Complement component C1 and the collectins: parallels between routes of acquired and innate immunity.
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Autoimmunity↗
Biomedical subjects
M G Colomb
Publications and source records attributed to M G Colomb.
Heat shock increases antigenic peptide generation but decreases antigen presentation.
The heat shock response is a universal and highly conserved cellular response to stress. We describe here the effect of elevated temperature on the capacity of B cells to present antigen. Heat shock markedly affects the ability of these cells to process and present tetanus toxin to class II-restricted T cell clones. Inhibition of antigen presentation is due neither to a modification of antigen capture nor to a variation of major histocompatibility complex (MHC) class II molecule synthesis and cell surface expression. Stressed and nonstressed B cells are able to present peptides loaded at the cell surface with the same efficiency. Nevertheless, heat shock leads to an increase of antigen peptide generation in subcellular compartments; an enhancement of cathepsin B activity is also observed. These data suggest that such a stress induces a failure in the intracellular peptide loading onto MHC class II molecules.
Amino Acid Sequence↗
The role of antigen-bound C3b in antigen processing.
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Animals↗
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.
B-Lymphocytes↗
Monoclonal IgG as antigens: reduction is an early intracellular event of their processing by antigen-presenting cells.
Murine mAb injected into patients behave as exogenous antigens and trigger a specific immune response characterized mainly by CD4+ T lymphocytes. They are recognized by T cells under a processed form and in a MHC class II-restricted fashion. IgG degradation which occurs in antigen-presenting cells (APC) has been studied in vitro. We have shown that partial reduction of this antigen is an early event which is significant for the generation of class II-restricted fragments presentable to antigen-specific T cells. Two different murine mAb were used as antigens and human monocytic U937 or antigen non-specific Epstein-Barr virus-transformed B cells as APC. Upon cellular internalization IgG are rapidly cleaved leading to 24-25 kDa fragments. One of the major and early events corresponds to partial reduction of IgG--the light chain is released from the intact molecule, heavy chains remaining bound together. Partial in vitro reduction of IgG followed by presentation by fixed B cells to specific T cells showed that only kappa light chain-specific T cell clones are stimulated, in contrast to heavy chain-specific T cell clones. The response to the kappa chains of specific T cells points to a significant role played by the early IgG partial reduction in the generation of kappa light chain class II binding fragments.
Antibodies, Monoclonal↗
Species specificity of anti-beta 2 glycoprotein I autoantibodies and its relevance to anticardiolipin antibody quantitation.
Some patients suspected of having antiphospholipid antibody syndrome (APS) were found to be positive for anti-beta 2 glycoprotein I (beta 2GPI) antibodies despite negative results for antibodies to cardiolipin (ACA). Since the major source of beta 2GPI in the ACA assay is animal (usually bovine) serum, we studied the influence on ACA quantitation of the species specificity of anti-beta 2GPI antibodies from patients with various autoimmune disorders, mostly systemic lupus erythematosus and primary APS. Ninety-seven sera were selected based on IgG (n = 76) or IgM (n = 64) positivity by ELISA using gamma-irradiated plates coated with human or bovine purified beta 2GPI. A higher proportion of IgM (43.7%) than IgG (7.9%) reacted to human, but not bovine, beta 2GPI. Furthermore, from the samples reactive to both proteins, the ratio of antibody level against bovine to that against human beta 2GPI was 1.08 +/- 0.58 for IgG and 0.58 +/- 0.3 for IgM (p < 10(-5)). IgG and IgM ACA were detected in 78 and 40 sera, respectively; concordance between the two ELISAs for ACA and anti-beta 2GPI antibodies was 94% for IgG and 75% for IgM. Out of 28 IgM showing recognition restricted to human beta 2GPI, 21 were missed by the ACA assay, possibly because of lower concentrations of beta 2GPI in those patients' sera. The antibody reactivity pattern towards human and bovine beta 2GPI of individual sera showed no variation with time and was related to the relative antibody avidity for each protein. A murine anti-human beta 2GPI monoclonal antibody, 9G1, that cross-reacts with bovine beta 2GPI, competed to a large extent with the patients' anti-beta 2GPI antibody binding sites whatever isotype involved or protein recognized. Therefore, anti-beta 2GPI antibodies of IgM isotype display a marked preference for human compared to bovine beta 2GPI responsible for frequent inconsistencies in the ACA assay.
