Complement and its receptor: a physiological transport system for circulating immune complexes.
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Antigen-Antibody Complex↗
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Role of complement receptor type three and serum opsonins in the neutrophil response to yeast.
Previous studies have suggested that neutrophil complement receptor type three (CR3) has two binding sites: (1) a site for fixed iC3b that does not trigger ingestion or a superoxide (O2-) burst, and (2) a function-triggering site for the beta-glucan component of yeast (Saccharomyces cerevisiae) cell walls. In the present study it was found that yeast (Y) coated with C3b (YC3b) or iC3b (YC3bi), prepared with purified complement in an IgG-free system, were avidly ingested ans stimulated a vigorous O2- burst, whereas sheep erythrocytes (E) bearing C3b or iC3b, were not ingested and did not give an O2- burst. YC3b and YC3bi contained an amount of fixed C3 that was approximately equal to serum-opsonized Y (OY), and produced O2- bursts comparable to OY. Experiments utilizing rabbit F(ab')2 anticomplement receptor type one (anti-CR1) to block fixed C3b binding to CR1, and monoclonal anti-CR3 (MN-41 or OKM1) to block fixed iC3b and Y cell wall binding to CR3, indicated that the O2- burst response to OY was primarily due to fixed iC3b and Y cell wall binding to CR3. Fixed C3b (that represented 33% of the fixed C3 on OY) and IgG anti-Y antibodies that bound to CR1 and Fc receptors, respectively, were found to contribute little to the response. Although YC3b did bind avidly to neutrophil CR1, the results suggested that the O2- burst response to YC3b was triggered after the initial YC3b binding by the secondary attachment of Y cell wall components to CR3. When neutrophils were treated with anti-CR3, 90% of neutrophils bound YC3b (via CR1), but phagocytosis and an O2- burst were completely absent. Similar findings were made with OKM1-treated neutrophils and YC3bi. Responses of OKM1-treated neutrophils were inhibited because only the iC3b-binding site of CR3 was ligated by the YC3bi. Thus, fixed C3b or iC3b on Y mediate avid binding of Y to neutrophils via CR1 or the iC3b-binding site of CR3, respectively, but ingestion and an O2- burst response are only triggered when glucans in the Y cell wall secondarily bind to neutrophils via the beta-glucan binding site of CR3.
Binding Sites↗
Origin of the fourth component of complement related Chido and Rodgers blood group antigens.
We have reviewed the relationship between C4 and its related blood group and discussed the mechanisms whereby a fragment of C4 could become attached to erythrocytes (E). We hypothesize that there is chronic fluid-phase activation of C4 by either C1 to form C4b or spontaneous cleavage of the thioester to form iC4. These activated molecules bind to E. Proteolytic degradation of the bound C4b or iC4 would leave a covalently attached fragment of C4 on E and thereby give rise to the Ch and Rg blood group antigens. This system is of further immunopathologic interest since this 'normal' activation or turnover of C4 is closely regulated. In patients deficient in regulatory proteins, this spontaneous or normal turnover of C4 and C3 may initiate a pathologic condition.
Animals↗
Common binding region of complement factors B, H and CR1 on C3b revealed by monoclonal anti-C3d.
Fab fragments of a murine C3d-specific monoclonal antibody, clone 4C2, strongly inhibited the binding of H, and up to about 80% that of B, to C3b on sheep erythrocytes, as shown by using radiolabelled purified proteins. The inhibition was also detected in the fluid phase. Conversion of C3 by preformed solid phase alternative pathway C3 convertase was not affected by 4C2. Also, it did not affect the decay rate of the convertase, but reduced the decay-accelerating effect of H on it. The results suggest that the binding sites of B and H on C3b are close to each other, and their competition for binding to C3b is focused around the C3d region. In addition, 4C2 partially blocked noncovalent binding of C3b to human erythrocytes, which suggests that CR1 also binds close to the H- and B-binding region in C3b.
Antibodies, Monoclonal↗
Application of protein A-rosette assay for screening of monoclonal antibodies to human complement regulatory proteins.
