Expression of complement receptor type 1 (CR1) on erythrocytes of paracoccidiodomycosis patients

Complement receptor type 1 (CR1) is a membrane glycoprotein that acts as a receptor for the C3b, iC3b and C4b fragments of complement. In primates, one function of erythrocytes is to promote safe clearance of immunocomplexes (IC) from the circulation through CR1. Theoretically, in diseases characterized by high levels of circulating IC, an erythrocyte CR1 (CR1/E) deficiency may favor IC deposition in tissues or facilitate inappropriate activation of leukocytes in the circulation. Depression of the cell immune response occurs in paracoccidioidomycosis (PCM), especially in the more severe cases, and is frequently associated with high serum IC levels. In the present study we quantified the number of CR1/E in patients with the acute and chronic forms of PCM before and after treatment and correlated it with serum IC levels and CD4+ and CD8+ T cell concentration in the peripheral blood of these patients. Patients with PCM, particularly those with active disease and who had received treatment for shorter periods of time, had low numbers of CR1/E. In addition, an increase in serum IC concentration and a reduction in the CD4+/CD8+ T cell ratio were observed. After treatment there was a significant increase in mean CR1/E number and a reduction in serum IC levels. In patients with the chronic form of the disease the CD4+/CD8+ T cell ratio tended to increase after treatment and was associated with increased CR1/E levels. These results suggest that the reduction in CR1/E observed in patients is a phenomenon acquired with the disease and that CR1 could play a role in the pathogenesis of PCM.This revised version was published online in October 2005 with corrections to the Cover Date.     

Distribution in clusters of complement receptor type one (CR1) on human erythrocytes

The distribution of CR1 on human E was studied using label-fracture and thin section electron microscopy. CR1 was found to be organized in clusters on unfixed cells and on cells that had been prefixed with paraformaldehyde or glutaraldehyde before labeling. The number of clusters/E ranged from 8 to 20 as estimated from the examination of freeze-fracture replicas of labeled cells. Clusters contained an average of 30 to 75 gold particles on cells from two donors which expressed 462 and 586 CR1 Ag sites/cell, as determined by flow cytometry. In thin section electron micrographs, gold complexes were seen surrounding an electron-dense material protruding from the membrane which represents compact aggregates of CR1. The maximal distance between gold particles and the membrane was 100 nm, which corresponds to the estimated length of the major allotypic form of CR1, as calculated from the primary DNA sequence of the molecule. The distribution in clusters of CR1 on the E membrane may provide the basis for an enhanced affinity of C3b-CR1 interactions on the plasma membrane of the cells and may explain the preferential binding of C3b-bearing immune complexes to E in vivo.     

Large-scale isolation of complement receptor type 1 (CR1) from human erythrocytes. Proteolytic fragmentation studies.

A large-scale procedure for the isolation of complement receptor type 1 (CR1, the C3b receptor) from human erythrocytes is described. Two of the four known phenotypes of CR1 are detectable in the isolated material. Amino acid and hexosamine analysis of the A phenotype (Mr 240 000) indicates a polypeptide chain length of about 2030 amino acids and a carbohydrate content of 8%. Both N- and O-linked sugars appear to be present. Trypsin digestion of isolated CR1 shows that it is degraded rapidly and extensively, and no stable products of Mr greater than 25000 are found. The ability of the receptor to bind to solid-phase ligand is destroyed after a single cleavage by trypsin. The capacity of the receptor to act as a cofactor for Factor I-mediated cleavage of soluble C3b is, however, only gradually decreased by proteolysis, and 30% of this activity remains after extensive degradation. The same pattern of loss of binding to solid-phase ligand, with partial retention of interaction with soluble ligand, is also characteristic of the complement proteins Factor H and C4bp, which are functionally related to CR1.     

