Immune function of C1q and its modulators CD91 and CD93.

C1q is a subcomponent of the first component of complement C1, which is a multimolecular complex comprising one molecule of C1q and two molecules each of the autoreactive proteases, C1r and C1s. This multimolecular complex triggers the classical pathway of complement. Advances in the past several years have provided a partial crystal structure of the C1q subunit. This, together with gene deletion of C1q, has allowed further insight into the multifunctional immune aspects of this molecule. Two C1q-mediated functions that have received intense scrutiny recently are C1q-mediated apoptotic clearance of cell debris and phagocytosis. This has led to a heightened search for specific receptors for the collagen-like region (CLR) as well as the globular heads. Two transmembrane proteins, CD91 and CD93, have been proposed to interact indirectly with the CLR of C1q, promoting apoptotic clearance and phagocytosis, respectively. The aim of this article is to provide an overview of the structural and functional information that implicates CD91 and CD93 in C1q-mediated functional effects.

Intracellular localization of the human receptor for the globular domains of C1q.

This study was performed to determine the localization of the recently described receptor for the globular domain of C1q, gC1qR. In contrast to previous reports, we were not able to detect significant surface expression of gC1qR on Raji cells, monocytes, neutrophils, human or rat mesangial cells, the endothelial cell line EA.hy 926, or HUVEC using FACS analysis. Only by using digoxigenin-conjugated Abs could some surface staining of gC1qR be observed on rat mesangial cells and neutrophils. However, after permeabilizing these cells with saponin, a strong positive intracellular staining for gC1qR was observed by FACS, fluorescence microscopy on coverslips, and confocal laser scanning microscopic analysis. By reflection contrast microscopy and electron microscopy on ultrathin sections of permeabilized Raji cells, it was shown that gC1qR is present in double membranous cytoplasmic vesicles located in the proximity of the plasma membrane. To determine whether certain conditions could induce surface expression of gC1qR, Raji cells were either stimulated with T cell growth factor, LPS, or driven to apoptosis by incubation with fenretinide or by serum depletion. None of the conditions resulted in significant surface expression of gC1qR. Our hypothesis that gC1qR is not a surface molecule but a soluble molecule that is secreted by cells is supported by the observation that gC1qR is found in significant concentrations in supernatants of several cultured cells and in normal human and rat sera. Our results suggest that the recently described gC1qR is not a cell surface receptor, but a soluble binding protein with affinity for the globular heads of C1q. Excreted gC1qR might act as a potential fluid phase regulator of complement activation.

High voltage immunoelectron microscopy of complement receptor type 3-mediated capping and internalization of group A streptococcal cell walls by human neutrophils.

The mechanism of human neutrophil clearance of peptidoglycan group A-specific polysaccharide polymers derived from streptococcal cell walls (PG-APS) was investigated by high voltage immunoelectron microscopy (HVEM) in order to determine how neutrophils process this highly inflammatory bacterial debris. Neutrophil monolayers were incubated from 5-30 min with serum-opsonized PG-APS. Cells were lightly fixed with 0.5% glutaraldehyde, and the PG-APS was localized on the neutrophil surface by immunogold using antibodies to N-acetyl-glucosamine and 15 nm colloidal gold coupled to goat anti-rabbit IgG. Neutrophils were viewed unsectioned by stereo HVEM. Patches of PG-APS were distributed randomly on the plasmalemma of well-spread neutrophils within 5 min. In polarized cells, PG-APS was densely localized on the uropod and retraction fibers. Within 15 min, PG-APS was predominantly concentrated into a large aggregate, measuring approximately 1 micron in diameter, near the cell margin or nucleus. The aggregate of PG-APS was engulfed in the vicinity of the indentation of the nucleus (hof). Intact microfilaments were required for aggregation and internalization of PG-APS. Binding of PG-APS was dependent upon complement fixation. Furthermore, PG-APS elicited an increase in density of complement receptor type 3 (CR3, C3bi receptor) on the neutrophil surface as determined by morphometry of immunogold labeled anti-CR3. When cells were stained for both PG-APS and CR3, co-localization was observed, and stereomicroscopy revealed clusters of CR3 in areas associated with phagocytosis. These data suggest that neutrophils use an efficient mechanism for removal of bacterial debris. Unlike whole streptococci which are phagocytosed at multiple sites, these bacterial cell walls are first collected into a large aggregate, or cap, which is then internalized at one site.

Complement receptors.

Recently, a number of exciting developments have increased our understanding of complement receptors. These advances include determination of the spatial organization of the short consensus repeat unit, analysis of active sites within short consensus repeats, downregulation in vivo of the complement system by a recombinant receptor, elucidation of the structure of mouse receptors and their relationship to human molecules, and the cloning of the human C5a receptor.

Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

Epstein-Barr virus (EBV) receptors on epithelial hybrid cells derived from nasopharyngeal carcinoma.

We previously established an Epstein-Barr virus (EBV) genome-positive epithelial hybrid cell line, designated NPC-KT, which was prepared by fusing primary epithelial cells derived from nasopharyngeal carcinoma (NPC) with an epithelial Ad-AH cell line. In this study, we measured the presence of the EBV receptors using radiolabelled EBV. It was determined that similar amounts of Burkitt's lymphoma (BL)-derived P3HR-1 or NPC-KT-derived EBV can attach BL-derived Raji cells, but that only P3HR-1 virus can attach to NPC-KT cells. In addition, the superinfection of NPC-KT cells with P3HR-1 virus could not be inhibited by pretreatment with a monoclonal antibody against C3d (OKB7). Raji cells adsorbed with OKB7 became much less susceptible to superinfection with P3HR-1 or NPC virus. The data suggest that EBV receptors unrelated to C3d receptor exist.

Direct evidence for the clustered nature of complement receptors type 1 on the erythrocyte membrane.

C receptors 1 (CR1) of human E are involved in the transport of C3b-coated immune complexes (IC) in the circulation. Many studies have suggested that the binding of IC to E is multivalent. This would require CR1 to be clustered on the cell membrane, but no direct evidence for such clustering is available. We studied the distribution of CR1 on human E by immunofluorescence and shadow-casting immuno-electron microscopy techniques with the use of a monoclonal anti-CR1 antibody followed by FITC- or gold-conjugated second antibodies, respectively. By immunofluorescence, CR1 appeared as small dots (clusters) on fixed and unfixed E prepared either at 4 degrees C or at 37 degrees C. In the same donor, the number of clusters varied extensively from cell to cell (e.g., 1 to 43 clusters/E for a donor with 520 CR1/cell), but the mean number of clusters per cell correlated significantly with the mean number of CR1/cell. These images contrasted with those obtained for Rhesus D (RhD) Ag used as controls (RhD Ag are known to be evenly distributed): only a faint uniform fluorescence was seen despite the presence of 10,000 antigenic sites. As determined by immunocytochemical method, more than 65% of the total gold particles were organized in clusters (2 to 15 gold particles/cluster) whether cells were prefixed or not. Quantitative determinations suggested that each gold particle corresponded to one CR1. The fraction of gold particles grouped into clusters of three or more receptors, the mean size of the clusters, and the maximal size of clusters correlated with the mean number of CR1 per cell. By contrast, RhD Ag were distributed homogeneously (less than 2% gold particles in clusters). These data are the first to demonstrate the preclustered nature of CR1 on E. Such distribution could explain the high binding efficiency of C3b-coated IC to E despite the low number of CR1 per cell.