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PubMed1988

Presence of the C3b complement receptor (CR1) antigen in the glomerular basement membrane of human fetal kidneys.

The distribution pattern of the C3b receptor (CR1) antigen, one of the earliest differentiation markers of the podocyte plasma membrane, has been studied during human renal ontogenesis by indirect immunofluorescence and immunoelectronmicroscopy using Fab'2 fragments of anti-CR1 antibodies. CR1 antigen has been detected at the open-stage glomerulus, which precedes constriction of the vascular pole, along the capillary wall, in a thick and homogeneous pattern of staining. By immunoelectronmicroscopy, CR1 antigen was present on the porocyte plasma membrane towards the glomerular basement membrane (GBM), and diffusely distributed within the GBM according to a decreasing intensity gradient from podocytes to endothelial cells. At a later stage of differentiation ('closed' glomerulus stage) when constriction of the vascular pole has occurred, the podocyte plasma membrane appeared diffusely labelled for CR1; the GBM was also stained with a decreasing intensity gradient from podocytes to endothelial cells. This result indicates that a plasma membrane receptor belonging to the cell coat of podocytes can be found in the extracellular space within the GBM. Thus the antigenic repertoire of the GBM of human fetal kidney comprises antigens of constituent molecules of the extracellular matrix and planted antigens originating from adjacent cells.

Basement Membrane↗
PubMed1976

Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b.

Animals↗
PubMed1987

Complement C3b receptors on erythrocytes in patients with juvenile rheumatoid arthritis.

Forty patients with juvenile rheumatoid arthritis (JRA) were examined for C3b receptor (CR1) levels on erythrocytes (by an enzyme-linked immunosorbent assay), levels of circulating immune complexes (IC) (by a polyethylene glycol precipitation-complement consumption method), C3d split products (by intermediate gel rocket immunoelectrophoresis), and HLA-A, B, and C (by microdroplet lymphocytotoxicity test). Lower CR1 levels were found predominantly among patients with JRA (mean 57%) compared with 40 age-matched controls (mean 68%) (P = 0.008). The CR1 levels differed when the JRA patients were grouped by mode of disease onset and by clinical state at the time of testing (levels in patients with pauciarticular disease were higher than those in patients with polyarticular disease, and levels in patients with polyarticular disease were higher than those in patients with systemic disease) or by sex (girls had higher levels than boys; P = 0.01). The levels of circulating IC and C3d were elevated in 25% and 40% of JRA patients, respectively, and were mutually correlated (P less than 0.05, tau = 0.20). A negative correlation was found between levels of C3d and the numbers of CR1 (P less than 0.01, tau = -0.28), but concentrations of circulating IC did not correlate with CR1 values. CR1 levels were the same in 6 HLA-B27 positive and 25 HLA-B27 negative patients. These findings do not represent conclusive evidence that the number of CR1 on erythrocytes serves as a predictor of disease severity or as an indicator of disease activity in patients with JRA.

Adolescent↗
PubMed1982

Antisera with anti-pan C3 receptor reactivity which block complement C3b, C3bi and C3d receptors.

Antisera directed at human C3 receptors were prepared by 2 different methods. In method I rabbits were immunized with EAC coated with C3 receptors of tonsil cells (AC3RS type I). In method II rabbits were immunized with insoluble aggregates of tonsil membrane constituents containing significant amounts of C3 receptors. After appropriate absorptions both types of AC3RS showed a selective reactivity with those cells known to express C3 receptors. With the EAC rosette inhibition test it is demonstrated that both types of AC3RS contain antibodies against the binding sites of C3b, C3bi and C3d receptors. The antibody titer was consistently higher in AC3RS of type II.

Animals↗
PubMed1986

Erythrocyte complement C3b receptor (CR1) levels and immune complex-induced manifestations in patients with primary Sjögren's syndrome.

To assess the importance of low C3b receptor (CR1) numbers on erythrocytes for the development of clinical features in patients with primary Sjögren's syndrome (SS), two groups of patients were selected for investigation--one without (n = 13), and one with (n = 21) extraglandular, possibly immune complex-induced, clinical manifestations during the disease course. A preponderance (p less than 0.01) of low CR1 levels was found in patients with primary SS (mean 57%) as compared with normal controls (mean 70%), but the CR1 levels did not differ between the two groups of patients. The CR1 values did not correlate with the levels of circulating immune complexes and C3d. Although the preponderance of low CR1 levels is pathogenetically intriguing, it appears to be of little help in the clinical assessment of patients with primary SS.

