Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP

The receptor-mediated binding of C1q to human phagocytic cells was investigated in this study. By using a C1q binding assay, we determined that purified, elutriated monocytes expressed an average of 4.6 X 10(5) C1q receptors (C1qR) per cell, with an equilibrium binding constant (Keq) of 0.91 X 10(7) (M-1). When analyzed in an identical manner, the polymorphonuclear leukocytes (PMN) expressed an average of 4.2 X 10(5) C1qR per cell, with a Keq for C1q of 1 X 10(7) (M-1). Fluorescent flow cytometric analysis showed that C1q was bound by 98% of the monocytes studied. Further, the pattern formed by these cells was consistent with a normal log distribution, indicating that this was a homogeneous population of cells. When PMN were assayed with flow cytometry, however, we found that C1q was bound by an average of only 45% of the PMN analyzed. Further, these PMN were not dispersed in a normal log distribution, indicating some heterogeneity among the cells that bind C1q. We examined the abilities of the chemoattractant N-formylmethionylleucylphenylalanine (fMLP) and the phorbol ester phorbol dibutyrate (PDBu) to modulate expression of C1qR as compared to the receptor for C3b (CR1). Pretreatment of the monocytes and the PMN with either 10(-6)M fMLP or 10 ng/ml of PDBu significantly increased cell surface CR1 expression, as reported previously by other investigators. In contrast, no significant increase in the number of C1qR on the monocytes or the PMN was observed with any of the concentrations of fMLP or PDBu used during pretreatment. However, with certain pretreatment doses of these agents, some reduction was noted in the amount of 125I-C1q bound to the monocytes and the PMN. This study characterizes the binding of C1q to purified monocytes and confirms previously published values for PMN. The distribution of cells expressing C1qR is shown to be significantly different between identically treated populations of monocytes and PMN. Finally, the abilities of fMLP and PDBu to modulate the binding of C1qR are examined. Our results indicate that the control of C1qR expression differs markedly from that of CR1.     

Platelet C1q receptor interactions with collagen- and C1q-coated surfaces

We recently described specific binding sites for C1q on human blood platelets. Structural similarities between the amino-terminal of C1q and collagen have suggested that receptors for both molecules on platelets might be the same. The present study thus compared the interaction of purified C1q receptors (C1qR) and whole platelets with collagen- and C1q-coated polystyrene surfaces. Surfaces coated with BSA or gelatin served as controls. Purified 125I-labeled C1qR recognized both C1q- and collagen-coated surfaces in a divalent, cation-independent manner. This adhesion was inhibited by polyclonal or monoclonal (II1/D1) anti-C1qR antibodies. Although C1qR adhered preferentially to C1q-coated surfaces, adhesion to bovine and human type I collagen, as well as to human type III and V collagen, was also noted. In parallel studies, 51Cr-labeled platelets bound equally well to collagen- or C1q-coated surfaces, albeit in a magnesium-dependent manner. Partial inhibition of platelet adhesion was observed in the presence of RGDS, despite the inability of RGDS to modify C1qR interaction with C1q or collagen. Moreover, anti C1qR antibodies selectively inhibited platelet adhesion to C1q-coated surfaces, whereas antibodies specific for the GPIa/IIa collagen receptor (6F1) preferentially inhibited platelet collagen interactions. These data support the presence of distinct platelet membrane C1qR, which may cross-react with collagen, and suggest that C1qR are necessary but not sufficient for platelet adhesion to C1q-coated surfaces. Additional divalent cation and/or RGD-sensitive binding sites may participate.     