Overexpression of complement receptor type I (CR1, CD35) on erythrocytes in patients with hemoplasma infection

In the present study, 12 patients with fever of undetermined origin, anemia and icterus were diagnosed with hemoplasma infection by light microscopy, transmission electron microscopy examination and PCR assay after being excluded from other usual febrile diseases. Complement receptor type I (CR1, CD35) expression on the surface of erythrocytes was assessed by flow cytometry using mouse anti‐human CD35 antibody. Compared with healthy volunteers, the level of CD35 was significantly elevated in patients with severe hemoplasma infection at diagnosis, and decreased sharply after treatment. However, in latent infection cases without clinical manifestations, CD35 expression showed an ascending trend but had no statistical difference compared to the healthy controls. The present study demonstrated that hemoplasma infection can induce high levels of expression of CR1 on the membrane of red blood cells, which may be a reaction to the immunity challenge.     

Human complement receptor type 1 (CR1) binds to a major malarial adhesin

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a major adhesin molecule expressed on Plasmodium-falciparum-infected erythrocytes, interacts with several receptors on endothelial cells and uninfected erythrocytes. This 'stickiness', known as rosetting, is a strategy used by the parasite to remain sequestered in the microvasculature to avoid destruction in the spleen and liver. Erythrocyte rosetting causes obstruction of the blood flow in microcapillaries. Recent data suggest a direct interaction between PfEMP1 and a functional site of complement receptor type 1 (CR1; CD35) on uninfected erythrocytes. Consistent with the hypothesis that CR1 is important in malaria pathogenesis is a 40-70-fold increase in the frequency of two CR1 blood-group antigens (at least one of which might rosette less efficiently) in malaria-exposed African populations. Furthermore, structural differences in erythrocyte CR1 between human and non-human primates are probably explained by the selective pressure of malaria.     

Roles of the complement receptor type 1 (CR1) and type 3 (CR3) on phagocytosis and subsequent phagosome-lysosome fusion in Salmonella-infected murine macrophages

Abstract The receptors involved in the recognition of Salmonella typhimurium and S. typhi by murine macrophages were identified, and their relevance to phagosome-lysosome fusion was also investigated. Phagocytosis of S. typhimurium by murine macrophages was dependent on the opsonization with normal fresh serum, although the opsonin had no triggering activity in phagosome-lysosome fusion. In contrast, the opsonization of S. typhi with normal fresh serum efficiently triggered both phagocytosis and following phagosome-lysosome fusion. Anti-murine CR1 antibody suppressed phagocytosis of S. typhimurium by 36%, whereas anti-CR3 antibody, mannan, and advanced glycosylation endproducts (AGE)-BSA all failed to prevent phagocytosis of S. typhimurium , suggesting that CR1 may only contribute to the recognition of S. typhimurium and may possibly play a minor role. Other receptors involved may also influence the outcome phagocytosis in terms of phagosome-lysosome fusion. In the case of S. typhi , only anti-CR3 antibody significantly inhibited not only phagocytosis of S. typhi but also following phagosome-lysosome fusion. Treatment with K76COONa, an inhibitor of C3bINA (I factor), resulted in a marked inhibition of phagosomelysosome fusion in S. typhi -infected macrophages, although no significant inhibition was observed on phagocytosis of S. typhi . These results suggest that S. typhimurium and S. typhi may be recognized at least in part by CR1 and CR3, respectively, and that the recognition by CR3 but not CR1 is functionally associated with subsequent phagosomelysosome fusion in murine macrophages.     

Distribution and quantitative expression of the complement receptor type 1 (CR1) on human peripheral blood T lymphocytes.

