Complement ligand-receptor interactions that mediate biological responses.
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Animals↗
Biomedical subjects
D T Fearon
Publications and source records attributed to D T Fearon.
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↗
Endocytosis of the C3b receptor of complement within coated pits in human polymorphonuclear leukocytes and monocytes.
The distribution and endocytosis of the C3b receptor by human polymorphonuclear leukocytes and monocytes were visualized by both fluorescent and electron microscopic examination of cells that had been labeled with monospecific F(ab')2 anti-C3b receptor and anti-F(ab')2 conjugated with rhodamine or ferritin. When prefixed or unfixed cells that were labeled at 0 to 4 degrees C were examined, the receptor was distributed within clusters on the plasma membrane. After the cells had been warmed to room temperature or to 37 degrees C for 5 minutes, the fluorescently labeled receptors appeared to enter the cells, and the ferritin-tagged receptors often occurred within coated endocytic pits and coated vesicles within the cytoplasm. After incubation at 37 degrees C for 20 minutes, the C3b receptor-antibody complexes were largely cleared from the cell surface, and much of the label was found within lysosomes. These results indicate that C3b receptors may directly mediate endocytosis within coated pits, thus utilizing a mechanism shared by a variety of other receptors for the rapid, efficient, and selective internalization of extracellular ligands.
Cell Membrane↗
Expression of C3b receptors on human be cells and myelomonocytic cells but not natural killer cells.
We have examined natural killer (NK) cells, B cells and myelomonocytic cells at different stages of differentiation for the expression of surface C3b receptors (C3bR). Receptor presence was detected using affinity-purified F(ab')2 anti-C3bR antibodies in indirect immunofluorescence assays. NK cells, identified in fetal and adult tissues by the monoclonal antibody HNK-1, were C3bR- except for infrequent C3bR+ HNK-1+ cells in some blood samples. NK cells were not induced to express C3bR by exposure to interferon, target cells, or phorbol myristate acetate. B cells gradually acquired the ability to express C3bR with maturity: 15% of large pre-B cells were C3bR+, 35 to 48% of small pre-B, 60 to 80% of immature B, and 99% of mature B cells. Mature plasma cells were rarely C3bR+. Myelomonocytic cells acquire C3bR relatively late during their development, with neutrophils beginning to express C3bR during the band stage of differentiation. All adult blood myelomonocytic lineage cells, identified by the monoclonal antibody MMA and by morphology, were C3bR+.
Animals↗
Characterization of human T lymphocytes that express the C3b receptor.
The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.
Animals↗
Generation of C3a anaphylatoxin from human C3 by human mast cell tryptase.
Tryptase, the dominant neutral protease of human pulmonary mast cell secretory granules, has the capacity in vitro to generate C3a anaphylatoxin from purified human C3. Only the alpha-chain of C3 is cleaved, and major fragments with apparent m.w. of 105,000, 39,500, 34,000, 29,000, and 9000 are detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing conditions. Fragments of 34,000 and 9000 m.w. are detected without reduction. A portion of the 9000 m.w. protein corresponds to C3a by virtue of its co-migration in SDS polyacrylamide gels with purified C3a and with trypsin-generated C3a, by its detection in a radioimmunoassay for C3a, and by its contractile activity on the guinea pig ileum bioassay. In the presence of heparin, another component of the mast cell secretory granule, the rate of appearance and the distribution of C3 cleavage fragments as assessed in SDS polyacrylamide gels are not appreciably changed with the exception that no C3a material can be detected in the SDS polyacrylamide gels or by radioimmunoassay and bioassay of the unresolved reaction mixture. Enhanced catabolism of authentic C3a by tryptase occurs in the presence of heparin and by analogy when C3a is generated from C3 by tryptase in the presence of heparin. Whereas tryptase secreted by activated human mast cells may generate C3a, a potentially important additional mediator of immediate hypersensitivity events, the concomitant release of heparin may serve to down-regulate C3a irrespective of its mechanism of generation.
Anaphylatoxins↗
Increased expression of C3b receptors on polymorphonuclear leukocytes induced by chemotactic factors and by purification procedures.
