Third component of human complement: C3.
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Avidin↗
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Complement receptor enhancement by chemotactic factors.
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Chemotactic Factors↗
Zymosan-induced leukotriene B4 generation by human neutrophils is augmented by rhTNF-alpha but not chemotactic peptide.
Tumour necrosis factor (TNF) is a mediator of inflammation that has been shown to enhance neutrophil responses to soluble and particulate stimuli. The release of leukotriene B4 (LTB4) by human neutrophils stimulated by unopsonized zymosan was measured in the presence or absence of recombinant human TNF-alpha (rhTNF-alpha) preincubation. There was a threefold increase in the LTB4 response at an optimal TNF concentration of 10(-9) M and an optimal preincubation time of 10-20 min. A similar time and dose dependency was observed for CR3 receptor expression and for the release of the secondary lysosomal granule marker, vitamin B12-binding protein. In contrast, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), although stimulating an increase in both CR3 receptor number and in particle phagocytosis, failed to induce an increase in LTB4 release in response to zymosan. In addition, the present study demonstrated that, unlike FMLP, the exocytotic mechanism for secondary granule release by rhTNF-alpha functioned in the absence of a rise in cytosolic free calcium. Furthermore, it was independent of changes in cyclic nucleotide concentrations and did not require an intact cytoskeleton. Thus the capacity of rhTNF-alpha to amplify the neutrophil response to zymosan through the CR3 receptor appears to be related to the amplification of post-membrane events as well as to an increase in the number of functionally active receptors.
Antigens, CD↗
An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors.
CD11b/CD18 is a heterodimeric leukocyte surface receptor which functions in both C3bi-ligand binding and homotypic and heterotypic cell adherence. We have examined the effect of several anti-CD11b/18 mAb on phagocytosis of IgG (EIgG) or complement (EC4b) opsonized erythrocytes by polymorphonuclear leukocytes (PMN) and monocytes. F(ab')2 of two mAb (IB4, an anti-beta-chain mAb and Mo-1 an anti-alpha-chain mAb), inhibited both phagocytosis of EIgG and phorbol ester-stimulated phagocytosis of EC4b by PMN and monocytes. These F(ab')2 inhibited the binding of EIgG to monocytes, but they had no effect on binding of EIgG to PMN, or EC4b to either phagocyte. In addition, IB4 inhibited phorbol-ester stimulated phagocytosis of sheep E opsonized with C component 3bi (EC3bi) without inhibiting rosetting of these same targets. These data separate the anti-phagocytic effect of these mAb from effects on phagocyte-target adherence. When PMN were adherent to an anti-CD11b/CD18 F(ab')2-coated surface, EC3bi binding was abolished, but phagocytosis of EIgG or EC4b was unaffected. Subsequent addition of fluid- phase IB4 or Mo-1 F(ab')2 inhibited phagocytosis of EIgG or EC4b by the adherent cells. This suggested that the CD11b/CD18 involved in C3bi rosetting were mobile in the membrane, whereas those involved in phagocytosis of EIgG or EC4b were not. Cytochalasin treatment of PMN during adherence to F(ab')2-coated plates decreased both apical expression of CD11b/18 and subsequent ingestion of EIgG by 70%, suggesting that microfilaments are important in maintaining immobile CD11b/18 on the apical PMN surface. We conclude that there are functionally distinct populations of CD11b/CD18 on monocytes and PMN: one involved in C3bi rosetting and another involved in the process of phagocytosis mediated via several different receptors. CD11b/18 is not required for optimal target binding in all cases, but is always required for ingestion. As with several other integrins, the CD11b/18 molecules involved in phagocytosis have a functional association with the cell cytoskeleton.
Antibodies, Monoclonal↗
Characteristics of a novel low affinity complement C3dg-binding protein of human platelets.
