Search

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, transports immune complexes, and participates in humoral and cellular immune responses. CR1 is present on the surface of many cell types, including erythrocytes, and exhibits polymorphisms in length, structure (Knops, or KN, blood group), and density. The average density of CR1 per erythrocyte (CR1/E) is 500 molecules per erythrocyte. This density varies from one individual to another (100-1,200 CR1/E) and from one erythrocyte to another in the same individual. We present here a robust flow cytometry method to measure the density of CR1/E, including in subjects expressing a low density, with the help of an amplifying immunostaining system. This method has enabled us to show the lowering of CR1 erythrocyte expression in diseases such as Alzheimer's disease (AD), systemic lupus erythematosus (SLE), AIDS, or malaria.

Accepted manuscript version, licensed CC BY-NC-ND 4.0. Aim - To study the role of complement receptor 1 (CR1) for binding of Escherichia coli (E. coli) to erythrocytes, for leukocyte phagocytosis, oxidative burst and complement activation in human whole blood from a CR1 deficient (CR1D) patient and healthy controls with low, medium and high CR1 numbers. Methods - Alexa-labelled bacteria were used to quantify erythrocyte-bound bacteria, free bacteria in plasma and phagocytosis using flow cytometry. Complement activation in plasma was measured by enzyme-linked immunosorbent assay. The CR1 numbers as well as C3bc and C4bc deposition on erythrocytes were measured by flow cytometry. Cytokines were measured using multiplex technology, and bacterial growth was measured by colony forming units. CR1 was blocked using the anti-CR1 blocking mAb 3D9. Results - Approximately 85% of E. coli bound to erythrocytes after 15 min incubation in donor blood with high and medium CR1 numbers, 50% in the person with low CR1 numbers and virtually no detectable binding in the CR1D (r2 = 0.87, P P E. coli-induced phagocytosis and oxidative burst were significantly enhanced by the anti-CR1 mAb 3D9 and in the CR1D and the donor with low CR1 numbers. E. coli-induced complement activation in plasma, C3bc and C4bc deposition on erythrocytes, and bacterial growth were similar in all four cases. Conclusions - CR1D and low CR1 numbers prevented E. coli binding to erythrocytes, increased free bacteria in plasma, phagocytosis and oxidative burst, but did not affect plasma or surface complement activation and bacterial growth.

The complement receptor 1 (CR1) gene was shown to be involved in Alzheimer&rsquo, s disease (AD). We previously showed that AD is associated with low density of the long CR1 isoform, CR1*2 (S). Here, we correlated phenotype data (CR1 density per erythrocyte (CR1/E), blood soluble CR1 (sCR1)) with genetic data (density/length polymorphisms) in AD patients and healthy controls. CR1/E was enumerated using flow cytometry, while sCR1 was quantified by ELISA. CR1 polymorphisms were assessed using restriction fragment length polymorphism (RFLP), pyrosequencing, and high-resolution melting PCR. In AD patients carrying the H allele (HindIII polymorphism) or the Q allele (Q981H polymorphism), CR1/E was significantly lower when compared with controls carrying the same alleles (p &lt, 0.01), contrary to sCR1, which was significantly higher (p &lt, 0.001). Using multivariate analysis, a reduction of 6.68 units in density was associated with an increase of 1% in methylation of CR1 (estimate &minus, 6.68, 95% confidence intervals (CIs) &minus, 12.37, &minus, 0.99, p = 0.02). Our data show that, in addition to inherited genetic factors, low density of CR1/E is also acquired. The involvement of CR1 in the pathogenesis of AD might be linked to insufficient clearance of amyloid deposits. These findings may open perspectives for new therapeutic strategies in AD.

International audience; Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (Aβ) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation.