that the loss of CR1 was not due to a conformational change, and E CR1 levels returned to normal in 2–3 weeks, suggesting that the return was associated with synthesis of new E. Our findings suggest that the key step in the clearance mechanism requires recognition (possibly by Fc receptors) of IC-like material associated with E CR1, and this leads to loss of CR1 and uptake of the CR1-IC substrate by liver phagocytic cells.

Complement-Dependent Binding of C-Reactive Protein Complexes to Human Erythrocyte CR1

C-reactive protein (CRP) is an acute phase serum protein that binds to phosphocholine (PC) on phospholipids and polysaccharides and to protein components of chromatin and small nuclear ribonucleoproteins. Complexes between CRP and ligands activate complement and bind to receptors on phagocytic cells. Although complement is required for CRP-mediated clearance or phagocytosis of ligand-coated erythrocytes, the participation of complement and complement receptors in clearance of soluble CRP complexes has not been examined. We have used PC-conjugated BSA to prepare complexes containing either IgG antibody or CRP. We found similar complement-mediated binding of both types of complexes to human erythrocyte complement receptors (CR1, CD35). We also found that serum deficient in C4A or C4B supported binding of CRP and IgG complexes to erythrocytes. These findings indicate that complexes between CRP and soluble ligands may be cleared by the erythrocyte CR1 pathway described for soluble immune complexes.

Reduction in erythrocyte complement receptor 1 (CR1, CD35) and decay accelerating factor (DAF, CD55) during normal pregnancy

RBC-membrane CR1 has been assayed in pregnancy and at 48 h postpartum. It has been shown that RBC CR1 is reduced as pregnancy progresses, reaching a nadir in the third trimester and that it returns to levels approaching normal within 48 h postpartum. Further, there is a reduced expression of RBC decay accelerating factor (DAF) during pregnancy but no change in expression of RBC CD59. We suggest that the reduction in RBC CR1 and DAF may reflect increased levels of circulating immune complexes and consequent increased complement activation in pregnancy.

Low expression of erythrocyte complement receptor type 1 in chronic hepatitis C patients

Low expression of erythrocyte complement receptor type 1 in chronic hepatitis C patients

Low expression of erythrocyte complement receptor type 1 in chronic hepatitis C patients.

Primate erythrocyte complement receptor type 1 (CR1) plays an essential role in complement-associated immune complex clearance by transporting complexes to macrophages in the liver and/or spleen. Antibody-bound hepatitis C virus, which consists of immune complexes, is observed in patients with chronic hepatitis C. The aim of this study was to clarify the pathophysiological roles of erythrocyte CR1 in hepatitis C virus-infected individuals. We quantified the expression of erythrocyte CR1 with a fluorescence-activated cell sorter system in 57 chronic hepatitis C and 37 chronic hepatitis B cases and 20 normal volunteers. Complement-bound immune complexes were quantified by means of an enzyme-linked immunosorbent assay using anti-C1q and anti-C3d antibodies. Hepatitis C virus-infected patients showed lower erythrocyte CR1 and higher C3d immune complex levels than volunteers (P < 0.01 and P < 0.05, respectively). An inverse correlation was observed between the erythrocyte CR1 and C3d immune complex levels in hepatitis C virus infection (r = -0.300, P = 0.032). The erythrocyte CR1 levels in hepatitis C virus infection were lower in patients with severe liver inflammation, cirrhosis, or hepatocellular carcinoma than in those with mild inflammation, whereas the levels did not differ regardless of the disease stage in hepatitis B virus infection. These findings demonstrate that the expression of erythrocyte CR1 is related to immune complex quantity and the severity of liver disease in hepatitis C virus infection.

The baboon erythrocyte complement receptor is a glycophosphatidylinositol-linked protein encoded by a homologue of the human CR1-like genetic element.

The human erythrocyte CA receptor (E-CR) is the type 1 complement receptor (CR1), the most common form of which is a 220,000 Mr integral membrane glycoprotein composed of 30 short consensus repeats (SCRs). The E-CR of many nonhuman primates is a smaller receptor of unknown genetic origin. Recently, we identified a chimp cDNA, termed CR1b, which represented transcription of a homologue of the human genetic element, CR1-like. The purpose of this study was to identify CR1b in the baboon and, if present, determine whether it encodes the 65,000 Mr baboon E-CR. Baboon bone marrow cDNA was amplified by PCR using primers specific for the signal peptide-encoding region of human CR1 and the 3' region of chimp CR1b. This amplification yielded a CR1b sequence predicted to encode seven SCRs followed by a hydrophobic region, with an N terminus homologous to the N terminus of baboon E-CR. Expression of baboon CR1b yielded a membrane protein that reacted with an anti-CR1 mAb, was identical in size to baboon E-CR, and, like baboon E-CR, could bind baboon C3 linked to activated thiol-Sepharose (C3i-ATS), but not human C3i-ATS. Phosphatidylinositol-specific phospholipase C (PIPLC) released CR1b from Chinese hamster ovary cells and E-CR from baboon erythrocytes, demonstrating that both of these proteins are glycophosphatidylinositol linked to the membrane. Thus, the data indicate that baboon CR1b, a homologue of the human CR1-like genetic element, encodes a glycophosphatidylinositol-linked protein that is the baboon E-CR.

Substitution of two amino acids confers C3b binding to the C4b binding site of CR1 (CD35). Analysis based on ligand binding by chimpanzee erythrocyte complement receptor.