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Chemotactic Factors↗
Mode of inheritance of decreased C3b receptors on erythrocytes of patients with systemic lupus erythematosus.
The erythrocytes of patients with systemic lupus erythematosus have been shown to have a decreased number of receptors for the major cleavage fragment of the third component of complement (C3b). We studied the basis for this decreased number of C3b receptors by measuring the uptake of anti-C3b-receptor antibody on cells from 113 normal subjects, 38 patients with systemic lupus erythematosus, 14 of their spouses, and 47 of their relatives. The normal subjects had 5014 +/- (mean +/- S.E.M.) receptor sites per cell, but the patients and their relatives had significantly fewer sites--2809 +/- 241 and 3167 +/- 196, respectively (P less than 0.0005). The number of sites in the patients' spouses did not differ from normal (P greater than 0.3). Three phenotypes, indicated by the numbers of receptors, occurred in the normal population; a high phenotype (HH, with 5500 to 8500 sites per cell), an intermediate phenotype (HL, with 3000 to 5499), and a low phenotype (LL, less than 3000). These three phenotypes were present in 34, 54, and 12 percent, respectively, of the normal subjects; in contrast, 5, 42, and 53 per cent of patients had these respective phenotypes. Pedigree analyses indicated that these phenotypes were inherited in an autosomal codominant manner. We conclude that the decreased number of C3b receptors in lupus is inherited, not acquired.
Adult↗
Lipid monolayer-coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils.
We have used epifluorescence and photobleaching techniques to study the lateral distribution and motion of fluorescein-conjugated Fab fragments of anti-C3b receptor antibody bound to human neutrophils when the cells rest on various solid supports (microscope slides or cover slips). Supports composed of quartz, glass, or alkylated glass induced cellular adhesion, spreading, and an extensive lateral redistribution of C3b receptors (but not HLA antigens). The neutrophil C3b receptors become patchy, and the patches apparently undergo nonrandom translational motion. Many patches are found on the upper surfaces of the cells removed from the region of cell membrane-glass contact. In contrast, neutrophils supported by lipid monolayer-coated glass do not adhere or spread, and the C3b receptor remains uniform and diffuses freely (D approximately equal to 2 X 10(-10) cm2/s).
Antibodies, Monoclonal↗
Immunohistochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal diseases: loss of C3b receptor antigen in focal hyalinosis and in proliferative nephritis of systemic lupus erythematosus.
The presence and distribution of C3b receptors in normal human kidneys and in biopsies from 75 patients with renal disease were investigated by immunohistochemical techniques using monospecific rabbit antibody to the 205,000-mol wt glycoprotein that is the C3b receptor of human peripheral blood cells. Anti-C3b receptor bound exclusively to podocytes in normal renal cortex, and was homogeneously distributed on the plasma membrane of these cells. Biosynthesis of the receptor by the podocyte was suggested by the presence of antigenic activity in the Golgi apparatus. Although occupancy of receptor sites following the interaction of kidney sections with aggregated IgG preincubated with normal serum inhibited binding to glomeruli of C3b coated cells, the C3b receptor remained accessible to anti-C3b receptor antibody. No staining of podocytes was found in extra-capillary proliferating cells in rapidly progressive glomerulonephritis (GN). Segmental loss of staining was found in focal hyalinosis, nodular diabetic glomerulosclerosis, and amyloidosis while no detectable C3b receptor antigen was found in severe proliferative nephritis of systemic lupus erythematosus (SLE). Normal staining of podocytes was found in other nephropathies with endocapillary proliferation such as acute GN and mesangial GN and in renal diseases associated with immune deposits containing C3 such as mesangial proliferative and membranous SLE nephritis, idiopathic membranous GN, membranoproliferative GN types I and II, mesangial GN with IgA or C3 deposition and Henoch Schönlein's purpura. Loss of C3b receptor antigen in the diffuse proliferative nephritis of SLE distinguishes it both from nonproliferative lupus nephritis and other immunologically mediated proliferative GN.
Complement C3↗
Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system.
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Animals↗
Antibody restores human alternative complement pathway activation by mouse erythrocytes rendered functionally deficient by pretreatment with pronase.