The breakdown products of the complement protein C3 function in receptor-mediated immune clearance. The catabolism of the C3 molecule, mediated by factors I and H, results in the generation of the fragments C3c and C3dg. C3dg binds to human platelets in a specific and saturable manner. The direct interaction of platelets with soluble C3dg may contribute to immune-mediated platelet destruction. More importantly, platelets may interact with opsonized pathogens or complement-activating immune complexes via C3dg. In this report, we investigated the interaction of C3dg with platelets and calculated the Ka to be 3.2 x 10(6) M-1 with 1100 to 2200 specific binding sites/platelet. In the presence of 5 mM calcium, both the Ka and the number of specific binding sites were modestly decreased to Ka = 2.8 x 10(6) M-1 with 1400 to 2400 specific binding sites/platelet. The Scatchard plots demonstrated a curvilinear character. On labeling C3dg with peroxidase and visualizing platelet-bound C3dg by electron microscopy, it was shown that binding sites for C3dg were restricted to the platelet plasma membrane. Using a cell attachment assay, platelet adhesion to C3dg was readily detectable; attachment to C3dg-coated plates was not blocked by fibrinogen or fibronectin. We have characterized the C3dg-binding protein of platelets using the chemical cross-linkers, SASD and DSS, to cross-link C3dg to thrombin-stimulated platelets. Gel filtration of the 125I-labeled C3dg-platelet complex revealed the presence of a large protein complex that was absent when 125I-labeled C3dg alone was analyzed. SDS-PAGE of the radiolabeled cross-linked protein, followed by autoradiography, identified a 95-kDa membrane protein. The relationship of this C3dg-binding protein to other platelet membrane proteins has yet to be determined.
Blood Platelets↗
Classical complement pathway activation on nucleated cells. Role of factor H in the control of deposited C3b.
The restriction of alternative complement pathway activation in fluid phase or on nonactivator surfaces has been described as the major physiologic function of the complement regulatory protein factor H. In this study, we provide evidence that factor H is also a restriction factor of classical pathway activation on the surface of nucleated cells. We found that C3b was rapidly converted to inactivated C3b (iC3b) on human SK-MEL-93-2 melanoma cells after classical pathway activation with the murine monoclonal IgG3 Ab R24 directed against the disialoganglioside surface Ag GD3. The SK-MEL-93-2 cells are nonactivators of the alternative pathway and express neither CR1 (CD35) nor the C3b-cleaving protease p65. The cells are further characterized by the expression of only moderate amounts of DAF (CD55) and approximately 5 x 10(3) MCP (CD46) molecules/cell. FACS analysis and direct quantitation using [125I]factor H revealed high level binding of factor H to the melanoma cells (5.6 x 10(6) molecules/cell) during classical pathway activation. The binding of factor H could be inhibited under conditions that inactivate the classical complement pathway (EGTA and heat treatment), but not by factor B depletion of the serum, demonstrating that classical pathway activation was responsible for factor H binding. Treatment of factor B-depleted serum with neutralizing concentrations of polyclonal anti-factor H resulted in the prolonged presence of intact C3b on the cells and a significantly reduced generation of iC3b. The increased amount of C3b on these cells correlated with a 2.65-fold greater rate of cell death. In contrast, the increase in cell death effected by neutralizing concentrations of anti-CD46 or anti-CD55 Ab was only 0.13- or 0.35-fold, respectively. In addition, the supplementation of serum with purified factor H decreased the extent of lysis of the cells. Collectively, these data provide experimental evidence that factor H, through its cofactor activity for C3b degradation, is involved in the restriction of the classical pathway of complement on the surface of nucleated cells, a function that to date has been exclusively attributed to the membrane regulatory proteins CD35 and CD46.
Complement Activation↗
Ligand binding determines whether CD46 is internalized by clathrin-coated pits or macropinocytosis.