Animals↗
Neutrophil activation by anti-beta 2 glycoprotein I monoclonal antibodies via Fc gamma receptor II.
Murine monoclonal antibodies (mAbs) to human beta 2-glycoprotein I (beta 2GPI), a plasma protein required for the binding of some antiphospholipid antibodies, have been shown to possess lupus anticoagulant properties and to activate platelets via Fc gamma receptor (Fc gamma R) crosslinking. Here we investigated their ability to induce polymorphonuclear leukocyte (PMN) functional responses. The six mAbs (IgG1 isotype) tested in combination with beta 2GPI led to a concentration-dependent activation of human PMNs as appreciated by granule release, H2O2 production, and cytosolic Ca2+ increase. This activation process was accompanied by the enhancement of PMN-mediated heparan sulfate loss from the endothelial cell line EA.hy 926 without evidence for cell lysis or detachment. F(ab')2 fragments of one of the mAbs bound to PMNs in a beta 2GPI-dependent manner but were devoid of activating effects. Carbamylated beta 2GPI was unable to mediate PMN-antibody binding and subsequent activation. In addition, cationization of beta 2GPI or removal of its sialic acid groups led to higher efficiency in binding to the PMN surface and triggering activation in comparison with the untreated protein. Thus, the process of PMN activation depends on mAb binding to these cells through both Fab (via beta 2GPI) and Fc domains, as confirmed by the suppression of all responses upon treatment with an anti-Fc gamma RII, but not anti-Fc gamma RIII, antibody. Our data suggest a model of cellular activation by beta 2GPI-dependent antiphospholipid antibodies.
Antibodies, Monoclonal↗
Anti-beta 2-glycoprotein I antibodies.
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Animals↗
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.
Antigen Presentation↗
Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants.
Some lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect anti-prothrombin autoantibodies in patient serum or plasma, no or very low signal was observed using human prothrombin immobilized on plain polystyrene plates. In contrast, the same LA-positive samples bound specifically to prothrombin coated on gamma-irradiated plates, depending on the radiation dose, in the absence of added calcium and phospholipid. Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was ascertained by competition using liposome-bound prothrombin, since fluid-phase prothrombin competed poorly. Seventy-seven of 139 patients (55.4%) with LA related to a variety of underlying diseases possessed anti-prothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either isolated or more often associated with anti-beta 2 glycoprotein I (beta 2GPI) antibodies. These included 67-71% of the patients with systemic lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19-20% of those with infection or malignancy (p < 0.001). As previously shown for anti-beta 2GPI antibodies, IgG2 was the predominant IgG subclass reactive with prothrombin. Thus, autoimmune patients with LA have a high incidence of antibodies to beta 2GPI and prothrombin, the binding of which could similarly require high antigen density and/or exposure of cryptic epitopes resulting from protein interaction with an irradiated (i.e. more anionic) polystyrene surface.
Autoantibodies↗
Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells.
Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigen-presenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,kappa and IgG2a,kappa murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and purified in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of kappa light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigen-presenting cells.
Animals↗
C3b covalently associated to tetanus toxin modulates TT processing and presentation by U937 cells.