Identification and partial characterization of human platelet C1q binding sites

We recently showed that human blood platelets bind the complement component, C1q, and mAb directed against lymphoblastoid C1q receptors in a specific and saturable manner. To identify and further characterize platelet C1q binding sites, human platelets were washed, solubilized in Triton X-100 and either subjected to SDS-PAGE and Western blotting by using monoclonal (II1/D1) and polyclonal antibodies recognizing C1qR on lymphoblastoid cells, or applied to a C1q-Sepharose affinity column under low ionic strength conditions (20 mM NaCl). Adherent proteins were eluted with buffer containing 300 mM NaCl. Western blotting with monoclonal and polyclonal antibodies directed against C1qR showed exclusive reactivity with a 67,000 m.w. protein possessing intrachain disulfide bonds. SDS-PAGE of C1q-Sepharose eluates also revealed the presence of a 67,000 protein which was accompanied to varying degrees by a 94,000 constituent. Because similar m.w., 125I-labeled proteins were recovered from C1q-Sepharose columns to which lysed, surface-labeled platelets had been applied, both 94,000 and 67,000 components appear to be platelet membrane constituents. The 94,000 and 67,000 species, however, appear to be antigenically distinct. The 94,000 protein was immunoprecipitated by polyclonal antibodies against platelet membrane glycoprotein IIIa but not polyclonal antibodies against C1qR, whereas the 67,000 protein was immunoprecipitated exclusively by the polyclonal anti-C1qR antibody. The 67,000 protein thus appears to represent platelet C1q binding sites resembling C1qR on lymphoblastoid cells.     

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.     

Characterization of the C1q receptor on a human macrophage cell line, U937.

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.     

The saturable high affinity association of factor X to ADP-stimulated monocytes defines a novel function of the Mac-1 receptor

Initiation of the coagulation protease cascade as it assembles on cell surfaces requires limited proteolytic activation of the zymogen factor X. Not previously suspected to be the ligand of an organizing receptor on cell surfaces, we now describe that factor X specifically associates with cells of monocyte lineage and we identify the high affinity receptor for this zymogen. Following stimulation with ADP (10 microM), or with the ionophore ionomycin (1 microM), isolated human monocytes bind 125I-factor X in a saturable fashion with a dissociation constant (Kd) of 21.8-44.9 nM. Equilibrium binding analyses indicate that the reaction is optimal at room temperature, requires Ca2+ ions, and saturates at 128,500 +/- 21,300 molecules of 125I-factor X specifically associated with the cell surface. Molar excess of unlabeled factor X inhibits and reverses the binding, whereas the homologous gamma-carboxylated coagulation proteins factors II, VII, IX, IXa, and Xa are without effect. Similarly, chelation of divalent ions immediately dissociates bound 125I-factor X. The monoblast cell line U 937 and the monocytic cell line THP-1 when stimulated with ADP or ionomycin, bind 125I-factor X with characteristics similar to monocytes. Receptor identity was explored using antibodies to the leukocyte adhesive receptors Mac-1, LFA-1, and p150.95. Monoclonal antibodies specific for the alpha subunit of Mac-1 (M 1/70, LM 2/1) or for the common beta subunit (TS 1/18, 60.3) bound equally to resting and ADP- or ionomycin-stimulated cells and also completely blocked the binding of 125I-factor X to stimulated monocytes, U 937, or THP-1 cells. To distinguish between modulatory effects of the monoclonal antibodies and direct spatial hindrance binding of 125I-factor X to Mac-1 was analyzed directly. OKM10 anti-alpha subunit of Mac-1 monoclonal antibody immunoprecipitated 125I-factor X chemically cross-linked to its receptor on stimulated cells. In addition, the complement protein fragment C3bi, which is a recognized ligand for Mac-1, competitively inhibited the association of 125I-factor X. These findings indicate that human blood monocytes and less differentiated cells of this lineage possess an inducible receptor specific for factor X; and also support the conclusion that the heterodimeric leukocyte adhesive receptor Mac-1 functions as the specific receptor structure. We suggest that the novel properties of this receptor may be of importance in the organization and regulation of certain coagulation protease cascades on the monocyte surface.     

Lamellar body membrane turnover is stimulated by secretagogues

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. (125)I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.     

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.     

Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C1q-mediated enhancement of phagocytosis

C1q binds to and elicits cellular responses by several cell types, including monocytes, macrophages, neutrophils, B cells, and fibroblasts. The cell-binding domain is located within the collagen-like pepsin-resistant region of the C1q molecule (C1q tails). An affinity matrix of C1q tails coupled to Sepharose was used to select C1q-binding proteins from detergent extracts of surface-iodinated human monocytes, polymorphonuclear leukocytes, and the U937 cells. The major radiolabeled polypeptide eluted specifically from the ligand affinity column had an apparent molecular mass (Mr) of 126,000. Minor iodinated components eluted from Sepharose-tails migrated with Mr of 216,000 and 55,000. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions no change in the migration of any of these polypeptide bands was detected. None of these polypeptides reacted with antibodies directed against the integrins alpha 5 beta 1 (fibronectin receptor) or alpha v beta 3 (vitronectin receptor), LFA-1, or to several other cell adhesion molecules. The Mr 126,000 band was found to contain more than one polypeptide. Lectin binding properties, susceptibility to glycosidases and proteases, and immunoreactivity with the monoclonal antibody L-10, indicated that CD43 (sialophorin/leukosialin) is a component of this band. However, further data show that a monoclonal antibody, generated by immunization with the isolated Clq-binding fractions, recognizes a cell surface sialoglycoprotein distinct from CD43 and inhibits the C1q-mediated enhancement of phagocytosis in monocytes. These latter observations provide the first definitive connection between a specific phagocytic cell surface protein and a known C1q-mediated function. While these proteins contain sialic acid, binding assays and functional assays using neuraminidase-treated cells demonstrate that the functional interaction between C1q and the cell surface is not via sialic acid. The data taken together indicate either that the functional C1q receptor on phagocytic cells is a multi-subunit complex or that multiple proteins can interact with the fragment of C1q containing the cell-binding domain, at least one of which is involved in the C1q-mediated enhancement of phagocytosis.     

Mesenchymal stromal cells derived from umbilical cord blood migrate in response to complement C1q

Background aimsMesenchymal stromal cells (MSC) have great potential for tissue regeneration and cellular therapy. They migrate preferentially to sites of inflammation and tissue injury, but the molecular signals that guide them to their target tissue remain to be elucidated. We have shown that complement component 1 subcomponent q (C1q) enhances the homing-related response of hematopoietic stem/progenitor cells.MethodsIn this study, we investigated whether C1q elicits directional signals that could influence the migration of MSC to injured tissues/organs.ResultsWe found that C1q chemoattracted human umbilical cord blood-derived MSC in a dose-dependent manner and that the receptor for the global domains of Clq (gC1qR) is present on the surface of MSC. Specific gC1qR antibody blocked the chemotactic response of MSC to C1q, indicating that the C1q/gC1qR interaction may be responsible for the C1q-mediated migration of MSC. Further, we found that C1q enhanced/primed the migration of MSC across reconstituted basement membrane Matrigel towards a low gradient of the chemokine stromal cell-derived factor-1 (SDF-1), which is also present at sites of injury, partly as a result of an increase in surface expression of the SDF-1 receptor CXCR4. Moreover, C1q increased the secretion by MSC of matrix metalloproteinase (MMP)-2 and induced the phosphorylation of ERK1/2.ConclusionsThese results indicate that C1q mediates the migration of MSC in two ways: first, by acting as a chemoattractant, and second, by priming chemotactic responses to SDF-1. Our findings suggest new molecular mechanisms of MSC migration that may contribute to their clinical application in tissue repair.     

C1q receptor on murine cells

Different cells and cell lines of murine origin were tested for their capacity to bind the C subcomponent C1q by using biotinylated human C1q and streptavidin-FITC. Cytofluorometric analysis of splenocytes and thymocytes shows that the majority of C1q-reactive cells reside in the population of B cells and macrophages. There is a significant difference in the C1q-binding capacity of in vitro activated cells; although more than half of the B cell blasts bind the C subcomponent, T cell blasts are virtually negative. It is shown that pre-B lymphomas and cell lines of myeloid origin bind C1q strongly (90 to 98%), whereas in the case of mature B cell lymphomas, plasmocytomas, and the tested T cell lines, the percentage of C1q binding cells varies from 0 to 56. C1q affinity chromatography of the detergent extracts from P388D1 and WEHI-3 cells followed by SDS-PAGE of the eluted proteins under reducing conditions reveals a band at approximately 80 kDa. Analysis of splenocytes shows two additional minor C1q-binding molecules with apparent molecular masses of 50 and 45 kDa, whereas in the case of B cell blasts three bands of similar density are seen at approximately 95, 50 and 45 kDa. C1q-receptors of murine cells are shown to be antigenically related to their human counterpart, because a polyclonal antibody (266A) raised against the human C1q receptor reacts with them.     