CD46 is a ubiquitous human cell surface receptor for the complement components C3b and C4b and for various pathogens, including the measles virus and human herpes virus 6. Ligand binding to CD46 affects (i) protection of autologous cells from complement attack by breakdown of complement components, (ii) intracellular signals that affect the regulation of immune cell function, (iii) antigen presentation, and (iv) down-regulation of cell surface CD46. Recent evidence indicates that CD46 signaling can link innate and acquired immune function. The molecular mechanisms for these processes and the importance of intracellular trafficking of the receptor have not yet been elucidated. We demonstrate here that, in nonlymphoid cells, CD46 is constitutively internalized via clathrin-coated pits, traffics to multivesicular bodies, and is recycled to the cell surface. However, cross-linking of CD46 at the cell surface, by either multivalent antibody or by measles virus, induces pseudopodia that engulf the ligand in a process similar to macropinocytosis, and leads to the degradation of cell surface CD46. Thus, we have elucidated two pathways for CD46 internalization, which are regulated by the valence of cross-linking of CD46 and which utilize either clathrin-coated pits or pseudopodial extension. This has important implications for CD46 signaling, antigen presentation, CD46 down-regulation, and engulfment of pathogens.
Antibodies↗
Demonstration in human plasma of a form of C3 that has the conformation of "C3b-like C3".
Disruption of the thioester in native C3 yields a C3 molecule that functionally resembles C3b. It has been proposed that this C3 molecule (iC3) plays a key role in initiation of the alternative pathway of the C system. However, its presence in plasma has never been demonstrated. We investigated the presence of iC3 in plasma, using mAb that recognize iC3 as well as C3 activation products but not native C3. One of these mAb, anti-C3-5, which binds iC3 via its C3a moiety, was used together with polyclonal 125I-anti-C3c to develop a RIA for iC3. Plasma incubated with methylamine yielded a strong response in this RIA, whereas neither fresh plasma nor serum in which the C system had been activated by incubation with aggregated IgG, did show this strong response. The specificity of this RIA was further demonstrated by additional experiments including experiments with purified preparations of the various forms of C3. Mean level of iC3 in freshly obtained plasma samples from 10 normal donors was 27 nmol/liter, which is 0.49% of total C3. Analysis by SDS-PAGE of C3 species that had been immunoprecipitated by mAb antiC3-5, revealed that some iC3 consisted of C3 molecules with an intact alpha-chain whereas another part consisted of iC3 molecules with an alpha-chain that had been cleaved by factor I. Thus, this study shows that fresh human plasma contains a C3 species with the conformation of "C3b-like C3" (iC3).
Antibodies, Monoclonal↗
p65: A C3b-binding protein on murine cells that shares antigenic determinants with the human C3b receptor (CR1) and is distinct from murine C3b receptor.
Affinity-purified rabbit antibody to the human C3b receptor (CR1) cross-reacted with an antigen that was expressed on murine splenocytes, lymph node cells, thymocytes, peritoneal macrophages, erythrocytes, and L929 cells, as assessed by flow cytofluorographic analysis of indirectly stained cells. The cell surface antigen recognized by antihuman CR1 had a Mr of 60,000 to 65,000 on each cell type, and the cross-reactive epitope(s) was sensitive to reduction with dithiothreitol but not to oxidation with NaIO4. Analysis by affinity chromatography of murine splenic B lymphocytes identified two cell surface proteins capable of binding to guinea pig C3b (C3bgp). The larger polypeptide had a Mr of 210,000, was not present in L929 cells, and may represent the murine CR1. The smaller polypeptide of 65,000 Mr was also present on L929 cells and was shown to constitute the cross-reactive antigen. Adsorption of detergent lysates of L929 cells with C3bgp-Sepharose depleted by 44% the antigen recognized by anti-human CR1; the C3bgp-binding protein and the cross-reactive antigen exhibited similar patterns on two-dimensional gel electrophoresis; and the isolated C3b-binding protein could be immunoprecipitated with anti-human CR1. Thus, the murine cell surface protein, termed p65, that is antigenically cross-reactive with human CR1 shares a capacity for binding to C3b in its detergent-solubilized form but is distinct from murine CR1 in its lower Mr, wider cellular distribution, and inability to mediate the adherence of C3b-coated particles in its native, membrane-associated form.
Animals↗
Induction of C3b-mediated phagocytosis in macrophages by distinct populations of lipopolysaccharide-stimulated lymphocytes.