Complement protein C3, like C4 and alpha 2-macroglobulin (alpha 2M), is a potentially bivalent ligand: (1) its proteolytic fragment, C3b, is able to interact covalently with antigens, and (2) this bound fragment is able to interact non-covalently with specific complement receptors of antigen presenting cells (APC). The formation of antigen-C3b complexes frequently occurs in vivo at inflammatory sites during the early stages of an immune response. Tetanus toxin (TT)-C3b covalent complexes, prepared from purified proteins, were used to study how C3b association influences the handling of TT by U937 cells used as APC. TT-specific T cell proliferation following TT-C3b processing was observed at a concentration when TT alone was inefficient. Whereas TT pinocytic uptake was low, TT-C3b uptake, through the help of complement receptor CR1, was three times higher. Free TT was rapidly transported to the lysosomes where it was proteolysed, whereas TT-C3b complexes were first retained in the endosomes and underwent only limited proteolysis. While the ester link of the complexes was fairly stable in the endosomes, it was gradually hydrolysed in the lysosomes with ensuing efficient proteolysis of the two proteins. This reflects the fact that associated C3b escorts TT during intracellular trafficking in the APC, and influences antigen processing. A triple role of C3b escorting antigen residues at the level of antigen uptake, routing, and proteolysis inside U937 cells, thus modulating antigen-dependent T cell proliferation.
Antigen Presentation↗
IgG2 subclass restriction of anti-beta 2 glycoprotein 1 antibodies in autoimmune patients.
The IgG subclass and light chain distribution of antiphospholipid antibodies (aPL) occurring in autoimmune patients were determined by means of two radioimmunoassays using either cardiolipin- or beta 2 glycoprotein 1 (beta 2GP1)-coated microtitre plates and mouse MoAbs. Of 50 sera selected for positivity of anticardiolipin antibodies (ACA) of the IgG isotype, 32 (64%) possessed anti-beta 2GP1 antibodies and their presence was closely associated with clinical features of the antiphospholipid syndrome. Good correlations were found between ACA and anti-beta 2GP1 antibodies when considering antibody level and patterns of light chain and IgG subclass, suggesting that, overall, the same antibodies were being measured. Light chain analysis showed the polyclonal origin of these antibodies and, in most sera, a trend towards use of lambda chain. Among sera positive for anti-beta 2GP1 antibodies, IgG2 was the major subclass reactive with beta 2GP1 and cardiolipin (87% and 74% of the IgG antibody activity, respectively). In contrast, in the group of 18 sera lacking anti-beta 2GP1 antibodies, ACA were largely restricted to IgG3, with a lesser contribution by IgG1. A few selected sera from the anti-beta 2GP1-positive group were shown to contain mixtures of antibodies that required beta 2GP1 (restricted to IgG2 present in large amounts) and did not require this cofactor (restricted to IgG3 and/or IgG1 present in low amounts) for their reactivity with cardiolipin. There was no contribution of glycosylation to the epitopes recognized by anti-beta 2GP1 antibodies, even though human anti-carbohydrate antibodies are restricted to the IgG2 subclass. These findings further emphasize the intra- and interindividual heterogeneity of aPL, and should help to discriminate clinically relevant specificies.
Adolescent↗
Non-coordinated biosynthesis of early complement components in a deficiency of complement proteins C1r and C1s.
We report on a 60-year-old woman with systemic lupus erythematosus and a total (95%) C1r and a partial (36%) C1s deficiency. The patient complained about cutaneous lesions on forearms and legs without other systemic involvement. Elevated anti-nuclear, anti-native DNA and anti-SSA antibodies were present. The finding of persistently depressed levels of haemolytic complement activity (CH50) on both serum and plasma, associated with normal levels of C3, C4 and C2 components, and normal alternative pathway haemolytic activity showed a deficiency of an early component of the classical pathway. Indeed C1r component was below the limits of detection whereas C1s component was lowered (36%). The depressed CH50 was only corrected by purified C1r. Biosynthesis of C1r and C1s by patient's monocytes was spontaneously normal but not up-regulated by interferon-gamma for C1r alone, whereas the biosynthesis of C1s, but also of interleukin-6, was increased, indicating a specific disregulation of C1r. The deficiency was associated with a lupus syndrome and a fatal assumed septic shock. This is in agreement with other reported cases.
Cells, Cultured↗
[Antiphospholipid and anti beta 2-glycoprotein I antibodies].