Surface expression of complement receptor gC1q-R/p33 is increased on the plasma membrane of human spermatozoa after capacitation

Evidence is increasing that complement components might play a role in fertilization. C1q, the first component of the classical complement cascade, has the ability to promote sperm agglutination in a capacitation-dependent manner as well as an effect on sperm-oolemma binding and fusion. We have previously detected gC1qR, the receptor for the globular head portion of C1q, on the surface of capacitated sperm. In this study, we examined the expression of gC1qR in both fresh and capacitated human spermatozoa. We performed immunoprecipitation for gC1qR and analyzed biotinylated sperm membrane by Western blot to illustrate an increase in receptor density after overnight capacitation. These results were confirmed by flow cytometric analysis of spermatozoa using fluorescein isothiocyanate-labeled monoclonal anti-gC1qR antibody. Confocal, indirect immunofluorescence microscopy revealed an increase in receptor expression over the rostral portion of the sperm head after capacitation. In addition, the ability of live spermatozoa to bind to monoclonal anti-gC1qR antibody-coated microtiter wells was also increased after capacitation. These results suggest that gC1qR may play a role in human fertilization.     

分子佐剂C3d上调Raji细胞协同刺激分子B7-1和B7-2的表达(简报)

我们在以往研究中,引入选择性增强体液免疫效应的新型分子佐剂C3d,成功构建了重组避孕疫苗hCGB-C3d3,通过免疫Th2型优势的 BALB/c小鼠和,Th1型优势的C57BL/6小鼠,显示分子佐剂C3d在不同品系小鼠均使免疫效应从Th1型细胞免疫向Th2型体液免疫偏倚。     

Interactions of the extracellular matrix proteoglycans decorin and biglycan with C1q and collectins

Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.     

Direct binding of hepatitis C virus core to gC1qR on CD4+ and CD8+ T cells leads to impaired activation of Lck and Akt

Complement plays a pivotal role in the regulation of innate and adaptive immunity. It has been shown that the binding of C1q, a natural ligand of gC1qR, on T cells inhibits their proliferation. Here, we demonstrate that direct binding of the hepatitis C virus (HCV) core to gC1qR on T cells leads to impaired Lck/Akt activation and T-cell function. The HCV core associates with the surface of T cells specifically via gC1qR, as this binding is inhibited by the addition of either anti-gC1qR antibody or soluble gC1qR. The binding affinity constant of core protein for gC1qR, as determined by BIAcore analysis, is 3.8 x 10(-7) M. The specificity of the HCV core-gC1qR interaction is confirmed by reduced core binding on Molt-4 T cells treated with gC1qR-silencing small interfering RNA and enhanced core binding on GPC-16 guinea pig cells transfected with human gC1qR. Interestingly, gC1qR is expressed at higher levels on CD8(+) than on CD4(+) T cells, resulting in more severe core-induced suppression of the CD8(+)-T-cell population. Importantly, T-cell receptor-mediated activation of the Src kinases Lck and ZAP-70 but not Fyn and the phosphorylation of Akt are impaired by the HCV core, suggesting that it inhibits the very early events of T-cell activation.     

Antibody-independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4.

The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 X 10(5) molecules per bacterium, with a Ka of 1.4 X 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 X 10(5) molecules per bacterium, and a Ka of 0.25 X 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.     