Antigen-Antibody Complex↗
PubMed1986

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↗
PubMed1985

Circulating immune complexes and B lymphocytes in diabetic patients with and without microangiopathy.

Circulating immune complexes (CIC) were evaluated in 57 diabetic patients: 28 were insulin-dependent (IDD) and 29 were non insulin-dependent (NIDD) subdivided according to the presence or absence of microangiopathy. The following techniques were used: 1) binding to human red blood cells through C3b complement fraction and posterior radioimmunoassay with 125I labeled anti human IgG (HRBC RIA test); and 2) CIC precipitation with 3.5% polyethylenglycol (PEG test). The percentage of circulating B lymphocytes was evaluated simultaneously in the same patients, using a) direct immunofluorescence techniques (surface IgC cells) and b) cells with complement C3b fraction receptors (EAC rosettes). Twenty normal donors were studied simultaneously as controls. Our results showed that CIC levels were significantly higher in both groups of patients when compared to normal controls. Values for IDD and NIDD were 48.55 +/- 5.97 and 34.68 +/- 3.08 microgram/ml, respectively, for HRBC RIA test and 0.53 +/- 0.07 and 0.41 +/- 0.04 O.D., respectively, for PEG test, while control values were 26.63 +/- 2.12 microgram/ml for HRBC RIA test and 0.26 +/- 0.03 O.D. for PEG test. IDD patients with microangiopathy presented higher CIC levels as measured by HRBC RIA test than IDD without microvascular complications, while no difference was found within the NIDD group. An increase in the proportion of cells bearing surface IgG was observed in IDD and NIDD patients when compared to controls. By contrast, no difference was observed when EAC rosettes-forming cells were evaluated.

Antigen-Antibody Complex↗
PubMed2002

An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab.

We investigated deposition of the complement protein fragment C3b and its breakdown products (collectively designated as C3b(i)) on CD20-positive cells treated with rituximab (RTX) in the presence of normal human serum (NHS). Radioimmunoassay (RIA) demonstrates that about 500 000 C3b(i) molecules deposit per cell, and fluorescence microscopy reveals that C3b(i) colocalizes with bound RTX. Use of mAb 3E7, specific for C3b(i) bound to substrates, enhances C3b(i) deposition; > 1 million C3b(i) deposit when cells are incubated with NHS, RTX and mAb 3E7. Treatment of Raji cells in NHS plus RTX leads to robust cell killing (95%) after 24 to 48 hours, and mAb 3E7 significantly enhances RTX-mediated killing of Raji and DB cells. A cynomolgus monkey model based on intravenous infusion of RTX followed by mAb 3E7 demonstrated that RTX rapidly binds to B cells and promotes complement activation and C3b(i) deposition; fluorescence microscopy analyses revealed the same pattern of colocalization of C3b(i) on cell-bound RTX in vivo as observed in vitro. Preliminary in vitro studies with blood samples from patients with chronic lymphocytic leukemia lead to similar findings. These experiments suggest that complement plays a key role in the mechanism of action of RTX; moreover, the in vivo molecular form of RTX (and possibly other antitumor mAbs) in the circulation or in tissues may include C3b(i) molecules covalently bound to the therapeutic mAb, thus allowing it to interact with cells containing both Fc and complement receptors.

Animals↗
PubMed1991

Effect of complement-protein-C3b density on the binding of complement factor H to surface-bound C3b.

Various amounts of the activation fragment C3b of the complement (C) protein C3 were coupled to Sepharose 4B by catalysis with the C3 convertase of the alternative pathway of C. The binding of radioactively labelled C proteins B and H (= factor H) to the C3b-carrying particles was assayed. It was found that the relative binding of H, but not of B, fell rapidly with decreasing densities of solid-phase C3b, suggesting a sigmoidal relationship between C3b density and binding of H. To study the phenomenon in more detail, preformed C3b was coupled to activated thiopropyl-Sepharose 6B at various densities. By using this model system, it was shown that the binding of H/unit amount of C3b was positively correlated to C3b density up to a C3b concentration of about 0.5 mg/ml of gel, whereas binding of B was independent of C3b density. The results show that accumulation of high densities of C3b on a surface creates high-affinity binding sites for H. Because H has recently been shown to form dimers in solution, the interaction of dimeric H with neighbouring C3b molecules is a likely explanation for the phenomenon. The C3b density effect may be a regulatory mechanism keeping the activation of the alternative pathway of C on activating surface within reasonable limits.