The complement receptor, type 1 (CR1) is expressed on a variety of cell types including primate erythrocytes, phagocytic cells, and B lymphocytes. On these cells, CR1 plays a role in a diverse spectrum of biological activities including the clearance of immune complexes from the circulation, down-regulation of the complement system, recognition of complement-coated microorganisms, and cellular activation. CR1 is also expressed by some, but not all, T lymphocytes. The present study was undertaken in order to examine the distribution of CR1 on normal human T cell subsets by flow cytometry and to quantify the expression of T cell CR1 by radioimmunoassay. Data presented here indicate that, in a panel of 19 normal individuals, a mean of 9.7% of the overall peripheral blood lymphocyte population expressed CR1 and that, as assessed by two-color flow cytometry, 12.0% of CD3+, 13.0% of CD4+, and 20.0% of CD8+ cells expressed CR1. While single peaks of CR1 staining were observed within the CD3 and CD4 subsets, a biphasic pattern of staining was evident within the CD8 subset in which relatively high-intensity CR1 staining was detected within the subpopulation of "dull" CD8+ cells, whereas a lower intensity of CR1 staining was observed within the subpopulation of "bright" CD8+ cells. Duplicate analyses performed over a relatively short time frame suggested that, while the overall percentage of cells that expressed CR1 varied considerably among normal individuals, in at least some individuals the percentage of cells expressing CR1 was relatively stable, especially within the CD4 subset. In cell suspensions enriched for T lymphocytes by rosetting with sheep erythrocytes, 10.0% of the cells were CR1+ and a mean of approximately 3700 CR1 were expressed per CR1+ cell. There was no apparent correlation between the number of CR1 per T cell and the number of CR1 expressed per erythrocyte in the same blood sample. The expression of CR1 on subpopulations within the CD3+, CD4+, and CD8+ lymphocyte subsets may play a role in both normal cell function and in the pathophysiology of disease states including the acquired immune deficiency syndrome (AIDS).     

Monoclonal antibodies to mouse complement receptor type 1 (CR1). Their use in a distribution study showing that mouse erythrocytes and platelets are CR1-negative

mAb to murine C receptor type 1 (CR1) were produced and three of them were characterized. One antibody, designated as 8C12, immunoprecipitated a protein of 190,000 Mr from a detergent extract of surface-labeled spleen cells and stained spleen B but not T lymphocytes in fluorescent flow cytometry. It inhibited both CR1-mediated rosette formation and the cofactor activity of CR1 for factor I-mediated cleavage of C3b, suggesting that it recognizes the ligand-binding site of CR1. The two other antibodies, designated as 7G6 and 7E9, recognized different epitopes from that recognized by 8C12, and they cross-reacted with a protein of 150,000 Mr that is present in a spleen extract. The distribution of CR1 in murine hemopoietic cells was studied by binding experiments with radiolabeled 8C12 and fluorescent flow cytometry. When CR1 was not detected by 8C12 alone, the two other antibodies were used in combination with 8C12 to confirm the negative results. Almost all B lymphocytes from the spleen, lymph nodes, and peripheral blood were CR1 positive. Most of the Thy-1-positive lymphocytes from these tissues were CR1 negative. Thymus lymphocytes were also CR1 negative. Peritoneal macrophages and chemotactic factor stimulated but not unstimulated peripheral blood granulocytes were CR1 positive. In contrast to human E, mouse E were CR1 negative. This pattern of distribution was consistent with previous results obtained by rosette assays. Although mouse platelets cause immune adherence hemagglutination with C3b-bearing SRBC, they are CR1 negative. Three other lines of evidence also indicated that platelets are CR1 negative. First, no band of CR1 was demonstrated by immunoprecipitation with 8C12 of an extract of surface-labeled platelets. Second, 8C12, which inhibited rosette formation by lymphocytes, alone or in combination with 7G6 and 7E9, did not inhibit immune adherence between platelets and C3b-bearing SRBC. Third, polyclonal rabbit IgG prepared from anti-mouse CR1 antiserum did not inhibit immune adherence by platelets. These results strongly suggest that the C3b-binding factor(s) on mouse platelets is different from CR1 and that processing of C3b-bearing immune complexes in mouse blood may be mediated by a new and as yet unidentified C3b-binding factor(s).     

Hypoxia-induced expression of complement receptor type 1(CR1, CD35) in human vascular endothelial cells

Reoxygenation of hypoxic human umbilical vein endothelial cells(HUVECs) increases protein expression of the complement regulators CD46and CD55. As the receptor for C3b is known to be present on injuredbovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expressionincreased 3.7 ± 0.6-fold as measured by ELISA on HUVECsfollowing hypoxia (48 h, 1%O₂). Colocalization of CD35 andvon Willebrand factor by confocal microscopy confirmed that CD35 waspredominantly intracellular. Lipopolysaccharide or tumor necrosisfactor- also significantly increased HUVEC CR1 proteinexpression. Western blot analysis of neutrophil or hypoxicHUVEC lysates revealed a 221-kDa CR1 band under nonreducingconditions. RT-PCR of hypoxic HUVEC mRNA revealed a singleband that, after sequencing, was identified as CD35. In situhybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA.Hypoxic HUVECs bound immune complexes and acted as a cofactorfor factor I-mediated cleavage of C3b. Thus hypoxia induces functionalHUVEC CR1 expression.     