Recent studies have shown that beta 2glycoprotein I (beta 2GPI), a plasma inhibitor of coagulation with affinity for anionic phospholipids, is frequently required for the formation of the epitopes recognized by some anti-phospholipid antibodies. Six murine monoclonal antibodies directed against human beta 2GPI were compared with anti-beta 2GPI antibodies associated with systemic lupus erythematosus and primary anti-phospholipid syndrome. The beta 2GPI-dependent binding properties to neutral and anionic phospholipids were studied, as well as the target epitopes on the beta 2GPI molecule and the effect of various beta 2GPI treatments to get rid of contaminating phospholipids or to block the beta 2GPI-phospholipid interaction. The results are in favour of a direct binding of patient's antibodies to beta 2GPI, in the absence of phospholipids. Anti-beta 2GPI monoclonal antibodies, two of which inhibited beta 2GPI recognition by the antibodies from patients, possessed lupus-like anticoagulant properties, thereby constituting an interesting model for a subset (beta 2GPI-dependent) of anti-phospholipid antibodies.
Antibodies, Antiphospholipid↗
Platelet activating properties of murine monoclonal antibodies to beta 2-glycoprotein I.
Previously developed murine monoclonal antibodies (MAbs) to human beta 2-glycoprotein I (beta 2 GPI), a plasma protein required for the binding of anti-phospholipid antibodies, were studied for anti-platelet reactivity and influence on platelet function. The six MAbs (IgG1 isotype) tested interacted with both intact and fixed platelets in a beta 2 GPI-dependent manner. Carbamylated beta 2 GPI was still recognized by MAbs but was unable to mediate platelet-antibody binding. MAbs induced aggregation and secretion responses of platelets in platelet-rich plasma (PRP) and whole blood, provided subthreshold concentrations of weak agonists (i.e. ADP or adrenaline) were added. When aggregation in PRP was evaluated by a counting technique instead of turbidometrically, the sole addition of MAbs led to a rapid fall in single platelets. Triggering gel-filtered platelets with MAbs together with beta 2 GPI, but not its carbamylated form, led to platelet activation after a lag time, as monitored by aggregometry, measurements of ATP and beta-thromboglobulin secretion and calcium mobilization. F(ab')2 fragments of one of the MAbs failed to activate platelets but inhibited the responses to the whole antibody. This process thus depends on MAbs binding to platelets through both Fab and Fc domains, as confirmed by the suppression of platelet responses upon pretreatment with the anti-Fc gamma RII MAb IV.3. Aggregation and secretion induced by MAbs plus beta 2 GPI did not require exogenous fibrinogen and were variably inhibited in the presence of acetyl salicylic acid, apyrase or Ca2+, depending on the concentrations used for the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Diphosphate↗
Disulfide linkage between C3b and tetanus toxin on tetanus toxin-specific EBV-transformed B cells.
EBV-transformed B cells specific for tetanus toxin were found to bind C3b in excess over the expected figures based on the number of complement receptors CR1. This was confirmed by analysis of cell extracts by SDS-PAGE giving evidence for C3b-membrane protein complexes that were disrupted upon reduction. Alkylation of C3b-free cysteine abolished formation of these complexes and only a noncovalent binding of C3b to CR1 was observed, which could be inhibited by mAb to CR1. When C3b was incubated with the same cells coated with tetanus toxin bound to their specific membrane Ig, preferential formation of disulfide-bonded complexes between tetanus toxin and C3b was observed. These observations correspond to a novel capacity of C3b to interact covalently through its cysteine 1010 with free SH groups of protein acceptors. One hypothesis is that the disulfide bond formation is catalyzed by a thioredoxin-like protein secreted and expressed on the membrane of EBV-transformed B cells. In the context of Ag processing and presentation by B cells, disulfide binding of chaperone C3b to Ag is likely to persist during transcytosis and to play a significant role in the modulation of the processing.
B-Lymphocytes↗
Associated complement C3b. Towards an understanding of its intracellular modifications.
Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.
Cathepsin B↗