Unelicited resident peritoneal macrophages do not significantly ingest erythrocytes coated with C3b. However, these resident macrophages can be induced to ingest via the C3b receptor when cocultured with peritoneal or splenic nonadherent cells obtained from mice previously injected with lipopolysaccharide. In this study, the extent of ingestion induced in resident macrophages was dependent on the number of stimulated nonadherent cells cocultured with the macrophages as well as on the amount of lipopolysaccharide injected in the mice from which the nonadherent cells were obtained. The ability of the stimulated nonadherent cells to convert resident macrophages to a state of C3b receptor-mediated ingestion was not abrogated by the inclusion of polymyxin B in the cocultivation medium. To further characterize these nonadherent cells, different lipopolysaccharide-stimulated cells were obtained by either nylon-wool filtration, depletion of C3b receptor-bearing cells, or depletion of Thy 1.2-positive cells. None of these populations by themselves were capable of inducing resident macrophages to ingest via the C3b receptor, whereas unfractionated cells were. However, coculture of resident macrophages with recombinations of splenic nylon-wool effluent (T cell-enriched) or bound (B cell-enriched) nonadherent cells from lipopolysaccharide-injected mice reconstituted the ability to induce ingestion via the C3b receptor. Taken together, these results suggest that one means by which lipopolysaccharide can induce C3b receptor-mediated ingestion by macrophages is through the cooperative effects of stimulated T and B lymphocytes.
Animals↗
[Activity of the red-cell C3b receptor and the circulating immuno-complex in patients of herpes simplex keratitis].
The activity of red-cell C3b receptor and the level of circulating immuno-complex in 35 cases of herpes simplex stromal keratitis were determined, with the results that during the stage of acute attack, the former was significantly lowered, while the level of the latter became markedly higher than that of the controls, and after treatment, the indicators returned to normal values. There was a negative correlation between the red-cell C3b receptor activity and the circulating immuno-complex in serum, indicating that the immuno-function of red-cells in patients with herpes simplex stromal keratitis was impaired and the depression of red-cell C3b receptor activity was one of the reasons for the elevated concentration of circulating immuno-complex in serum.
Adolescent↗
Differential mechanisms of microparticle transfer toB cells and monocytes: anti-inflammatory propertiesof microparticles.
Microparticles are small vesicles released from the plasma membrane of various cell types independently of apoptosis or cell death, are transferred between cells, and carry membrane proteins from one cell to another. We have studied the mechanism of uptake of microparticles by monocytes and B cells. The transfer of microparticles to B cells was almost completely dependent on complement. Incubation of microparticles with serum resulted in opsonization of microparticles with the complement cleavage product iC3b. The subsequent transfer to B cells was mediated by the complement receptor CR2. The interaction between iC3b-opsonized microparticles and B cells reduced the activation of B cells as measured by expression of MHC class II, CD86 and CD25. In contrast, transfer of microparticles to monocytes was only partially complement dependent, but involved calcium and annexin V, and was found to change the cytokine profile of monocytes towards a reduced release of the pro-inflammatory cytokines GM-CSF and TNF-alpha and an increased release of the anti-inflammatory cytokine IL-10. These data show that microparticles are taken up by B cells and monocytes by different mechanisms and modulate the activation of monocytes and B cells towards an anti-inflammatory phenotype. Microparticles might be involved in counterbalancing pro-inflammatory signals arising from tissue injury or inflammation.
Annexin A5↗
The influence of complement receptors on the localization of lymphocytes in vivo.
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Animals↗
Restoration of complement function in vivo by plasma infusion in factor I (C3b inactivator) deficiency.
The serum complement activities of a 1-year-old infant with recurrent life-threatening bacterial infections and persistent C3-Coombs-positive red blood cells were investigated. The patient's serum had a depressed serum level of CH50, C3, factor B, and factor H as well as undetectable antigenic or functional factor I. The complement profile of the parents was normal, with the exception of factor I, which was approximately 50% of normal in each parent. The Coombs positivity of the patient's red blood cells could be reversed in vitro by incubation with normal serum containing factor I. Infusion of normal plasma into the patient resulted in increased levels of CH50, with concomitant increases in serum C3, factor B, and factor H levels. The patient's red blood cells became transiently Coombs negative. At no time after plasma infusion was factor I detectable in the patient's serum. All complement functions and the C3 Coombs reactivity of the patient's red blood cells returned to preinfusion levels within 14 days. These findings are consistent with an inherited deficiency of factor I and emphasize the critical role this protein plays in the regulation of the alternative complement pathway. Plasma therapy may be an adjunct to the management of acute infection in patients with factor I deficiency.
