Cooperation of C1q receptors and integrins in C1q-mediated endothelial cell adhesion and spreading

The interaction of C1q with endothelial cells elicits a multiplicity of biologic responses. Although these responses are presumed to be mediated by the interaction of C1q with endothelial cell surface proteins, the identity of the participants is not known. In this study we examined the roles of two C1q binding proteins, cC1q-R/calreticulin and gC1q-R/p33, in C1q-mediated adhesion and spreading of human dermal microvascular endothelial cells (HDMVEC). When HDMVEC were cultured in microtiter plate wells coated with concentrations of C1q ranging from 0 to 50 microg/ml, a specific and dose-dependent adhesion and spreading was observed. The extent of adhesion and spreading was similar to the adhesion seen on collagen-coated wells. Spreading (68 +/- 12%) and to a moderate extent adhesion (47 +/- 9%) were inhibited by anti-gC1q-R mAb 60.11. Similar effects were noted with polyclonal anti-cC1q-R but not with control nonimmune IgG. The two Abs had a slight additive effect (75 +/- 13% inhibition) when mixed together in the proportion of 100 microg/ml anti-gC1q-R and 30 microg/ml anti-cC1q-R. More importantly, a 100% inhibition of spreading, but not adhesion, to C1q-coated wells was observed when HDMVEC were cultured in the presence of 30 microM of the peptide GRRGDSP but not GRRGESP. Furthermore, while anti-beta(1) integrin Ab blocked both adhesion and spreading, anti-alpha(5) integrin blocked only spreading and not adhesion. Ag capture ELISA of endothelial cell membrane proteins using polyclonal anti-gC1q-R showed the presence of not only beta(1) and alpha(5) integrins but also CD44. Taken together these results suggest that endothelial cell adhesion and spreading require the cooperation of both C1qRs and beta(1) integrins and possibly other membrane-spanning molecules.     

Participation of C1q and its receptor in adherence of human diploid fibroblast

C1q binds through its collagen-like domain to specific surface receptors of fibroblasts and to adhesive elements of extracellular matrix including fibronectin, collagens, proteoglycans, and laminin. To determine whether C1q participates in fibroblast adhesion, cells in serum-free medium were plated on surfaces coated with purified C1q at physiologic ionic strength and pH. Surfaces coated with fibronectin or collagen type I served as positive controls, and those coated with BSA were negative controls. Substratum-adsorbed C1q promoted fibroblast adhesion to a maximum of 73% of available cells within 90 min at 37 degrees C. Adhesion was C1q concentration dependent, saturable, specific, and dependent on the collagen-like domain of the molecule. De novo protein synthesis plays a role in adhesion: pretreatment of fibroblasts with cycloheximide reduced adherence about 50% of controls. Addition of exogenous fibronectin, collagen type I, or C1q as soluble mediators did not affect adhesion of the cycloheximide-treated cells to C1q substrate. Adhesion could be accounted for primarily, although not completely, by the C1q receptors. Antibodies raised against the Raji cell C1q receptors (alpha C1qR Ab) specifically inhibited fibroblast adhesion to C1q substrates about 60% of controls. The binding of fibroblasts to C1q substrates could be inhibited about 24% of controls with the GRGDTP cell recognition peptide. GRGDTP and alpha C1q Ab had an additive effect on adhesion that was inhibited 77 to 80% of controls. We conclude from these data that aggregated rather than monomeric C1q may be the natural ligand of the fibroblast C1q receptor, and the biologic function of the receptor in cells of the connective tissue may be cell adhesion.     