Activation of the human alternative pathway of complement (C) by surfaces requires the initial deposition of C3b by fluid-phase C3 convertase and sustained C3 cleavage by C3 convertases fixed to the surface. Nonactivating particles have previously been characterized by an inability to sustain the function of C3 convertases on their surfaces because these sites were susceptible to the regulatory action of the control proteins. Pronase converts the mouse erythrocyte (E) from an activator to a nonactivator by markedly decreasing the ability of the cell to affix C3b generated by a fluid-phase C3 convertase; this conversion is unrelated to the action of the control proteins on bound C3b. The capacity of antibody and its F(ab')2 or Fab' fragments to restore the alternative pathway-activating function of pronase-treated mouse E indicates that the contribution of antibody is mediated by its combining site without a requirement for bridging or for the Fc portion. The fab'-dependent activation of the alternative pathway of C relates to the deposition of C3b on the particle surface, which is a first and continuing step in alternative pathway activation. The antibody effect is not directed to the action of the regulatory proteins. Thus, particle-dependent activation of the alternative C pathway has been shown for the first time to be abolished and restored by cell surface-directed mechanisms that function independently of the regulatory proteins.
Animals↗
Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes.
C3b receptors on human polymorphonuclear leukocytes (PMN) were nonrandomly distributed in small clusters on the plasma membranes of these cells when assessed by indirect immunofluorescence at 0 degree C using monospecific rabbit Fab' or F(ab')2 anti-C3b receptor and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat IgG anti-F(ab')2. When PMN were incubated with the bivalent anti-C3b receptor at 37 rather than at 0 degree C, almost no immunofluorescence was observed, which indicates that the C3b receptor-F(ab')2 complexes had been rendered inaccessible to TRITC-IgG anti-F(ab')2. Endocytosis of the anti-C3b receptor ligand was quantitated by measuring the binding 131I-IgG anti-F(ab')2 by PMN that had previously taken up 125I-F(ab')2 anti-C3b receptor at 0 and at 37 degree C, respectively. There was a constant 2: 1 molar ratio of anti-F(ab')2 to anti-C3b receptor with PMN that had been incubated with the first antibody at 0 degree C. In contrast, when increments of F(ab')2 anti-C3b receptor were taken up by the cells at 37 degree C, there was a dose-related decline in this molar ratio to a minimum of 0.2 molecules of anti-F(ab')2 anti-F(ab')2 bound per molecule of PMN-associated anti-C3b receptor. 125I-F(ab')2 anti-C3b receptor taken up by PMN at 37 degree C was also inaccessible to release by proteolytic treatment of the cells with pronase. The rate of endocytosis of 125I-F(ab')2 anti-C3b receptor was rapid as the PMN-bound antibody fragment became inaccessible to 131I-IgG anti-F(ab')2 within 10 min during incubation of the cells at 37 degree C. In contrast to these findings, 125I-Fab' anti-C3b receptor that was taken up by PMN at 37 degree C remained accessible to both 131I-IgG anti-F(ab')2 and to proteolytic release by pronase, which suggests that monovalent interaction of ligand with C3b receptors was not sufficient for induction of endocytosis. The requirement for multivalency was also demonstrated using the C3b-OR, the normal ligand for the C3b receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell surface, as determined by its accessibility to pronase, unless it was cross-linked with F(ab')2 anti-C3. Although C3b receptors on PMN do not mediate internalization of adsorptive pinocytosis of soluble ligand indicates their potential for the clearance of C3b-bearing immune complexes without recruitment of other cell surface receptors.
Antigen-Antibody Complex↗
Clinical and biochemical effects of stanozolol therapy for hereditary angioedema.
