Inhibition of C3b and C3d receptor activity by xenoantisera prepared by immunization with cell-bound C3 receptor protein.
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Animals↗
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Bioactive complement fragments in immunoregulation.
Several fragments derived from complement components have been identified as potent effector substances in in vitro assays that measure cell proliferation and antibody synthesis. The anaphylatoxin C3a suppresses the immune response but fails to influence T- or B-cell proliferation. The factor C5a augments both antibody production and antigen-induced, but not mitogen-induced, T-cell proliferation. C3a-mediated suppression occurs through the activation of a suppressor T-cell cascade with macrophage collaboration. C5a-mediated enhancement, depending upon the in vitro system studied, acts at the level of the helper T cell and/or macrophage. A fragment generated from treating iC3b with kallikrein (c3d-K) has aided in defining a structural region of the C3b molecule that can influence the level of circulating leukocytes. The factor C3d-K is also capable of suppressing both specific and non-specific T-cell proliferative responses and mitogen-induced B cell growth. The mechanism of C3d-K action is defined as a direct effect on "activated" T cells, even though IL-2 synthesis of treated cells is diminished. The effect of C3d-K is long lasting, non-reversible and requires only a short exposure to the target cell.
Animals↗
Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).
Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.
Amino Acid Sequence↗
OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2.
OKB7, an IgG2 mouse monoclonal antibody, binds to the C3d receptor (CR2) of human B lymphocytes in or near the active site of the receptor. OKB7 precipitates the same molecule as monoclonal antibodies B2 and HB-5 which have previously been reported to react with the C3d receptor at epitopes distant from the active site. The epitope recognized by OKB7 is distinct from those bound by B2 and HB-5 as shown in competitive binding experiments and is near the complement binding site since OKB7 blocks EAC3d rosetting at concentrations as low as 1 microgram/ml in the absence of cross-linking antibodies.
Animals↗
Polymer microbeads bound to C3 fragments for detecting and labeling cells with C3 receptors.
Monodisperse microbeads (mbs) of a hydrophilic polymer with diameters of 4 microns and 0.2 micron were prepared. To these mbs, IgM or a sugar was bound covalently, and then the mbs were treated with fresh human or mouse serum. This led to the activation of complement and to the binding of C3 fragments to the mbs. C3 fragments fixed on mbs varied with the type of immobilized substance on the mbs and the incubation time of the mbs with fresh serum. Three kinds of mbs binding C3 fragments were prepared: mbs binding C3b, C3bi and C3d (C3hu-mbs); the ones binding C3bi and C3d (C3mu-mbs); and the ones binding only C3b (C3bhu-mbs). They formed rosettes with cells which had the corresponding receptor. When lymphocytes were incubated with C3hu- or C3bhu-mbs with a diameter of 0.2 micron, cap formation of each receptor was observed under a microscope. Once prepared, C3hu-, C3mu- and C3bhu-mbs were stable for more than 4 months.
Animals↗
Modulation of mouse endotoxic fever by complement.
It was recently reported that the complement system may be critically involved in the febrile response of guinea pigs to systemic, particularly intraperitoneally (i.p.) injected, lipopolysaccharides (LPS). The present study was designed to identify which component(s) of the complement cascade may be specifically critical. To this end, we used mice with C3, C5, and CR2 gene deletions. To assess preliminarily the suitability of mice for such a study, we replicated our earlier studies with guinea pigs. Thus, to verify initially whether complement is similarly involved in the febrile response of wild-type (C57BL/6J) mice to i.p. LPS (Escherichia coli, 1 microg/mouse), we depleted complement with cobra venom factor (CVF; 7 U/mouse, intravenously [i.v.]). These animals did not develop fever, whereas the core temperature (T(c)) of CVF vehicle-treated controls rose approximately 1 degrees C by 80 min postinjection and then gradually abated over the following 2.5 h, confirming the involvement of complement in fever production after i.p. LPS injection and the suitability of this species for these studies. C3- and C5-sufficient (C3(+/+) and C5(+/+)) mice also developed 1 degrees C fevers within 80 min after i.p. LPS (1 or 2 microg/mouse) injection. These fevers were totally prevented by CVF (10 U/mouse, i.v.) pretreatment. C3- and C5-deficient (C3(-/-) and C5(-/-)) mice were also unable to develop T(c) rises after i.p. LPS. Both CR2(+/+) and CR2(-/-) mice responded normally to i.p. LPS (1 microg/mouse). These data indicate that C5, but not C3d acting through CR2, may play a critical role in the febrile response of mice to i.p. LPS.
Acute-Phase Proteins↗
Binding kinetics, structure-activity relationship, and biotransformation of the complement inhibitor compstatin.