Mice were immunized with purified membrane cofactor protein (MCP) and its monoclonal antibodies were screened by protein A(PA)-rosette assay. In this assay, the culture supernatants of hybridoma cells were layered over fixed MCP-bearing cells, and after washing, PA-coated sheep erythrocytes were applied as an indicator to these MCP-bearing cells. No purified antigen was therefore required throughout the screening. More than 300 of the supernatants harvested were successfully examined within 6 h. Each resultant antibody consisted of a single subclass of IgG, and reacted only with MCP in both transblotted and surface-labeled materials. The sensitivity of this assay was then assessed with these purified antibodies. As little as 0.5 micrograms of IgG1 or 0.01 micrograms of IgG2a was found to be detectable with more than 30% rosette formation. There were variations among cell lines in the sensitivity to the PA-rosette assay and the sensitivity did not correlate with the quantity of MCP surface expression in any of the cell lines. K562 gave the lowest background (nonspecific rosette formation) and the best specificity for anti-MCP of the 20 MCP-positive cell lines tested. Cell lines suitable for the detection of monoclonal anti-decay-accelerating factor and anti-C3b/C4b receptor were also examined and CCRF-SB and HSB2, and peripheral blood granulocytes, were found to be proper cell lines for screening the decay-accelerating factor and C3b/C4b receptor, respectively. Clones for anti C3b/C4b receptor were successfully obtained using granulocytes by the PA-rosette assay. This method needs no purified antigen and facilitates the rapid screening and purification of positive clones against cell-surface complement regulatory proteins.
Antibodies, Monoclonal↗
Complement receptor type 1 (CR1, CD35) expression on peripheral T lymphocytes: both CD4- and CD8-positive cells express CR1.
T lymphocytes from 42 healthy blood donors were examined for expression of CR1 on the cell surface using a fluorescence-activated cell sorter. The fraction of CR1-positive T lymphocytes varied in the range from 1 to 8% (median 2.4%). The CR1-positive T lymphocytes constituted a homogeneous group of cells regarding both size and granulation; they seemed to be larger and more granulated than the average T lymphocytes. The CR1-expressing T lymphocytes were found both in the CD4- and in the CD8-positive subpopulations of T lymphocytes, and although the CD4-positive cells expressed CR1 with a slightly higher percentage than did the CD8-positive cells, this difference was not significant. The number of CR1-positive T lymphocytes did not correlate with the level of CR1 on erythrocytes. The presence of CR1 on a small T lymphocyte population suggests that CR1 on T lymphocytes might play a role in the immune regulation.
Antigens, Differentiation, T-Lymphocyte↗
Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors.
Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the membrane attack complex do not participate in the reaction. Besides affecting the size and solubility of circulating IC the interaction with C factors influences the reactivities of the complexes towards fluid phase reactants and mediates the reversible binding of IC to cellular C3 receptors. Our knowledge of the cellular localization, expression and structure of the C3 receptors, especially the C3b (CR1) receptor, has been considerably extended in the last few years, whereas our understanding of the physiological role of these receptors is still fragmentary. However, it is becoming increasingly evident that impaired solubilization of IC in patients with compromised C function may permit the complexes to deviate from their normal pattern of interaction with C3 receptors probably influencing both the organ distribution and clearance of IC and thereby also their phlogistic potentials.
Antigen-Antibody Complex↗
Enzyme-linked immunoassay to monitor the purification of, and to screen for monoclonal antibodies against, C3b receptor.
An enzyme-linked immunosorbent assay both to screen for monoclonal anti-bodies to the C3b receptor and to monitor its purification was developed. The test requires only purified C3, converted to either the hemolytically no longer active iC3 or C3b. An NP-40 lysate of tonsil cells can be used as a source of CR1 in this test which works best under hypotonic conditions facilitating the interaction of iC3/C3b and CR1. Two monoclonal antibodies to CR1 were produced using this test for the screening of hybridomas. The identity of the molecule recognized by these antibodies with CR1 is demonstrated by immunoprecipitation and Western blot studies, as well as immunofluorescence and immunoperoxidase staining of cells and tissues known to contain CR1. Fractions from lentil-lectin and DEAE-Sephadex columns containing CR1 can be identified using this test.
Antibodies, Monoclonal↗
Structural and functional studies on the human C3b/C4b receptor (CR1) purified by affinity chromatography using a monoclonal antibody.
A procedure was devised that has several advantages over previously described methods to purify CR1 from both erythrocytes (E) and the HL-60 promyelocytic cell line. Using a monoclonal antibody immunoaffinity column, CR1 was purified to homogeneity as assessed by silver staining and 2-D gel analysis. Protein purified by this method comigrates on SDS-PAGE with 125I surface-labeled CR1 isolated by immunoprecipitation or iC3-Sepharose affinity chromatograhy and can be specifically immunoblotted with a second monoclonal anti-CR1 antibody. Employing this method, CR1 can be purified to homogeneity in amounts adequate for both functional studies and biochemical microanalysis. Purified E CR1 is functionally active as assessed by its ability to specifically rebind to an iC3-Sepharose affinity column, act as a cofactor for I-mediated cleavage of C3b to C3c and C3d, g and to accelerate decay of both the classical and alternative pathway C3 convertases. Its specific activity is similar to that of CR1 purified by a method not employing the potentially denaturing washing and eluting conditions of immunoaffinity chromatography. The pIs of the two major E CR1 allotypes are both approximately 7.1. Using pooled human E CR1, an amino acid composition was derived which revealed a relatively high proline content. This has also been found in two functionally related and genetically linked complement-regulatory proteins, H and C4-binding protein, NH2-terminal sequencing of E CR1 and HL-60 CR1 was unsuccessful indicating that the NH2-terminus is blocked.
