A new role for complement C3: regulation of antigen processing through an inhibitory activity.

Increasing evidence underlines the involvement of complement component C3 in the establishment of acquired immunity which appears to play a complex role and to act at different levels. As antigen proteolysis by antigen presenting cells is a key event in the control of antigen presentation efficiency, and consequently in the quality of the immune response, we investigated whether C3 could modulate this step. Our results demonstrate for the first time that C3 can interfere with antigen proteolysis: (i) proteolysis of tetanus toxin (TT) by the lysosomal fraction from a human monocytic cell line (U937) is impaired in the presence of C3, (ii) this effect is C3-specific and involves the C3c fragment of the protein, (iii) C3c is effective even after disulfide disruption, but none of its three constitutive peptides is individually accountable for this inhibitory effect and (iv) the target-protease(s) exhibit(s) a serine-protease activity. The physiological relevance of our results is demonstrated by experiments showing a subcellular colocalisation of TT and C3 after their uptake by U937 and the reduction of TT proteolysis once internalised together with C3. These results highlight a novel role for C3 that broadens its capacity to modulate acquired immune response.

Prion protein activates and fixes complement directly via the classical pathway: implications for the mechanism of scrapie agent propagation in lymphoid tissue.

C1q-deficient and complement depleted mice are highly resistant to intraperitoneal scrapie infection. The molecular mechanisms of complement involvement in scrapie pathogenesis remain unclear. Previous detailed studies have indicated mouse prion protein interactions with human C1q but the question of subsequent complement activation has remained unaddressed. In this investigation, murine prion protein, both recombinant and also from diseased tissue sources, directly activated and fixed complement via the classical but not the alternative pathway. The importance of complexed cupric ions was observed. In addition, evidence of IgG-independent C4 fixation by prion proteins was also shown. Surface plasmon resonance binding studies using variously clustered immobilized recombinant mouse prion protein indicated strong interactions with both purified mouse C1q and also mouse Factor H. Binding, especially by C1q, was dependent upon the volume of immobilized prion protein, suggesting a threshold of clustering density required to support strong interactions. Furthermore, clustered immobilized prion protein appeared capable of promoting polymerization of soluble-phase monomeric prion protein. Direct covalent attachment of complement components to prion proteins via classical pathway activation illustrates a potential mechanism underpinning their trafficking to, and subsequent propagation within, lymphoid tissues.

C1q binding and complement activation by prions and amyloids.

C1q binds to many non-self and altered-self-materials. These include microorganisms, immune complexes, apoptotic and necrotic cells and their breakdown products, and amyloids. C1q binding to amyloid fibrils found as extracellular deposits in tissues, and subsequent complement activation are involved in the pathology of several amyloid diseases, such as Alzheimer's disease. Prion diseases, such as scrapie also involve formation of amyloid by polymerization of the host prion protein (PrP). Complement activation is likely to contribute to neuronal damage in the end stages of prion diseases, but is also thought to participate in the initial infection, dissemination and replication stages. Infectious prion particles are likely to bind C1q and activate the complement system. Bound complement proteins may then influence the uptake and transport of prion particles by dendritic cells (DCs) and their subsequent proliferation at sites such as follicular DCs.

Complement receptors and B lymphocytes.

Most of the biological processes depend on cell-to-cell and protein-to-cell interactions, which take place through receptors present on the cell surface. Various physiological systems are linked by such interactions, as is the case for innate and adaptative immune response. There is increasing evidence that two of the main actors involved in host defense, namely, the proteins of the complement system (nonspecific response) and the B lymphocytes (specific response), are strongly connected through the complement receptors displayed on the B-cell surface. Many parameters account for the importance of these molecules: (1) their diversity in terms of binding specificity allows them to interact with different fragments resulting from complement activation and C3 component proteolysis; (2) the structures of their extra- and intracytoplasmic domains differ from one receptor to another, controlling their interactions with other nall surface molecules as well as pathogens and regulating cell signaling; (3) their expression on the majority of the cells involved in immune response, especially B lymphocytes, make them an essential link between specific and nonspecific immune responses. This review deals with these different aspects, taking into account the most recent data.

Relationship between humoral response against hepatitis C virus and disease overcome.