Complement C3-C5 Convertases↗
PubMed1983

Stimulation of prostaglandin E and thromboxane synthesis in macrophages by purified C3b.

Complement cleavage product C3b was examined for its effect on macrophages. Albumin-elicited guinea pig peritoneal macrophages kept in short-term culture were challenged with purified homologous C3b, and release of oxygen and arachidonic acid metabolites was studied. C3b at concentrations ranging from 20 to 80 micrograms/ml induced synthesis of large quantities of the arachidonic acid cyclooxygenation products thromboxane B2 (TXB2) and prostaglandin E (PGE) but failed to trigger an oxidative burst. Our findings link together macrophages, complement, and arachidonate metabolites as major participants in inflammatory responses.

Animals↗
PubMedDate not supplied

Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor.

INTRODUCTION: Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme. METHODS: CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively. RESULTS: About 200 microg of CVF was purified from 100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found. DISCUSSION: The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact.

Animals↗
PubMed1993

Disulfide bridges in human complement component C3b.

The disulfide bridges of human complement component C3b, derived from C3 by removal of the 77-residue C3a, have been determined. The 10 bridges are Cys537-Cys794, Cys605-Cys640, Cys851-Cys1491, Cys1079-Cys1136, Cys1336-Cys1467, Cys1367-Cys1436, Cys1484-Cys1489, Cys1496-Cys1568, Cys1515-Cys1639, and Cys1615-Cys1624. Including the 3 bridges in C3a (Cys670-Cys698, Cys672-Cys705, and Cys685-Cys706) previously determined by high-resolution X-ray crystallography [Hoppe-Seyler's Z. Physiol. Chem. 361 (1980) 1389-1399] all disulfide bridges of C3 are localized. C3 and the strongly related C4 and C5 are members of the alpha 2-macroglobulin superfamily. The predicted bridge patterns of C4 and C5 are discussed and compared with that of alpha 2-macroglobulin.

Amino Acid Sequence↗
PubMed1981

The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.

The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.

Binding, Competitive↗
PubMed2004

Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b.

The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.

Binding Sites↗
PubMed1982

A general method for affinity purification of complement component C3b using factor H-sepharose.

Complement component C3b has been purified from human, rabbit and bovine serum by affinity chromatography on human factor H-Sepharose after preliminary fractionation by poly(ethylene glycol) and DEAE-Sepharose. The yields are high (35--40%) and the whole process is rapid (3 days). Binding of C3b to factor H-Sepharose is equimolar, has a sharp optimum pH at 7.6 and is quite sensitive to ionic strength.

Animals↗
PubMed1990

Complement fragments C3b and iC3b coupled to latex induce a respiratory burst in human neutrophils.

The complement fragments C3b and iC3b were purified from human serum by affinity chromatography with Sepharose-coupled monoclonal antibody against the C3d region of C3. The resulting preparations were more than 95% pure and contained less than 0.1% native IgG. Purified C3b and iC3b were coupled to latex beads (0.8 micron diameter) by means of F(ab')2 fragments of monoclonal antibodies against the beta chain or the C3d region of C3, thus orienting the C3b and the iC3b on the latex with the C3b- and iC3b-specific regions outwards. These particles were found to activate the respiratory burst of freshly isolated human neutrophils to 20-30% of the maximal capacity. Latex particles randomly coated with C3b or iC3b were about 3 times less stimulatory. C3b, iC3b and IgG coupled to latex in an oriented fashion were about equally effective in stimulating the respiratory burst. Neutrophils from a patient with a total deficiency of CR3 responded normally to C3b-coated latex but did not respond to iC3b-coated latex. A monoclonal antibody against the alpha chain of CR3 inhibited the activation by iC3b-coated latex and a polyclonal antibody against CR1 partially inhibited the activation by C3b-coated latex. We found an additive effect between IgG-coated latex and C3b-coated latex, regardless of the presence of IgG and C3b on the same particle or on different particles. Thus, binding of ligands to either CR1 or CR3 per se is sufficient to induce an activating signal to the NADPH oxidase in human neutrophils.

Antigens, CD↗