Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

Abstract

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heatinactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component Clq markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component Clq was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of Clq in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100-to 126-kDa Clq-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa Clq receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be Clq.Vascular endothelium plays a central role in inflammation by expressing specific adhesion molecules including P-selectin, E-selectin, and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1) that attract and localize leukocytes to inflamed sites (reviewed in ref. 1). The recent observation that endothelial expression of E-selectin is required for development of inflammatory injury after intravascular injection of immune complexes (ICs) suggests an active role for endothelium in IC-mediated vasculitis (2). However,

Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium

“…This was not surprising since these receptors are found predominantly inside the cell and are expressed on resting ECs in a low number that increases upon cell exposure to pro-inflammatory stimuli, such as LPS, TNF␣ and IFN␥ (Guo et al, 1999) although this remains a controversial issue (Oroszlan et al, 2007). In addition, C1q has been shown to bind to C1qRs with high affinity only when immobilized on polystyrene plate, heat-treated or bound to ICs, suggesting that the conformational changes of the molecule may favour its interaction with the ligand (Lozada et al, 1995;Steino et al, 2004;van den Berg et al, 1998). Heparan sulphates present on the surface of ECs offer an alternative to C1qRs in binding C1q, as suggested by our data showing that treatment of DECs with heparinase removes a substantial portion of bound C1q.…”

“…Binding of C1q to C1qRs has been shown to induce diverse biologic effects including cell adhesion and spreading, expression of the adhesion molecules E-selectin, ICAM-1 and VCAM-1 (Lozada et al, 1995) and production of IL-6, IL-8 and monocyte chemoattractant protein-1 (van den Berg et al, 1998). However, cC1qR and gC1qR are unlikely to be involved in the binding of C1q because we failed to identify bands corresponding to either of the two C1qRs in western blot analysis of C1q purified from DECs by affinity chromatography and to obtain positive signals when the same preparation was examined by a sandwich capture ELISA using antibodies to C1q and to each C1qR.…”

“…Data collected from various groups over the last decade have shown that immune complexes (IC)-bound C1q stimulates the expression of adhesion molecules on EC (Lozada et al, 1995) and also that free C1q induces cell adhesion and spreading (Feng et al, 2002) and production of IL-8, IL-6 and monocyte chemoattractant protein-1 (van den Berg et al, 1998). The effects mediated by free C1q have been shown to involve the interaction of C1q with C1q receptors expressed on the surface of ECs (Peerschke et al, 1993;Peerschke et al, 1996).…”

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia

“…It has been suggested that CRP can directly bind to C1q, 25 which we confirm by SPR, where C1q bound CRP captured by anti-CRP antibody (supplemental Figure 6A-B). However, CRP binding to C1q is unlikely to explain the mechanism of CRP enhancement of phagocyte responses toward IgG1-opsonized platelets, as the effect was observed with purified CRP in the absence of serum ( Figure 3B-E) and after serum heat inactivation, for which C1q is sensitive 26 ( Figure 2A). In further support of this, we did not find C1q binding to spotted IgG1 to facilitate additional CRP binding (supplemental Figure 6B-C), nor did captured C1q by spotted antiC1q antibodies provide a platform for CRP binding (supplemental Figure 6B-C).…”

Cooperation of C1q Receptors and Integrins in C1q-Mediated Endothelial Cell Adhesion and Spreading

“…Among these molecules, receptors for the collagen-like (cC1q-R or collectin receptor, a homolog of calreticulin (CR) 3 ) (1-3), and globular domains (gC1q-R/p33 (also known as p32)) (4,5), of the complement component C1q, have been identified and implicated in various ligand-mediated functions. In addition, endothelial cells have been shown to express C1qRp, a highly glycosylated, membrane-spanning molecule implicated in the enhancement of Fc-and C3b-mediated phagocytosis (6,7). Although both cC1q-R/CR and gC1q-R/p33 lack a transmembrane segment and have been thought to be found predominantly inside the cell, it is now believed that both molecules have a multicompartmental localization, including on the cell surface (8 -11).…”