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.     

Cellular distribution of complement receptor type 4 (CR4): expression on human platelets

Neutrophils have been shown to express a receptor for C3dg that is distinct from CR2 and is termed complement receptor type 4 (CR4). In the present study, other peripheral blood cell types were examined by indirect immunofluorescence and flow cytometry for the presence of C3dg binding activity. Specific uptake of C3dg occurred with neutrophils, platelets, and B lymphocytes, but not with eosinophils or T lymphocytes. Monocytes, contained within a mixed cell population of peripheral blood mononuclear cells and platelets, also bound C3dg, whereas purified monocytes did not. Binding of 125I-labeled glutaraldehyde-cross-linked C3dg to platelets was saturable, with an average of 1940 C3dg molecules bound per platelet at saturation (n = 8), ranging in number from 660 to 3930 molecules bound. Activation of platelets with thrombin did not consistently cause an increase in the expression of CR4 sites. 125I-C3dg binding to platelets was competitively inhibited equally well by unlabeled C3dg and iC3b, and approximately fourfold less well by C3b. The addition of platelets to elutriated monocytes generated C3dg binding activity on these cells by the formation of platelet-monocyte complexes. Thus, the CR4 on platelets accounted for the C3dg binding activity initially observed with partially purified monocytes. The adherent property of platelets may enable them to confer on certain other cell types the ability to localize C3dg-coated immune complexes or particles.     

Solubilization of active complement receptor type 1 (CR1) from human erythrocyte stroma with trypsin and its purification

Mild trypsinization of human erythrocyte stroma solubilized CR1 (complement receptor type 1, C3b/C4b receptor) without significant loss of decay-accelerating activity to C5 convertases on hemolytic intermediate cells (EAC 1-3b, P). The solubilized CR1 was purified using DEAE-Sephacel, C3-Sepharose, and anti-CR1-Sepharose column chromatographies. The purified material showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, and its molecular weight was determined to be 175K, about 20K smaller than native CR1. Because the purified sample was separated into the several segments by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the molecule is considered to be nicked and those segments are associated by disulfide bonds. These results mean that a large portion of the CR1 molecule is present outside of the plasma membrane of erythrocytes, and the intramembranous and cytoplasmic domains are not necessary for decay-accelerating activity.     

Functional properties of membrane-associated complement receptor CR1

It was previously shown that membrane receptors for C3b (CR1) purified from human erythrocytes were powerful inhibitors of the complement cascade and that they encompass the regulatory functions of the serum proteins beta 1H (H) and C4-binding protein (C4bp). In the present report we study the functional properties of membrane-associated CR1. When tonsil lymphocytes, which contain between 30 and 60% of CR1-bearing B cells, are incubated with the red cell complement intermediate EAC14oxy2lim or EAC14oxy23lim, they inhibit both C42 and C423 in a dose-dependent manner. These effects are mediated by membrane-associated molecules. Indeed, mild trypsinization of the lymphocytes abolishes their activity, and formaldehyde-fixed cells are as effective as viable cells. The inhibitory effects are in part mediated by CR1. The lymphocyte activities are reversed about 60% if monoclonal antibodies to CR1 or fluid phase C3b are present in the incubation medium. Moreover, upon addition of C3b-inactivator (l), lymphocytes release C3c fragments from EAC14oxy23b. The release of C3c was also abolished by antibodies to CR1. These results support the idea that CR1, as well as other molecules from the lymphocyte membrane, can function as inhibitor(s) of complement activation in their vicinity.     

AML1, the target of chromosomal rearrangements in human leukemia, regulates the expression of human complement receptor type 1 (CR1) gene.