ABSTRACT: Hepatitis C virus infection leads to liver disease whose severity can range from mild to serious lifelong illness. However the parameters involved in the evolution of the disease are still unknown. Among other factors, the virus-elicited antibody profile is suspected to play a role in the outcome of the disease. Analysis of the relationship between anti-virus antibodies and disease state requires the analysis of a large number of serums from patients (hepatitis C virus+) and of epitopes from the viral proteins. Such a study would benefit from microarray-based screening systems that are appropriate for high-throughput assays. We used a method combining peptide chips and surface plasmon resonance imaging previously shown to be suitable for analyzing complex mediums and detecting peptide-protein interactions. 56 peptides covering the entire viral proteome were grafted on chips and their interaction with antibodies present in the 68 injected serums from infected and non-infected donors was measured. Statistical analyses were conducted to determine a possible relationship between antibodies (specificity and amount) and disease states. A good discrimination between infected and non-infected donors validated our approach, and several correlations between antibodies profiles and clinical parameters have been identified. In particular, we demonstrated that ratios between particular antibodies levels allow for accurate discrimination of patients according to their pathologic states. CONCLUSION: Humoral response against hepatitis C virus linear epitopes is partly modified according to the disease state. This study highlights the importance of considering relative quantities of antibodies with different specificities rather than the amount of each antibody.

Human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice.

Multiple sclerosis (MS) is a complex multifactorial disease of the central nervous system (CNS) for which animal models have mainly addressed downstream immunopathology but not potential inducers of autoimmunity. In the absence of a pathogen known to cause neuroinflammation in MS, Mycobacterial lysate is commonly used in the form of complete Freund's adjuvant to induce autoimmunity to myelin proteins in Experimental Allergic Encephalomyelitis (EAE), an animal model for MS. The present study demonstrates that a protein from the human endogenous retrovirus HERV-W family (MSRV-Env) can be used instead of mycobacterial lysate to induce autoimmunity and EAE in mice injected with MOG, with typical anti-myelin response and CNS lesions normally seen in this model. MSRV-Env was shown to induce proinflammatory response in human macrophage cells through TLR4 activation pathway. The present results demonstrate a similar activation of murine dendritic cells and show the ability of MSRV-Env to trigger EAE in mice. In previous studies, MSRV-Env protein was reproducibly detected in MS brain lesions within microglia and perivascular macrophages. The present results are therefore likely to provide a model for MS, in which the upstream adjuvant triggering neuroinflammation is the one detected in MS active lesions. This model now allows pre-clinical studies with therapeutic agents targeting this endogenous retroviral protein in MS.

Biosensor for direct cell detection, quantification and analysis.

Microarrays are promising tools for cell isolation and detection. However, they have yet to be widely applied in biology. This stems from a lack of demonstration of their sensitivity and compatibility with complex biological samples, and a lack of proof that their use does not induce aberrant cellular effects. Herein, we characterized and optimized a recently developed technology associating antibody microarrays with surface plasmon resonance imaging (SPRi). Using a murine macrophage cell line we demonstrate the binding specificity of our antibody-microarrays and the correlation between SPRi signals and both the number of bound cells, and the level of expression of cell surface markers. Confocal microscopy reveals that cell binding to the chip through antibody-antigen interactions underwent morphological changes reflecting the density of the relevant cell surface marker without affecting cell viability as shown by fluorescent microscopy. The detection threshold of the microarray-SPRi system is lowered 10-fold by applying a polyethylene oxide film to the gold surface of the chip. This increased sensitivity allows the detection of cells representing as little as 0.5% of a mixed population. The potential of this method is illustrated by two applications: characterization of ligand-cell receptor interactions, allowing determination of receptor specificity, and analysis of peripheral blood mononuclear cells, demonstrating the suitability of this tool for the analysis of complex biological samples.

Clinically related protein-peptide interactions monitored in real time on novel peptide chips by surface plasmon resonance imaging.

BACKGROUND: Developing rapid, high-throughput assays for detecting and characterizing protein-protein interactions is a great challenge in the postgenomic era. We have developed a new method that allows parallel analysis of multiple analytes in biological fluids and is suitable for biological and medical studies. METHODS: This technology for studying peptide-antibody interactions is based on polypyrrole-peptide chips and surface plasmon resonance imaging (SPRi). We generated a chip bearing a large panel of peptide probes by successive electro-directed copolymerizations of pyrrole-peptide conjugates on a gold surface. RESULTS: We provide evidence that (a) the signal produced by antibody binding is highly specific; (b) the detected signal specifically reflects the antibody concentration of the tested solution in a dose-dependent manner; (c) this technique is appropriate for analyzing complex media such as undiluted sera, a novelty with respect to previous techniques; and (d) correlation between classic ELISA results and the SPRi signal is good (P = 0.008). We also validated this system in a medical model by detecting anti-hepatitis C antibodies in patient-derived sera. CONCLUSION: Because of its characteristics (easy preparation of the peptide chip; high-throughput, label-free, real-time detection; high specificity; and low background), this technology is suitable for screening biological samples and for large-scale studies.

Overexpression of cellular prion protein induces an antioxidant environment altering T cell development in the thymus.

Cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein whose roles are still widely discussed, particularly in the field of immunology. Using TgA20- and Tg33-transgenic mice overexpressing PrP(C), we investigated the consequences of this overexpression on T cell development. In both models, overexpression of PrP(C) induces strong alterations at different steps of T cell maturation. On TgA20 mice, we observed that these alterations are cell autonomous and lead to a decrease of alphabeta T cells and a concomitant increase of gammadelta T cell numbers. PrP(C) has been shown to bind and chelate copper and, interestingly, under a copper supplementation diet, TgA20 mice presented a partial restoration of the alphabeta T cell development, suggesting that PrP(C) overexpression, by chelating copper, generates an antioxidant context differentially impacting on alphabeta and gammadelta T cell lineage.

Normal differentiation and functions of mouse dendritic cells derived from RAG-deficient bone marrow progenitors.

Dendritic cells (DC) mature upon infectious agent detection to elicit immune responses. It has been suggested that T cells influence peripheral DC function. However, it is not known if lymphocytes influence DC progenitors. Therefore, we determined the ability of bone marrow progenitors from T and B cell-deficient mice to generate functional DC. We report that bone marrow-derived DC from RAG-2(-/-) mice differentiate and proliferate normally. Moreover, such generated DC efficiently internalize particles, mature in response to various Toll-like receptor engagement, and activate allogenic T cells. This work strongly supports that early signals delivered during DC ontogeny by mature lymphocytes do not influence the functional differentiation of DC progenitors.

Improvement of long-lasting response and antibody affinity by the complexation of antigen with complement C3b.

Complement protein C3 plays a major role in cell regulation and immune response. This last point is mainly due to C3's capacity to act as a bifunctional link between antigen and immunocompetent cells. In a previous work, we have reported the adjuvant effect produced by linking C3 fragments to antigen. In this paper, we characterize the long-term secondary antibody response induced by C3b-antigen complexes. Mice were immunized using either a protein (hen egg lysozyme) or an ovalbumin-derived peptide as antigen. We compared the secondary response elicited by these antigens covalently linked to C3b or emulsified in complete Freund's adjuvant (CFA). Our results provide evidence that C3b linkage induces better long-term adjuvant effect than CFA, resulting in the production of a higher specific IgG titer with a better affinity.

B cell receptors and complement receptors target the antigen to distinct intracellular compartments.

The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation.

Influence of complement C3 amount on IgG responses in early life: immunization with C3b-conjugated antigen increases murine neonatal antibody responses.

Complement component C3, which plays an important role in both the innate and adaptative immune response, is present at low level in human infants. We show here that: (i) serum C3 amount is weak also in infant mice, (ii) these young animals fail to upregulate C3 to adult levels following tetanus toxoid immunization, (iii) neonatal macrophages have a limited capacity to synthesize C3 upon LPS exposure, (iv) conjugation of antigen to C3b significantly enhances antibody response elicited in 1-week-old mice--although it does not increase primary IgG response in adult mice. Altogether, this identifies C3 as one of the factors limiting early life antibody response and emphasizes the potential interest of immunization strategies overcoming this limitation.

Cryptic O2- -generating NADPH oxidase in dendritic cells.

All the components of the O(2)(-)-generating NADPH oxidase typically found in neutrophils, namely a membrane-bound low potential flavocytochrome b and oxidase activation factors of cytosolic origin, are immunodetectable in murine dendritic cells (DCs). However, in contrast to neutrophils, DCs challenged with phorbol myristate acetate (PMA) can barely mount a significant respiratory burst. Nevertheless, DCs generate a substantial amount of O(2)(-) in the presence of PMA following preincubation with pro-inflammatory ligands such as lipopolysaccharide and pansorbin, and to a lesser extent with anti-CD40 or polyinosinic polycytidylic acid. We found that the virtual lack of the oxidase response to PMA alone is specifically controlled in DCs. Through the use of homologous and heterologous cell-free systems of oxidase activation, we showed the following: (1) a NADPH oxidase inhibitory factor is located in DC membranes; it exerts its effect on oxidase activation and not on the activated oxidase. (2) The inhibition is relieved by pretreatment of DC membranes with beta-octylglucoside (beta-OG). (3) The beta-OG-extracted inhibitory factor prevents the activation of neutrophil oxidase. (4) The inhibitory activity is lost after treatment of DC membranes with proteinase K or heating, which points to the protein nature of the inhibitory factor. Overall, these data indicate that the O(2)(-)-generating oxidase in DCs is cryptic, owing to the presence of a membrane-bound inhibitor of protein nature that prevents oxidase activation. The inhibition is relieved under specific conditions, including a prolonged contact of DCs with pro-inflammatory ligands from microbial origin, allowing a substantial production of O(2)(-), which may contribute to the response of DCs to a microbial exposure.

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells.

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.