We have previously identified a 13-residue cyclic peptide, Compstatin, that binds to complement component C3 and inhibits complement activation. Herein, we describe the binding kinetics, structure-activity relationship, and biotransformation of Compstatin. Biomolecular interaction analysis using surface-plasmon resonance showed that Compstatin bound to native C3 and its fragments C3b and C3c, but not C3d. While binding of Compstatin to native C3 was biphasic, binding to C3b and C3c followed the 1:1 Langmuir binding model; the affinities of Compstatin for C3b and C3c were 22- and 74-fold lower, respectively, than that of native C3. Analysis of Compstatin analogs synthesized for structure-function studies indicated that 1) the 11-membered ring between disulfide-linked Cys2-Cys12 constitutes a minimal structure required for optimal activity; 2) retro-inverso isomerization results in loss of inhibitory activity; and 3) some residues of the type I beta-turn segment also interact with C3. In vitro studies of Compstatin in human blood indicated that a major pathway of biotransformation was the removal of Ile1, which could be blocked by N-acetylation of the peptide. These findings indicate that acetylated Compstatin is stable against enzymatic degradation and that the type I beta-turn segment is not only critical for preservation of the conformational stability, but also involved in intermolecular recognition.
Amino Acid Sequence↗
Complement (C3b) interaction with the human granulocyte receptor: correlation of binding of fluid-phase radiolabeled ligand with histaminase release.
The interaction of C3b, the major cleavage product of C3, with its receptor on human granulocytes results in important biological functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have determined the binding kinetics and isotherm of trypsin-generated fluid-phase dimeric C3b at both 0 degrees C and 37 degrees C using human polymorphonuclear leukocytes (PMN). At 37 degrees C the apparent number of receptors per cell was threefold greater than number at 0 degrees C (66,000 vs 21,000), and the affinity (Ka) was slightly less (3.5 X 10(7) M-1 vs 6 x 10(7) M-1). C3b dimer binding was specifically inhibited by a F(ab,)2 anti-C3b receptor antibody. C3b dimer induced histaminase release in a dose-dependent fashion at a concentration of 1 to 4 X 10(7) molecules/cell, whereas at lower concentrations a dose-dependent inhibition of opsonized zymosan-induced release was noted. These effects were independent of IgG. In contrast, C3b monomer failed to demonstrate measurable direct binding or to induce histaminase release. Histaminase release, however, did occur after incubation of PMN with affinity-linked monomer (anti-C3 F(ab')2-C3b complexes). Monovalent complexes did not affect release. Monomeric C3, C3c, and C3d were without effect; however, affinity-linked C3 and C3c did cause release.
Amine Oxidase (Copper-Containing)↗
Cross-linking of at least three binding sites mediates signal transduction in a CR2-positive Burkitt lymphoma derived cell line (Raji).
In this study we demonstrate that Raji cells, a CR2-positive Burkitt lymphoma-derived cell line, during cell growth, need the cross-linking of multiple OKB7 binding sites or C3d determinants to mediate signal transduction. The loss of one of these affects the cellular response. Moreover, OKB7, the anti-CR2 MoAb, recognizes C3d determinants on the cell surface and inhibits signal transduction induced by anti-C3d polyclonal antibody. Since Raji cells are always CR2 positive during cell growth, we suppose that at least another protein, along with CR2, may be involved in setting up a cell surface complex able to receive and transduce the signal triggered by OKB7. In our experimental system the protein that offers a third binding site to OKB7, may be represented by a 33 kDa protein bearing C3d determinants.
Binding Sites↗
A trypsin method for coating human red blood cells with plasma C3 and/or C4.
Simple procedures are described for the production of human red blood cells coated with C3 and/or C4 by brief trypsin treatment of cell-plasma mixtures at room temperature. These cells ('EC3/4'-try) were unusual in that their agglutinability by anticomplement sera, as compared to sucrose-water RBC, was similar when tested by AutoAnalyzer but was markedly reduced in manual testing, especially against anti-C4. In spite of this, both 'EC34'-try and 'EC3'-try were found to be suitable indicator cells for determining the adequacy of anti-C3c and/or anti-C3d activities in reagents used for manual antiglobulin tests.
Antibodies, Anti-Idiotypic↗
Receptor-binding sites on C3 and C3b.
Human erythrocytes carry C3b receptors: Daudi lymphoid cells carry exclusively C3d receptors. With these two types of cells, it could be shown that isolated, uncleaved human C3 and soluble C3b possess two stable binding sites: SBS1-specific C3b receptors, and SBS2-specific for C3d receptors. Upon binding of freshly cleaved C3b (nascent C3b) via its labile binding site (generated on C3b through cleavage of C3) to the C3 acceptors on cell surfaces, the SBS2 becomes concealed. Kinetic experiments show that immediately after the action of fetal calf serum or partially purified C3b-inactivator on EAC1423b the SBS2 is accessible again. The reappearance of SBS2 does coincide with cleavage of surface-bound C3b into C3c and C3d but not with the release of C3c from the cell; C3c remains attached and is released only with delay. Concomitant with the cleavage event the number of SBS1 is reduced, stressing the importance of an unaltered steric configuration of the C3b structure for the expression of SBS1. An alternative explanation might be that the SBS1 is located at the site connecting C3c and C3d, so that it becomes altered upon dissection of the two fragments.