Amino Acid Sequence↗
Normal distribution of complement C3b receptor (CR1) numbers on erythrocytes in blood donors: low levels are associated with HLA-B27.
Blood donors were examined for C3b receptor (CR1) levels on erythrocytes by a microtiter plate enzyme-linked immunosorbent assay and for HLA-A, -B, and -C antigens, C3 phenotypes and blood groups. The CR1 levels among 150 donors varied in the range from 24 to 130% (mean +/- 1 SD = 69.9 +/- 22.9) of an erythrocyte standard, and in accordance with a polygenic model of inheritance the distribution of CR1 was well approximated by a normal curve (chi-square goodness-of-fit test nonsignificant, p = 0.66). Low CR1 levels were associated with HLA-B27 (n = 11, p = 0.05), which was confirmed by investigation of further 20 HLA-B27-positive donors (p = 0.05). The CR1 levels were not associated with a certain C3 phenotype. Possibly, the blood group phenotype M-M + S-s+ is associated with high and the S antigen with low CR1 numbers. The relation between low CR1 numbers and HLA-B27 might be important for the understanding of the association between HLA-B27 and certain inflammatory rheumatological diseases.
Blood Donors↗
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↗
Soluble complement receptor type 1 inhibits the complement pathway and prevents contractile failure in the postischemic heart. Evidence that complement activation is required for neutrophil-mediated reperfusion injury.
BACKGROUND: Complement-mediated neutrophil activation has been hypothesized to be an important mechanism of reperfusion injury. It has been proposed that soluble complement receptor 1 (sCR1), a potent inhibitor of both classical and alternative complement pathways, may prevent the complement-dependent activation of polymorphonuclear leukocytes (PMNs) that occurs within postischemic myocardium and thereby inhibit PMN-derived free radical generation and prevent postischemic contractile failure. Therefore, we performed studies to determine the effects of sCR1 on contractile function, PMN adhesion, complement deposition, and PMN-derived free radical generation in the postischemic heart. METHODS AND RESULTS: Studies were performed in an isolated rat heart model in which the isolated effects of given cellular or humoral factors could be determined. Plasma and PMNs were present to study the effects of sCR1 on contractile function, coronary flow, leukocyte adhesion, complement deposition, and PMN-derived free radical generation. Isolated rat hearts were perfused by the method of Langendorff (n = 10 in each group) and subjected to 20 minutes of global ischemia and reperfusion with PMNs and plasma in the presence or absence of sCR1. Left ventricular developed pressure (LVDP), coronary flow (CF), left ventricular end-diastolic pressure (LVEDP), and rate-pressure product (RPP) were measured during the preischemic period, during 1-minute control infusion of PMNs and plasma, and on reflow following 20 minutes of global ischemia. During the preischemic control infusion, no significant alterations in the physiologic parameters were observed, and there was no measurable free radical generation. Reperfusion with sCR1 markedly improved the recovery of postischemic contractile function. LVDP after 45 minutes of reperfusion was 76 +/- 9.8% compared with 32 +/- 6.2% (P < .001). In addition, significant improvements in LVEDP, RPP, and CF were observed in hearts treated with sCR1. Additional experiments were also performed to determine the effect of sCR1 on complement-mediated PMN activation. Measurements of PMN-derived free radical generation were performed in both isolated PMNs and the coronary effluent of hearts using electron paramagnetic resonance spectroscopy (EPR) with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR measurements in both isolated PMNs and coronary effluent demonstrated that sCR1 blocked complement-mediated free radical generation from the PMNs. Increased accumulation of PMNs was observed both in hearts treated with sCR1 and in those not treated with sCR1. Immunohistochemical staining of the postischemic myocardial tissue demonstrated marked complement deposition on the endothelial surface of small arterioles and capillaries, which was prevented by sCR1 treatment. Thus, sCR1 did not prevent PMN adhesion but did prevent complement deposition with activation of the PMN oxidative burst. CONCLUSIONS: The potent complement inhibitor sCR1 was found to be effective at preventing postischemic myocardial contractile dysfunction and enhancing the recovery of coronary flow. This study demonstrated that complement activation occurs in postischemic myocardium and is necessary for activation of the neutrophil oxidative burst with the generation of reactive oxygen free radicals. The process of neutrophil adhesion, however, was not affected by sCR1 and was independent of complement factors. These findings demonstrate the sCR1 is a highly potent agent at preventing complement-mediated PMN activation and secondary free radical generation in the postischemic heart. This genetically engineered protein appears to be a promising therapeutic agent in the prevention of myocardial reperfusion injury.