Afibrinogenemia↗
Structure-function analysis of the active sites of complement receptor type 1.
Two functionally distinct but homologous sites in complement receptor type 1 (CR1) (CD35) were further characterized by homologous substitution mutagenesis of two CR1 derivatives, each containing one site. In both sites, reducing negative and/or increasing positive charge augmented interaction with iC3/C3b and C4b, supporting a role of ionic forces in the binding reaction. In one case, substitution of Asp at the end of complement control protein repeat (CCP) 2 with an Asn transformed the protein, with negligible cofactor activity and iC3 binding, into a mutant with activities similar to native CR1. Consequently, this protein, one-fourth the size of CR1, is a therapeutic candidate for a complement inhibitor. Another important observation is that the residues between two CCPs contribute to activity, probably because they influence positioning of one CCP relative to the next. The initial characterization of the third CCP of an active site led to identification of three peptides necessary for binding. In line with earlier findings for the first two CCPs, interactions with iC3/C3b are similar but not identical to those with C4b, implying overlapping but distinct binding domains. Moreover, changes in cofactor activity usually, but not always, parallel alterations in binding, indicating that these two activities are separable. We also mapped epitopes for a blocking and a function enhancing monoclonal antibody. Their effects can be explained by epitope location. The first antibody binds near functionally important residues. The second may shield inhibitory (negatively charged) residues. These results represent a comprehensive analysis of the active sites of CR1, which is built of modules found in more than 50 mammalian proteins.
Amino Acid Sequence↗
Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes.
We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co-localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.
Antibodies↗
The murine complement receptor gene family. Analysis of mCRY gene products and their homology to human CR1.
The mouse genome contains two sets of gene sequences which are highly homologous to the gene encoding the human C3b/C4b receptor (CR1). These genes, termed murine CRY (mCRY) and murine CRX (mCRX) reside on murine chromosomes 1 and 8, respectively. Analysis of cDNA isolated by using these sequences as probes indicates that there are two related but distinct mRNA which are expressed in a wide variety of murine tissues including spleen, liver, lung, and brain. Both of these transcripts encode proteins which should contain a signal sequence for membrane insertion, a transmembrane/cytoplasmic tail region for membrane anchoring, and five extracellular domains made up of 60 amino acid consensus repeat sequences. The difference between the two is the presence of an additional exon of 129 bp immediately 3' of the signal sequence. This additional exon does not encode a 60 amino acid repeat. The sizes of the mature proteins predicted from the cDNA sequences are 43,998 Mr and 48,680 Mr; however, antisera raised against carboxy-terminal sequences detects a 70,000 Mr protein from murine fibroblasts suggesting a high degree of post-translational modification of the mature protein. A comparison of these murine gene sequences with a partial human CR1 sequence suggests that the human CR1 gene evolved by direct duplication of the ancestral coding sequences contained within these murine genes including those sequences important for membrane anchoring and cytoplasmic protein attachment.
Amino Acid Sequence↗
[Expression of the surface membrane receptors of neutrophils in myeloproliferative diseases].
For 147 patients with myeloproliferative diseases, a study was made of the expression of Fc-receptors to immunoglobulins IgC, IgA, and that of FcH-receptor and receptors to C3-components of the complement in peripheral blood neutrophils. The data obtained show the lower level of neutrophils having membrane receptors in patients with acute myeloblastic leukemia and chronic granulocytic leukemia at the stage of blast transformation. A decrease in expression of membrane receptors of neutrophils was shown in patients with chronic granulocytic leukemia and benign subleukemic myelosis. The finding of a higher level of neutrophils having surface receptors revealed in patients with real polycythemia is close to the data obtained in the study of expression of membrane receptors in patients with chronic myeloproliferative diseases and healthy persons.
Adolescent↗