Binding Sites, Antibody↗
Specificity of three anti-complement factor 3 monoclonal antibodies.
Monoclonal antibodies which recognize specific C3 fragments may be used to distinguish C3 cleavage products bound to organisms. We defined the specificity of three commercially available monoclonal antibodies by Western immunoblot analysis, enzyme-linked immunosorbent assay, and a quantitative flow cytometric technique. Two monoclonal antibodies with specificity for (i) an erythrocyte-bound C3d epitope or (ii) an erythrocyte-bound C3c epitope retained their specificity in all assays. However, the third monoclonal antibody with selectivity for erythrocyte-bound C3bi failed to retain specificity for C3bi bound to non-erythrocyte surfaces in each of our assays; binding instead to all C3 fragments containing the C3g domain. We postulate that erythrocyte C3b-binding surface proteins may alter the availability of certain C3b epitopes and influence observed anti-C3 monoclonal antibody specificity. We conclude that the specificity of monoclonal antibodies for C3 fragments should be confirmed with assays which do not employ erythrocytes or other surfaces bearing C3 receptors. Also, our quantitative flow cytometric technique is a potentially valuable tool for the enumeration of particle-bound C3 fragments.
Adult↗
Mapping of the properdin-binding site in the third component of complement.
The properdin-binding site in the human third complement component (C3) was mapped by using isolated C3b, C3c, alpha- and beta-chains of C3 and C3 polypeptide fragments and an enzyme-linked-immunosorbent-assay procedure. The C3 chains and the polypeptide fragments were purified to homogeneity by preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The alpha-chain polypeptides included a 68 kDa and a 43 kDa polypeptide, which were generated by cleavage of C3b with factors I and H, and a 40 kDa, 33 kDa (C3d) and 27 kDa polypeptide, which were generated by cleavage of C3b with porcine elastase. It was shown that properdin binds to C3b, C3c, alpha-chain, and to the 43 kDa (factor-I + H-derived), as well as to 40 kDa (elastase-derived) alpha-chain fragment, but not to the beta-chain 68 kDa, 33 kDa (C3d) and 27 kDa alpha-chain fragments. Thus the binding site for properdin resides on the 40-43 kDa C-terminal alpha-chain fragment of C3.
Binding Sites↗
Phototoxic erythema following PUVA treatment: independence of complement.
The effect of PUVA treatment on normal human serum (NHS), on isolated PMN, or on C3-deficient guinea pigs and congenic (C3-competent) control animals was tested. At a concentration of 0.1 or 1 mM/l 8-MOP and UVA doses of 5-30 J/cm2, PUVA failed to induce any detectable C3-cleavage in NHS. Furthermore, when the complement (C) activation in NHS had been induced before or after PUVA treatment by various methods. PUVA did not modulate the extent of C3-cleavage. PUVA did not affect the viability of isolated PMN, nor did it induce a release of LDH or elastase. No differences between C3-deficient and C-competent guinea pig skin exposed to PUVA were observed in erythema or histologic responses. Immunohistologic examination of specimens from normal guinea pigs revealed C3b and C3d deposits on necrotic keratinocytes, findings restricted to the PUVA-treated areas. Necrosis of keratinocytes was present in skin specimens of C3-deficient animals from PUVA-treated sites to a similar extent. However, deposits of C3-related antigens were completely absent there. From these observations, we suggest that the induction of phototoxic erythema following PUVA treatment is independent of complement.
Animals↗
Coupling of C3b to erythrocytes by disulfide bond formation: preparation of EC3b for hemolytic and complement receptor assays.
We describe a new method of preparing C3-coated erythrocytes by coupling C3 to thiol-activated erythrocytes. The procedure involves three steps. Firstly, sheep erythrocytes were treated with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to introduce 3-(2-pyridyldithio) propionyl residues into membrane proteins. Secondly, C3 was cleaved with trypsin or CoVF, Bb enzyme to obtain C3b exposing the SH group (C3b-SH). Finally, the C3b-SH was coupled to the thiol-activated erythrocytes (TA-E) through thiol/disulfide exchange to form the TA-EC3b conjugate. E coated with C3d was prepared by treating TA-EC3b with KSCN inactivated serum and plasmin. Studying the rosette formation between TA-EC3b or TA-EC3d and cells expressing C3b (CR1) and C3d (CR2) receptors and the inhibition thereof with anti-CR1 and anti-CR2 antibodies as well as with C3-sheep E membrane protein complexes, we found that TA-EC3b and TA-EC3d bound exclusively to CR1 and CR2, respectively. In addition, TA-EC3b like EAC1423b bound factors B and H as tested by hemolytic and direct binding assays. The advantage of TA-EC3 for complement receptor and hemolytic assays are the simplicity of the preparation method and the general applicability of the TA-EC3.