Animals↗
Inhibition of complement activation decreases airway inflammation and hyperresponsiveness.
Studies in murine models have suggested the involvement of the complement anaphylatoxins (C3a and C5a) in the development of allergic asthma. We investigated the effects of inhibiting complement activation after sensitization but before allergen challenge on the development of allergic airway inflammation and airway hyperresponsiveness. To prevent complement activation, we used a recombinant soluble form of the mouse membrane complement inhibitor complement receptor-related gene y (Crry) fused to the IgG1 hinge, CH2 and CH3 domains (Crry-Ig), which has decay-accelerating activity for both the classic and alternative pathways of complement as well as cofactor activity for factor I-mediated cleavage of C3b and C4b. C57BL/6 mice were sensitized (Days 1 and 14) and challenged (Days 24-26) with ovalbumin. Crry-Ig was administered after allergen sensitization either as an intraperitoneal injection or by nebulization before allergen challenge. Crry-Ig significantly prevented the development of airway hyperresponsiveness, decreased airway and lung eosinophilia as well as the numbers of lung lymphocytes, decreased levels of interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and decreased serum ovalbumin-specific IgE and IgG1. These results suggest that prevention of complement activation may have a therapeutic role in the treatment of allergic airway inflammation and asthma in sensitized individuals.
Animals↗
Complement activation is critical to airway hyperresponsiveness after acute ozone exposure.
Ozone (O3) can induce airway hyperresponsiveness (AHR) and neutrophilic inflammation. We evaluated the role of complement in development of AHR and inflammation after acute O3 exposure in mice. Mice were exposed to O3 at 2 ppm for 3 hours, and airway responsiveness to methacholine was measured 8 hours after O3 exposure. Complement was depleted or inhibited by intraperitoneal injection of cobra venom factor (CVF) or complement receptor-related gene y (Crry)-Ig, a potent C3 convertase inhibitor; neutrophils were depleted using an antineutrophil monoclonal antibody. CVF attenuated the development of AHR by O3. Administration of Crry-Ig also prevented the development of AHR. Bronchoalveolar lavage (BAL) fluid neutrophilia after O3 exposure was significantly decreased by administration of either CVF or Crry-Ig. Increased BAL fluid total protein after O3 exposure was lowered by depletion or inhibition of complement. In contrast to the effects of complement inhibition or depletion, depletion of BAL neutrophil counts by more than 90% with the monoclonal antibody did not affect the development of AHR after O3 exposure. These data indicated that activation of the complement system follows acute O3 exposure and is important to the development of AHR and airway neutrophilia. However, this neutrophil response does not appear necessary for the development of AHR.
Animals↗
The role of the type 3 complement receptor in the induced recruitment of myelomonocytic cells to inflammatory sites in the mouse.
The type 3 complement receptor (CR3), initially identified as the leukocyte cell surface receptor for iC3b, is now known to form part of the extended integrin family of cell adhesion molecules that mediate both cell-cell and cell-extracellular matrix interactions. The identification of a heritable deficiency of human leukocyte adhesion together with the advent of monoclonal antibodies has shed some light on the central role of CR3 in the transendothelial migration of macrophages and neutrophils to sites of inflammation. We review the general structural features of CR3 and then examine our understanding of its role in both nonspecific and T cell-dependent inflammatory processes based on our murine in vivo experiments. CR3-dependent inflammation seems to contribute to the pulmonary response to some stimuli (lipopolysaccharide) but not to others (bacillus Calmette-Guerin). These studies highlight the potential therapeutic benefits, as well as the significant risks of potentiating acute bacterial infections, of CR3 blockade in vivo.
Animals↗
Genetic regulation of a structural polymorphism of human C3b receptor.