Binding and catabolism of aggregated immunoglobulins containing C3b by U937 cells

We studied Fc receptor and C3b receptor (CR1) function on U937 cells, a human monocyte cell line. C3b was incorporated into stable soluble heat aggregates of 125I-IgM (A-IgM) and 125I-IgG (A-IgG) by using functionally pure classical pathway components. C3b incorporation was verified by the ability of aggregates to bind to human red cells and by cosedimentation of 125I and 131I during ultracentrifugation. Cell uptake and degradation of A-IgG X C3b was increased up to twofold compared with A-IgG not containing C3b molecules. However, A-IgG X C3b bound to CR1 after Fc receptors were blocked with nonradiolabeled A-IgG were also not endocytosed and catabolized. Moreover, A-IgM X C3b was bound but not degraded by U937 cells. As expected, uptake of A-IgM without C3b was negligible. CR1-mediated binding of A-IgM X C3b was specifically inhibited both by a murine monoclonal antibody against the human CR1 that blocks C3b binding and by C3b oligomers generated by trypsin activation of C3, but not by monoclonal antibodies against the iC3b receptor (CR3). We conclude that CR1 on U937 cells cause increased binding of A-IgG, and this increased binding leads to increased Fc-mediated endocytosis and catabolism of model immune complexes. However, binding of soluble ligands by CR1 alone, even when binding is multivalent, does not lead to endocytosis and degradation of soluble ligands bearing C3b.     

Protruding disordered loop of gC1qR is specifically exposed and related to antiapoptotic property in germ cell lineage

We established a monoclonal antibody (MAb), 5G9, with the use of a fixed seminoma tissue from an archival paraffin-embedded specimen, as an immunogen. Without antigen retrieval, positive 5G9-immunohistochemical staining was confined mostly to primordial germ cells, spermatogonia and various germ cell tumors. 5G9 recognized a mitochondrial 32-kD protein with an isoelectric point of pH 4.2, identified as a multifunctional ubiquitous protein, receptor for globular head of C1q (gC1qR), whose epitope was mapped in a disordered loop connecting the β3 and the β4 strands. Reflecting the ubiquitous distribution of gC1qR, with antigen retrieval, 5G9 was found reactive to a wide range of normal and tumor tissues. Since several co-precipitated and phosphorylated bands were observed in various human cell lines but not in germ cell tumor cell lines by in vitro phosphorylation assay, we speculate that the epitope of gC1qR is specifically unmasked in the germ cell lineage. By reducing gC1qR by siRNA, a significant increase was observed in the number of apoptotic cells in ITO-II and TCam-2 cell lines, but to a lesser extent in the Colo201 colon cancer cell line, showing an antiapoptotic property of gC1qR in the germ cells. Since protein–protein interaction is partially preserved by fixation, archival paraffin-embedded specimens can be a valuable source of immunogens for generating monoclonal antibodies (MAbs) that recognize tissue-specific protein conformation.     

Neutralization of hepatitis A virus (HAV) by an immunoadhesin containing the cysteine-rich region of HAV cellular receptor-1

Hepatitis A virus (HAV) infects African green monkey kidney (AGMK) cells via the HAV cellular receptor-1 (havcr-1), a mucin-like type 1 integral-membrane glycoprotein of unknown natural function. The ectodomain of havcr-1 contains an N-terminal immunoglobulin-like cysteine-rich region (D1), which binds protective monoclonal antibody (MAb) 190/4, followed by an O-glycosylated mucin-like threonine-serine-proline-rich region that extends D1 well above the cell surface. To study the interaction of HAV with havcr-1, we constructed immunoadhesins fusing the hinge and Fc portion of human IgG1 to D1 (D1-Fc) or the ectodomain of the poliovirus receptor (PVR-Fc) and expressed them in CHO cells. These immunoadhesins were secreted to the cell culture medium and purified through protein A-agarose columns. In a solid-phase assay, HAV bound to D1-Fc in a concentration-dependent manner whereas background levels of HAV bound to PVR-Fc. Binding of HAV to D1-Fc was blocked by treatment with MAb 190/4 but not with control MAb M2, which binds to a tag epitope introduced between the D1 and Fc portions of the immunoadhesin. D1-Fc neutralized approximately 1 log unit of the HAV infectivity in AGMK cells, whereas PVR-Fc had no effect in the HAV titers. A similarly poor reduction in HAV titers was observed after treating the same stock of HAV with murine neutralizing MAbs K2-4F2, K3-4C8, and VHA 813. Neutralization of poliovirus by PVR-Fc but not by D1-Fc indicated that the virus-receptor interactions were specific. These results show that D1 is sufficient for binding and neutralization of HAV and provide further evidence that havcr-1 is a functional cellular receptor for HAV.