The chimpanzee (Ch) E complement receptor type 1 (CR177) appears to be an alternatively spliced product of the Ch CR1 gene transcript. Its cDNA-derived amino acid sequence contains complement protein-repeating modules (CP) 1-6, 28, 29, and 30 in tandem and is 98.8% homologous to the corresponding regions of human (Hu) CR1. It differs from the C4b binding site of Hu CR1 only by two amino acids, Tyr for Ser37 in CP 1 and Asp for Gly79 in CP 2. However, in addition to binding C4b, Ch E binds C3b. As homologous substitution of one of these amino acids (Tyr for Ser37) was previously shown to not confer C3b binding, we reasoned that either single substitution of the other amino acid or a combination of the two amino acid changes would be required for C3b binding. To test this, using a truncated form of Hu CR1 that has a binding site only for C4b, we made these additional constructs. Single substitution of either amino acid did not affect the ligand binding or cofactor activity. However, the double substitution induced C3b binding and increased cofactor activity for C3b without changing the C4b-binding property. Of interest, these two amino acids are also found in the homologous positions of CP 9 and 16, which form part of the C3b binding site of Hu CR1. Thus, Ch E CR177, one-third of the size and with only a single ligand binding site, by acquiring key amino acid substitutions, binds C3b and C4b and functions analogous to Hu E CR1.

Do plasma anti phospholipid antibodies in patients with autoimmune haemolytic anaemia correlate with reduced levels of erythrocyte complement receptor 1?

Anti phospholipid antibody (APA), of both IgG and IgM class, was assayed in plasma obtained from patients with autoimmune haemolytic anaemia (AHA) and active haemolysis. Although plasma APA was elevated in some AHA patients, when compared to a normal control population, there was no apparent correlation with levels of erythrocyte complement receptor 1 (RBC-membrane CR1) in this patient group. However, there appeared to be a correlation between RBC-membrane CR1 and plasma IgG APA in a subset of the AHA patients with low RBC-membrane CR1. The potential significance of this observation was complicated by the majority of these patients having a plasma APA within the normal range. These results suggest that the reported inverse correlation between APA and RBC-membrane CR1 may not be definitive.

Primate erythrocyte (E) complement receptor (CR1) as an anchor site for bispecific-based therapies to clear pathogens or autoantibodies safely from the circulation.

We have prepared cross-linked, bispecific complexes [heteropolymers (HP) and antigen-based heteropolymers (AHP)] that facilitate complement-independent binding of target model pathogens or autoantibodies to primate erythrocytes (E) via complement receptors (CR1). The method is based on using monoclonal antibodies (mAb) specific for CR1 that either are cross-linked to an mAb specific for a prototype pathogen (e.g., IgE) or are cross-linked to an autoantigen (e.g., dsDNA) that is recognized by circulating pathogenic autoantibodies in the autoimmune disease systemic lupus erythematosus (SLE). The underlying assumption in this research is that complexed ligands containing IgG bound to primate E CR1 should be recognized and processed via the same mechanism by which complement-opsonized immune complexes bound to E CR1 are cleared from the circulation and phagocytosed in the liver and spleen. Our work in experimental monkey models has demonstrated that binding of substrates to primate E via this method does indeed lead to the safe and rapid clearance of the target pathogens or autoantibodies from the circulation, without any lysis or loss of the E. Although a number of questions must still be resolved, it may be possible to generalize these findings and use this CR1-based approach to develop a simple noninvasive bispecific therapy that can be used to clear pathogens or autoantibodies from the circulation.

Low expression of erythrocyte complement receptor type 1 induces defective immune complex clearance in chronic hepatitis C patients: [formula omitted]. 1) 1st Dept. of Med., Osaka Univ. Med. School, Suita Japan. 2)Dept of Gastroenterol., Osaka National Hosp., Osaka, Japan. 3)Dept. of Med., Shin-Senri Hosp., Suita, Japan

Low expression of erythrocyte complement receptor type 1 induces defective immune complex clearance in chronic hepatitis C patients: [formula omitted]. 1) 1st Dept. of Med., Osaka Univ. Med. School, Suita Japan. 2)Dept of Gastroenterol., Osaka National Hosp., Osaka, Japan. 3)Dept. of Med., Shin-Senri Hosp., Suita, Japan

Binding to erythrocyte complement receptor type 1 of BSA/anti-BSA complexes opsonized by C4A3 or C4B1 in the presence of serum.

An in vitro model with human serum and human 0 Rh-negative erythrocytes was used for studies on preformed BSA/anti-BSA complex binding to complement receptor type 1 (CR1). The serum used was first depleted of Clq, factor D and properdin, then of C3, C4 or both and finally reconstituted with the desired proteins (serum reagent). With varying C4 concentrations and 100% C3 present, binding curves obtained for the two C4 isotypes were similar. When the serum reagent was not reconstituted with factor D and properdin there was no difference between the CR1 binding of normal serum and the partially reconstituted serum reagent, nor between the two C4 isotypes in this serum reagent. When C3 at 50% or 100% of normal concentrations was added to the serum reagent together with 100% C4A3 or C4B1, the C4B1-opsonized complexes showed more binding than the C4A3-opsonized complexes. At very low levels of C3 (< 25%) the binding could not be distinguished from the background. The results suggest that the binding of complement opsonized antigen/antibody complexes to erythrocyte CR1 is mediated mainly by C3, originating from activation of the classical pathway, and that the difference in properties between C4A and C4B does not have a major influence.