Two forms of the human C3b receptor (C3bR), which have relative molecular weights (Mr) of 250,000 and 260,000 and are designated F and S, respectively, have been identified in specific immunoprecipitates from erythrocytes and leukocytes by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both forms of the receptor were visualized on gels by autoradiography of 125I-labeled antigen and by silver nitrate staining. Individual donors expressed one of three possible patterns of C3bR, either the F or S form alone or both, and these patterns represented stable phenotypic characteristics of their erythrocytes and polymorphonuclear and mononuclear leukocytes. Removal of N-linked oligosaccharides by endoglycosidase-F treatment decreased the Mr of both forms but did not abolish the difference in their electrophoretic mobilities. That both forms of the receptor were functional was indicated by the capacity of all antigenic C3bR sites on erythrocytes from individuals having any of the three phenotypes to bind dimeric C3b with affinities ranging from 3 to 5 X 10(7) M-1. Analyses of the occurrence of the F and S forms of C3bR in 76 individuals from 15 families revealed that this polymorphism was regulated by two alleles transmitted in an autosomal codominant manner. Of 111 normal unrelated individuals, 64.9% were homozygous for the F form (FF), 1.8% were homozygous for the S form (SS), and 33.3% were heterozygotes (FS). This distribution did not differ from that calculated by the Hardy-Weinberg equilibrium based on two codominant alleles that regulate the expression of the F and S forms and that have frequencies of 81.5 and 18.5%, respectively. The locus regulating structural polymorphism of C3bR is designated C3BRM (M for mobility or Mr), and is distinct from the recently described locus regulating the quantitative expression of C3bR on erythrocytes.
Alleles↗
Abnormality of glycophorin-alpha on paroxysmal nocturnal hemoglobinuria erythrocytes.
To investigate the greater enzymatic activity of the alternative pathway convertase (and the subsequent greater fixation of C3b) on paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, we have examined the topography of binding of C3b to PNH and normal erythrocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the alpha-chain of C3b was found to bind via predominantly ester bonds to free hydroxyl groups on glycophorin-alpha, the major erythrocyte sialoglycoprotein. The pattern of binding of nascent C3b was the same for normal and PNH erythrocytes. Thus, although C3b binding to a different membrane constituent did not appear to account for the greater enzymatic activity of the alternative pathway convertase when affixed to PNH erythrocytes, it seemed possible that the glycoproteins to which C3b bound might be qualitatively abnormal on the PNH cells, and that structural differences in these molecules might impose modifications in the enzyme-substrate interactions of the alternative pathway convertase. Using methods for radiolabeling both protein and carbohydrate residues, we therefore compared the electrophoretic pattern of the cell-surface glycoproteins on PNH and normal erythrocytes. The glycophorin-alpha dimer was found to be qualitatively abnormal on the PNH cells as evidenced by its greater susceptibility to trypsin-mediated proteolysis. In addition, the abnormal erythrocytes from patients with PNH had fewer periodate oxidizable constituents than did normal erythrocytes, indicating a relative deficiency of cell-surface sialic acid. These investigations suggest that abnormalities in membrane glycoproteins may underlie the aberrant interactions of complement with the hematopoietic elements of PNH.
Complement C3-C5 Convertases↗
Increased efficiency of binding of nascent C3b to the erythrocytes of chronic cold agglutinin disease.
The pathogenesis of chronic cold agglutinin disease (CCAD) has been enigmatic. To determine if abnormal erythrocyte membrane constituents might provide the stimulus for antibody production, we compared the electrophoretic pattern of radiolabeled membrane glycoproteins of four patients with CCAD to that of normal control erythrocytes. For the CCAD erythrocytes, fluorographs revealed the appearance of an abnormal band whose molecular weight was estimated at 126,000 D. Using two-dimensional gel analysis and immunoblotting techniques, it was determined that the 126,000 D glycoprotein consisted predominately of polymeric glycophorin-alpha. Previous investigations had suggested that abnormalities in glycophorin-alpha influence the functional activity of the complement system. When purified complement (C)3 was activated in the fluid-phase by cobra venom factor complexes, CCAD erythrocytes bound nascent C3b 7-27 times more efficiently than normal erythrocytes. Normal erythrocytes could be induced to manifest the appearance of the 126,000 D band, and the increased efficiency of binding of nascent C3b by incubation with CCAD serum or with the purified cold agglutinin antibody plus autologous serum, but not with the purified antibody alone or the purified antibody plus EDTA-chelated autologous serum. These studies demonstrate that the interactions of IgM cold-reacting antibody and complement with glycophorin induce changes in the biophysical properties of the erythrocyte membrane which modify subsequent interactions with complement.
Anemia, Hemolytic, Autoimmune↗