Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine-transfected fibroblasts.

CR1 and CR2 are expressed as associated proteins on the B-lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1- and CR1-CR2-expressing cells bound C3b and C3b-dimer whereas CR2- and CR1-CR2-expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1-CR2-positive cells internalized two- to threefold more C3b and 1.5-fold more iC3b than CR1- and CR2-single-positive cells, respectively. Internalization of the anti-CR1 antibody J3D3, or C3de was at the same level, in both double-transfected and single-transfected cells. Furthermore, the internalization of C3b dimer by CR1-CR2 cells was impaired in the presence of OKB7, an anti-CR2-blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.

Different stimulating effects of complement C3b and complete Freund's adjuvant on antibody response.

Upon activation, complement C3 undergoes a conformational change and acquires the capacity to covalently bind to other proteins such as antigen and to interact with specific receptors; therefore, C3 is involved in cell mediated immune response. The adjuvant effect produced by linking C3-fragments to antigen has recently been described. We injected C3b-Ag complexes consisting of one molecule of C3b ester linked to one molecule of HEL to immunised mice, and we compared the C3b adjuvant activity with that of complete Freund's adjuvant. IgG titers elicited by HEL emulsified in CFA (HEL + CFA) were higher than those elicited by HEL-C3b, but decreased rapidly after a peak response around day 45 whereas HEL-C3b resulted in a continuous increase of anti-HEL response. Mice immunised with HEL + CFA then boosted with HEL-C3b gave significantly higher response than those boosted with HEL + CFA, indicating more efficient memory cell restimulation by C3b. HEL + CFA leads to better priming than HEL-C3b when mice are boosted with HEL-C3b. Thus, adjuvant effect of C3b is different from that of CFA, leading to more stable IgG production and better memory stimulation.

[Role of the complement C3 protein in the control of the specific immune response]. / Rôle de la protéine C3 du système complémetnaire dans le contrôle de la réponse immunitaire spécifique.

Whereas complement system was usually considered as a member of innate defence, one of its components (C3) is now thought to facilitate acquired immunity. This role is due first to its capacity to covalently bind to antigens and secondly to the interactions of its proteolytic fragments with different receptors expressed on most cells involved in the acquired immune response. After activation in the plasma, C3 is proteolysed in fragments which possess various biological activities, as a modification in cell activities occurred after binding to cell surface receptors. Injection of low amount of antigen results in a modified immune response in C3 deficient animals with a decrease in the level of specific antibodies and an absence of IgM/IgG switch. One of the fragments of C3 (C3d) and its receptor (CR2) seem particularly important : knock out animals in C3 or CR2 have similar phenotypes. Mice with a deficit in CR2 restricted to the B lymphocytes present a strong reduction in the number and the size of germinal centers. Moreover, expression of CR2 on follicular dendritic cells is necessary for the generation of a strong memory response. C3 is also involved in the control of the intracellular processing of the antigen as the use of covalent complex (C3b-antigen) instead of free antigen increases the amount of stable MHC class II molecules at the antigen presenting cells surface. In summary, C3 fragments increase cell to cell interactions, induce intracellular signalling after binding to their receptors and increases intracellular processing of antigens. A better knowledge of the different roles of C3 may be useful to modify the immune response and to promote the immune memory, a domain where C3 seems particularly important.

Amplification of the antibody response by C3b complexed to antigen through an ester link.

Complement C3 has been described as playing an important role in the cell-mediated immune response. C3b has the capacity to covalently bind Ag and then to stimulate in vitro Ag presentation to T lymphocytes. To verify this observation in vivo, we prepared and purified covalent human C3b-Ag complexes using lysozyme (HEL) as Ag. The characterization of these HEL-C3b complexes indicates that they are representative of those susceptible to be generated in physiological conditions. Mice were immunized with 0.1 to 0.6 microgram of either free HEL, HEL + C3b, HEL-C3b, or HEL + CFA. Response was assessed after two i.p. injections by quantification of specific Ab production. Immunization with either HEL-C3b complexes or HEL + CFA leads to anti-HEL IgG production whereas free HEL or HEL + C3b was ineffective. Either HEL-C3b or HEL + CFA immunizations led to a similar Ig subclass patterns, including IgG1, IgG2a, IgA, and IgM. Our experiments provide the first evidence for modulation of specific Ab response by C3b when it is bound to Ag through a physiological-like link. Taken together with previous data concerning Ab response following recombinant HEL-C3d immunization, cellular events such as processing of C3b-Ag by APC and recognition by T lymphocytes, this present result underlines the importance of C3b and its fragments in stimulation of the immune system, through the multiplicity and complementarity of its interactions.

Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells.

Modulation of antigen processing and presentation by covalently linked complement C3b fragment.

Ligands such as complement fragments (C3, C4), IgG or alpha 2-macroglobulin, which bind antigen (Ag) before their uptake by antigen-presenting cells (APC), are likely to modulate the different steps of Ag processing and presentation. These ligands contribute to internalization and endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatibility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha 2-macroglobulin family, an intrachain thiolester bond. Conformational alteration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to esterify hydroxyl groups of Ag. Ester-linked complexes including tetanus toxin (TT) and C3b were prepared to analyse the influence of bound C3b on TT processing and presentation by APC. Covalent binding of C3b to TT resulted in increased and prolonged stimulation of specific T-cell proliferation. This effect was observed with non-specific B cells, as well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endosome/lysosome-enriched subcellular fractions prepared from human Epstein-Barr virus (EBV)-transformed B cells, indicated a delay of TT proteolysis when TT was associated to C3b. Treatment of APC with protease inhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using purified cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolysis by cathepsin B was preserved. This finding and the absence of cathepsin B in endosomes may explain a delay in TT processing when it is associated to C3b. Confirming these data, presentation by formaldehyde-fixed cells of C3b-TT proteolysates showed higher stimulation of specific T-cell clones than formaldehyde-fixed TT proteolysates.

Involvement of the Zn-binding region of tetanus toxin in B and T recognition. Influence of Zn fixation.

Tetanus toxin contains a metal-binding site for zinc, located in its light chain. The sequence accounting for Zn fixation is part of a predicted amphipathic helical secondary structure and corresponds to a putative T cell epitope according to Rothbard and Taylor (EMBO J. 7, 93-100, 1988). In this paper, we analyse the antigenic properties of two synthetic peptides (233-248 = P12 and 225-243 = P13) containing the Zn binding sequence. Our results show that peptide P13 contains a B and T epitope. The B epitope seems to be immuno-dominant whether the T epitope is at least DR2 restricted. Zn binding on P13 leads to a decrease in its recognition by both antibodies and T lymphocytes.

Formation of covalent C3b-tetanus toxin complexes: a tool for the in vitro study of antigen presentation.

A novel method is described for the formation and purification of covalent complexes between the complement component C3b and an antigen (tetanus toxin, TT), using purified proteins in fluid phase. C3b is generated in situ by tryptic cleavage of C3 after co-precipitation of C3 and TT in the presence of polyethylene glycol. Various parameters were analysed to optimize complex formation; under conditions which minimized the formation of covalent C3b multimers, 30% and 8% respectively of C3b and TT were incorporated into covalent one-to-one complexes which were purified using gel filtration chromatography. The linkage was localized between the alpha' chain of C3b and either the H or L chain of TT; it required the in situ formation of C3b and was partially destroyed by 1 M hydroxylamine. Spontaneous dissociation of the complex could be partly avoided by HgCl2, a thiol reagent which inhibits the esterase-like activity of bound C3b. These findings suggest the involvement of the reactive carbonyl of nascent C3b with hydroxyl groups of TT. Such C3b-TT complexes provide a defined tool to analyse the influence of antigen-bound C3b on antigen addressing and intracellular processing by antigen-presenting cells.

Antibody-dependent cytotoxicity on chicken red blood cell targets mediated by the U937 cell line.

We have characterized a model system for the study of antibody-dependent cytotoxicity (ADCC) mediated by human macrophages and monocytes. The U937 cell line is used as a source of effector cells. We confirmed a previous report (Gidlund et al., 1981) that U937 can be activated using PMA to kill in ADCC, and the characteristics of the observed cytotoxicity are described. Activation of effectors was maximal after 20-hr preincubation in the presence of 10 ng/ml PMA. In these conditions, lysis was approximately 70% in 2 hr at an effector to target ratio of 5:1. Activation correlated with the expression of complement receptors CR1 and CR3. No antibody-independent cytotoxicity was observed. The effects of various inhibitors of oxygen species were investigated: the lysis obtained in the above conditions was inhibited at 50% in the presence of either 450 mM dimethyl sulphoxide or 30,000 U/ml catalase, but superoxide dismutase (up to 10,000 U/ml) or ferricytochrome c (up to 2 mM) had no effect. The same inhibition was observed with 40 mM desferrioxamine or with 1 mM 0-phenanthroline, which are both iron scavengers, or in the presence of 300 microM colchicine or 1.5 microM dihydrocytochalasin B, which are two inhibitors of cytoskeletal functions. An identical effect was obtained in the presence of 1 TIU/ml bovine pancreas trypsin inhibitor, whereas soya bean trypsin inhibitor, which is more specific, had no effect up to 5000 BAEE U/ml. No inhibition was seen with protein synthesis inhibitors as cycloheximide or puromycin at 40 micrograms/ml. The significance of these results is discussed.