Animals↗
An immunochemical study of the synovial fluid can contribute to the distinction between rheumatoid and non rheumatoid arthritis.
Synovial fluids from 102 patients affected by various joint diseases have been analyzed for their protein, immunoglobulin and beta 2-microglobulin contents, for the total and alternative pathway hemolytic activities of the complement components, for the presence of rheumatoid factors and Clq binding materials. The aim of this study was to verify whether an immunochemical analysis of the synovial fluid could help to distinguish rheumatoid arthritis from other arthropathies. With regard to this, we emphasize that the latex test, the Clq binding assay, as well as the measurement of C3d and beta 2-microglobulin concentrations in synovial fluids, were the most helpful assays. An immunochemical synovial score was calculated summing the results of these four tests, thus making the recognition of rheumatoid arthritis, among the various inflammatory joint diseases, easier.
Arthritis↗
Evidence for autoantibody-induced CD4 depletion mediated by apoptotic and non-apoptotic mechanisms in HIV-positive long-term surviving haemophilia patients.
It is believed that autoimmune phenomena and apoptosis contribute to CD4 depletion. We investigated 11 long-term (>20 years) HIV-infected haemophilia patients and 10 healthy controls. Using four-colour-fluorescence flow cytometry, we studied the proportions of CD3+CD4+ and CD3+CD4- blood lymphocytes that were CD95+, CD95L+, immune complex+ (IC+, consisting of IgM, IgG, C3d and/or gp120), and were viable or non-viable (propidium iodide+ = PI+). In addition, we studied viability of CD4+IgG+ patient lymphocytes using the apoptosis marker annexin and the permeability indicator 7-amino actinomycin D (7-AAD). HIV+ patients had a higher proportion of CD3+CD4+IgG+PI+ lymphocytes than healthy controls (median: 3.7%versus 0.3%; P = 0.00001). These non-viable IgG-coated lymphocytes might have been killed in vivo by ADCC or complement lysis; 9.1% of the circulating CD3+CD4+ blood lymphocytes were IgG+PI- (controls: 2.5%; P = 0.001). These viable IgG-coated lymphocytes might be targets for phagocytosis or anti-CD95 autoantibody-mediated apoptosis. Because HIV+ patients and healthy controls had similar proportions of PI+ or PI- CD3+CD4+ lymphocytes that carried CD95L on the surface, and because CD3+CD4+CD95L+ cells that were IgG+, C3d+ and/or gp120- were increased in HIV+ patients, the role of CD95L-induced apoptosis in long-term HIV-infected haemophilia patients remains unclear. The findings that HIV+ patients had higher proportions of CD3+CD4+CD95+ (PI+: 6.5%versus 1.4%; P = 0.00002; PI-: 55.8%versus 44.4%; P = 0.04) blood lymphocytes and that the proportion of CD4+IgG+Annexin+7-AAD- blood lymphocytes was associated inversely with peripheral CD4 counts (r = -0.636; P < 0.05) suggest that attachment of IgG to CD4+ blood lymphocytes (anti-CD95?) induces in some lymphocytes apoptosis with subsequent depletion of these IgG-coated apoptotic CD4+ lymphocytes from the circulation. We found supporting evidence for the contention that autoantibody-induced apoptotic and non-apoptotic mechanisms contribute to CD4 depletion in long-term HIV-infected haemophilia patients.
Adolescent↗
IgE-containing circulating immune complexes in Churg-Strauss vasculitis.
In five patients with vasculitis, hypereosinophilia, and elevated serum IgE levels a diagnosis of Churg-Strauss syndrome was established. To identify a possible role of IgE in pathogenic mechanisms leading to the vasculitis, we performed a sequential precipitation of the patients' sera with different concentrations of polyethylene glycol (PEG) 6000. Using a radio immunosorbent test, we tested the precipitates obtained for IgE. Considerable amounts of IgE were traced in the serum precipitates of all patients, especially after the second precipitation step (4.0% PEG). In contrast, no IgE-containing precipitates were detectable in sera from patients with different allergic diseases and high IgE serum levels. Together with an increase in C3d serum levels and the failure to demonstrate C1q-binding material in patients' sera, these data suggest the involvement of IgE-containing immune complexes in the pathogenesis of Churg-Strauss vasculitis, activating the complement via the alternate pathway.
Antigen-Antibody Complex↗