The human CR1 gene is expressed specifically in hematopoietic cells. It is suggested that some cell-type specific factors which involve in gene-specific activation or repression exist in cells according to the result that the gene expression varies differently depend on differentiation stage. Here, we demonstrate that the integrity of a polyomavirus enhancer core sequence, 5'-TGTGGT-3', is critical to the human CR1 promoter activity. AML1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. We show that the AML1 binds specifically to this site and activates the human CR1 promoter. Furthermore, we demonstrate that the Ets binding site (GGAA) located 2 bp upstream of the AML1 site is also involved in the regulation of the human CR1 promoter activity. Point mutations of either the AML1 or the Ets binding site that abolish the binding of the respective factors result in significant decreases of the human CR1 promoter activity. These results suggest that AML1 and Ets proteins direct the expression of the human CR1 promoter.     

Platelet-activating factor enhances complement-dependent phagocytosis of diamide-treated erythrocytes by human monocytes through activation of protein kinase C and phosphorylation of complement receptor type one (CR1)

Oligomerization of band 3 protein has been recently indicated as an early event in senescent or damaged red cell membrane followed by specific deposition of anti-band 3 antibodies and binding of complement C3 fragments. The band 3-anti-band 3-C3b complex is recognized by homologous monocytes, and phagocytosis ensues. This study shows that recognition of the anti-band 3-C3b complex by the monocyte C3b receptor type one (CR1) plays a crucial role in the process of removal of damaged red cells. Indeed, blocking of monocyte CR1 with an anti-CR1 monoclonal antibody abrogated phagocytosis of diamide-treated red cells. Platelet-activating factor (PAF) is a phospholipid mediator involved in inflammatory processes. Nanomolar (R)-PAF enhanced the CR1-dependent phagocytosis of diamide-treated human red cell and of sheep red cells coated with C3b, induced the fast translocation of protein kinase C to monocyte membrane compartment, and stimulated the phosphorylation of monocyte CR1. The biologically inert lyso-PAF and the enantiomer (S)-PAF were inactive. PAF receptor antagonists and inhibitors of protein kinase C blocked the enhancement of phagocytosis induced by PAF. Protein kinase C translocation, phosphorylation of CR1, and stimulation of this receptor to an active state capable of mediating phagocytosis represent a novel pathway by which PAF interferes with red cell homeostasis and possibly modulates inflammatory reactions and host mechanisms against infections.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1)

We identified on the membrane of mouse spleen cells a polypeptide of Mr 190,000 (S190), with binding affinity for the mouse third component of the complement system (C3). S190, purified by affinity chromatography on C3-Sepharose, has properties resembling those of the human C3 receptor type 1 (CR1). Thus, S190, like CR1, served as a cofactor for the C3b inactivator (I)-mediated cleavage of fluid-phase C3b into iC3b, and had cofactor activity comparable to that of serum factor H (H). S190 also acted as a cofactor for the cleavages of membrane-bound C3b or membrane-bound iC3b into C3c (Mr 140,000) and C3dg (Mr 40,000) by serum factor I. As is the case with CR1, the specific activity of S190 for the cleavages leading to C3c-C3dg formation was approximately 100-fold greater than that of H. We therefore conclude that S190 and CR1 are analogous proteins.     

Urokinase receptor (uPAR) regulates complement receptor 3 (CR3)-mediated neutrophil phagocytosis

Urokinase receptor (uPAR) associates in cis with complement receptor 3 (CR3). In the present study, we addressed whether this coupling regulates CR3-mediated phagocytosis. CR3-mediated attachment of iC3b-opsonized sheep red blood cells to human neutrophils and internalization of these cells were reduced by removal of cell-bound uPAR by phosphatidylinositol-specific phospholipase C and reconstituted in the presence of soluble uPAR. The attachment and internalization were suppressed in the presence of anti-uPAR polyclonal antibody, proteolytically inactive urokinase and saccharides that disrupt interaction of uPAR with CR3. Thus, uPAR acts as a cofactor for iC3b binding to CR3 and regulates CR3-mediated phagocytosis.