Human blood platelets possess specific binding sites for C1q

Although platelet interactions with C1q are implied by the inhibitory effect of C1q on collagen-induced platelet aggregation, specific receptors have not as yet been identified. To address the question of platelet receptors for free C1q, direct radioligand binding studies were performed by using human blood platelets and purified, 125I-labeled C1q, and a monoclonal antibody (II1/D1) (IgM, lambda) directed against C1q receptors on peripheral blood leukocytes. Washed platelets bound both purified 125I-labeled C1q and II1/D1 in a specific and saturable manner under physiologic ionic strength conditions. At equilibrium, approximately 4000 molecules of C1q bound per platelet with an apparent dissociation constant of 3.5 X 10(-7) M. Maximum C1q binding was achieved in 5 min and correlated well with inhibition of collagen-induced platelet aggregation. Equilibrium binding of 125I-labeled II1/D1 to washed platelets required an incubation period of 15 to 30 min and II1/D1 concentrations approaching 50 micrograms/ml. Approximately 2000 molecules of II1/D1 bound per platelet, with an apparent dissociation constant of 2.8 X 10(-8) M. II1/D1 binding could be inhibited by the collagenous tail of C1q (c-C1q), suggesting that platelet receptors for these ligands are either the same or in close proximity. The data demonstrate that human blood platelets possess specific and saturable binding sites for free C1q that may function as collagen receptors, and may antigenically resemble C1q receptors on peripheral blood leukocytes.     

Complement component C1q and its receptor are involved in the interaction of human sperm with zona-free hamster eggs

C1q is a component of the classical complement pathway that can react with the Fc-fragment of immunoglobulins and with other proteins, such as fibronectin, laminin, and a specific C1q receptor present on several cell types. Given its role in many adhesion systems, mainly related to phagocytosis, we tested the effects of C1q on the interaction between human spermatozoa and zona-free hamster eggs. The presence of C1q in the medium used for gamete coincubation resulted in promotion of sperm-oolemma adhesion and an inhibition of penetration. The number of adherent sperm per egg at 5 micrograms/ml concentration was 90 +/- 35 vs. 29 +/- 7 for the control (P less than 0.001). At 1 microgram/ml, the lower concentration at which C1q had an effect, the number of penetrating sperm/egg was 0.6 vs. 1.7 for the control without C1q (P less than 0.01), and the percent of penetrated eggs was 28% vs. 85%. At 50 micrograms/ml, the percent of penetrated eggs was 7%, with a penetration index of 0.07. The addition of C1q to the medium resulted in sperm agglutination, which varied between sperm donors. The presence of C1q receptors, as detected by anti-C1qR monoclonal antibodies (Mabs), was demonstrated both on zona-free hamster eggs by immunobead rosetting and on human spermatozoa by immunobead binding and indirect immunofluorescence. Mabs directed against different epitopes of C1qR had different effects on gamete interaction, with a partial inhibition of penetration mediated by some of them. The binding of C1q to antibody-free human spermatozoa was also demonstrated both by means of indirect immunofluorescence and utilizing 125I-C1q.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella spiralis: activation of complement by infective larvae, adults, and newborn larvae.

The ability of Trichinella spiralis to activate complement (C) has been addressed by several investigators. However, these investigators employed methods in which either detection of C fragments on the parasite surface or the adherence of leukocytes to the parasite was considered an indication of C activation. The present studies were undertaken to examine: (a) whether activation of C occurs via the classical and/or alternative pathway, (b) at which stage(s) of the parasite C activating capacity is acquired, and (c) what molecular entities of the epicuticle and/or cuticle are responsible for initiating C activation. Our studies indicate that T. spiralis activates C primarily via the alternative pathway (and weakly via the classical pathway) since incubation of parasites obtained from infected mice with either normal human serum (NHS) or Mg.EGTA-NHS, followed by incubation (1 hr, 37 degrees C) with antibody-sensitized sheep erythrocytes or rabbit erythrocytes, respectively, showed a time-and parasite number-dependent depletion of C. Although the three stages of T. spiralis, i.e., infective larvae, adults and newborn larvae, are capable of activating C, the newborn appears to be the most potent activator, especially when parasite number and size are taken into consideration. Further evidence of C activation is obtained from SDS-PAGE and Western blot analysis in which homogenates of parasites preincubated with NHS showed the presence of C3, C9, and C1q, whereas controls without serum were negative. Since isolated C1q was also capable of directly binding to the surface of adults and infective larvae, it is postulated that their cuticle and/or epicuticle may possess surface structures which serve as binding sites for C1q.(ABSTRACT TRUNCATED AT 250 WORDS)