Ultrastructure of human C4-binding protein: proposition for a new model.

The structure of human C4-binding protein (C4bp), a regulatory factor of the classical C3 convertase of complement, has been under investigation for several years, but remains poorly understood. For example, the number of subunits in the C4bp molecule has not been established. In this report, we use two different techniques (partial reduction and electron microscopy) to clarify the structure of the C4bp. Our results lead us to propose a structural model which is quite different to that suggested before, i.e. the C4bp molecule appears to be a decamer. In addition to the disulfide bonds which link each subunit to another, a second disulfide interaction leads to the association of the subunits in pairs. Each pair of subunits appears as a filament ending in a globular head at the N-terminal extremity. The pairs of subunits join to form a conical central domain (at the C-terminal extremity) linked by disulfide bonds. The proposed pentameric shape of the C4bp is consistent with the stoichiometry of the C4b-C4bp interactions. The proposed model indicates an overall structural homology between C4bp and other binding proteins.

Soluble C3 proconvertase and convertase of the classical pathway of human complement. Conditions of stabilization in vitro.

Soluble classical-pathway C3 convertase and proconvertase were prepared from purified C4b-C2ox complex in the presence of Ni2+; the two complexes, stable for at least 15 h at 4 degrees C, were isolated by sucrose-density-gradient ultracentrifugation. The C3 convertase alone was able to cleave C3, and its decay was accelerated in the presence of C4-binding protein. The individual roles of Ni2+ and I2 treatment of C2 in the stabilization of the complexes seemed to be different and additive. 63Ni2+ binding coupled to h.p.l.c. analysis showed that 63Ni2+ bound only to the C2ox proteolytic fragment a (1 mol/mol) with a Kd of 26 microM. Competition studies between Ni2+ and Mg2+ indicated that only half of the Ni2+ bound to the C3 convertase was removed by Mg2+, whereas, in the same conditions, Ni2+ bound to C2ox proteolytic fragment a was not displaced, suggesting the presence of two sets of sites on the convertase. EDTA prevented the formation of both C3 convertase and proconvertase; EDTA had no effect on the preformed C3 convertase, whereas it dissociated the preformed proconvertase.

Comparative study of the fluid-phase proteolytic cleavage of human complement subcomponents C4 and C2 by C1s and C1r2-C1s2.

The C3 convertase of the classical pathway of complement is composed of fragments C4b and C2a resulting from cleavage of C4 and C2 by activated C1. The limited proteolysis of these two different substrates by the same protease, C1s, has been studied in the fluid phase using purified proteins. The turnover numbers of C2 and C4 cleavage by C1s were affected to different extents, depending on whether C1s was alone or associated with C1r or with monoclonal antibodies to C1s. The binding of C2 to C4 favours the proteolysis of C2 by C1s, as revealed by the use of I2-treated C2.

A study of a covalent-like interaction between soluble nascent C4b and C4-binding protein.

In the classical pathway of complement, the interaction between C4b and C4bp can be considered as a control of the C3 convertase formation. Purified C4-binding protein (C4bp) interacts with soluble nascent C4b to form covalent-like complexes; the interaction is also possible with nascent C4b-like C4, but not with C4, C4b or C4b-like C4. Formation of the complexes upon incubation of C4bp, C4 and C1s appears to involve a single link between a subunit of C4bp and the alpha' chain of C4b, as observed by SDS-polyacrylamide gel electrophoresis in reducing conditions (160 000 dalton band). In non-reducing conditions, a mixture of C4b-C4bp complexes is observed as a function of the C4b:C4bp molar ratio, with apparent molecular weights differing by a value of 210 000 and reflecting different C4b-C4bp associations. A maximum of five molecules of C4b are bound per molecule of C4bp, which appears to consist of 10 subunits of apparent molecular weight 72 000. The link between C4b and C4bp is partially destroyed by 1 M hydroxylamine at pH 9.0; its formation is strongly inhibited by 3.5 mM hydroxylamine or 60 mM methylamine at pH 9.0. These findings suggest an ester or amide bond between the activated carboxyl group of the thioester bridge in the alpha' or alpha chain of nascent C4b or C4b-like C4 and a hydroxyl or amino group of C4bp. Thus, C4bp might compete with other C4b acceptors such as membranes or IgG.