Stanozolol, an inexpensive anabolic steroid with a 30:1 anabolic:androgenic ratio, was administered to 12 male and 15 female patients with biochemically proven hereditary angioedema over a 2-yr period to obtain a systematic assessment of the relationship between drug dosage and clinical response, incidence of side effects, and amelioration of complement abnormalities. All 27 patients attained the minimal effective dose, ranging from 0.5 to 2 mg daily, which controlled the frequency and intensity of symptoms with minimal side effects. At daily maintenance doses of 2, 1, and 0.5 mg the frequencies of attacks per weeks of therapy were 1/14.6, 1/7.2, and 1/8.2 wk, respectively. Side effects with maintenance therapy included menstrual abnormalities and virilization in four females and elevation of serum creatinine phosphokinase (CPK) in five males. In six patients on maintenance doses of stanozolol, serum levels of testosterone, free thyroxin (T4), and thyroxin binding globulin (TBG) (four males), and of estradiol, progesterone, T4, and TBG (two females) were normal. Slightly low serum levels of progesterone and TBG were found in two females who had normal menstrual cycles. Statistically significant elevations above pretherapy levels of serum inhibitor to the activated first component of complement function and C4 protein and function occurred when patients were on maintenance therapy, but these measurements remained below the lower limit of normal range. Higher doses of stanozolol (4 mg/day), which caused greater immunochemical responses, were unnecessary for control of clinical disease and were unjustified for chronic therapy because of more frequent side effects.
Adolescent↗
Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase.
The ability of heparin glycosaminoglycan to prevent formation of the properdin-stabilized amplification C3 convertase is independent of antithrombin binding activity and requires substitution of the amino sugar and a degree of oxygen (O)-sulfation which could be on the uronic acid or the amino sugar. Preparations of heparin glycosaminoglycan isolated by different techniques from different species (rat, human, and porcine) exhibited an equivalent capacity to inhibit generation of the amplification C3 convertase. Hyaluronic acid, which is devoid of O-sulfation, had no inhibitory activity; chondroitin 4-sulfate of rat and whale origins, chondroitin 6-sulfate of rat and shark origins, and dermatan sulfate from porcine skin are O-sulfated on the galactosamine and had minimal activity. Porcine heparin glycosaminoglycan, isolated on the basis of affinity for antithrombin III, had no greater anticomplementary activity than porcine glycosaminoglycan, which failed to bind antithrombin III and had essentially no anticoagulant activity. Nitrogen (N)-desulfation of porcine heparin reduced anticomplementary activity to the level of the other sulfated mucopolysaccharides, and both N-resulfation and N-acetylation restored the original activities, thereby indicating a requirement for N-substitution, but not N-sulfation. N-resulfation of N-desulfated and O-desulfated heparin did not restore any activity, thus indicating that O-sulfation and N-substitution represent independent, critical structural requirements for the anticomplementary activity of heparin glycosaminoglycan. Inasmuch as N-desulfated-N-acetylated heparin had no anticoagulant activity, the nature of the N-substitution completely distinguishes the plasma-protein effector pathway that is inhibited.
Acetylation↗
IgG on mouse erythrocytes augments activation of the human alternative complement pathway by enhancing deposition of C3b.
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Animals↗
Current concepts in immunology: the alternative pathway of complement--a system for host resistance to microbial infection.
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Bacteria↗
Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.
A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells. The F(ab')2 fragment of rabbit IgG anti-gp205 inhibited the formation of rosettes with sheep EC3b of human erythroeytes, B lymphocytes, monocytes and PMN in a dose-response manner; the 50 percent inhibitory doses were 0.13/mug/ml, 0.90 mug/ml, 1.25 mug/ml, and 1.20 mug/ml of F(ab')2, respectively. Anti-gp205 did not impair the formation of rosettes by monocytes and B lymphocytes with sheep EC3bi or with EC3d. Scatchard analysis of the number of specific (125)I-F(ab')(2) anti-gp205 binding sites/cell revealed 950 sites/erythrocyte, 21,000 sites/cell of B lymphocyte preparation, 57,000 sites/PMN, and 48,000 sites/monocyte, indicating that the higher concentrations of antibody that had been required for inhibition of rosette formation by the nucleated cells reflected larger numbers of receptors on these cells. Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In each instance, the antigenic material reacting with anti-gp205 represented a single protein with an apparent 205,000 mol wt. Thus, gp205 is the C3b receptor of human erythrocytes, PMN, B lymphocytes, and monocytes.
Animals↗