Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells

Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.     

Elevated NK-mediated lysis of Raji and Daudi cells carrying fixed iC3b fragments

Raji and Daudi cells were opsonized with C3b, iC3b, and C3d fragments by using purified complement components. The sensitivity of C3-opsonized cells to lysis mediated by low density blood lymphocytes was studied. Raji and Daudi cells carrying C3b or C3d fragments were lysed with similar efficiencies as the nonopsonized cells. The presence of iC3b on the target surface imposed elevated NK sensitivity. The iC3b-mediated enhancement of NK lysis was inhibited when iC3b fragments or rabbit anti-human C3 antibodies were included into the lytic assays. These results indicate that the iC3b fragments fixed on the targets bind to the CR3 on the lymphocytes. Results obtained in immobilized conjugate-lytic assays showed that iC3b-opsonized targets interact more readily with the lymphocytes. This was reflected by the elevated proportion of lymphocytes that were bound to the iC3b-carrying targets. The proportions of conjugates in which target damage occurred were similar with the control and with the iC3b-carrying cells. It seems therefore that opsonization of targets with iC3b leads to recruitment of effector lymphocytes due to contact with their CR3. However, once the effector-target contact is established, the triggering of lytic function does not seem to be influenced by the iC3b/CR3 bridge.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

Neutrophils express a receptor for iC3b, C3dg, and C3d that is distinct from CR1, CR2, and CR3

In the present study we examined human neutrophils for the expression of a receptor capable of binding C3dg and defined the relationship of this receptor to those that have been previously described, namely CR1, CR2, and CR3. C3dg was isolated from serum depleted of plasminogen, supplemented with 20 mM Mg++, and incubated at 37 degrees C for 6 to 8 days. The purified protein was homogeneous when analyzed by polyacrylamide gel electrophoresis and exhibited an apparent m.w. of 41,000. C3dg was polymerized by treatment with dimethyl suberimidate, and the dimer was isolated by gel filtration. Binding of both monomeric and dimeric 125I-labeled C3dg to neutrophils was saturable, and the latter ligand bound to an average of 12,400 sites/cell among nine normal individuals. At 4 degrees C, bound monomeric C3dg dissociated from neutrophils with an average t1/2 of 30 min, whereas dimeric C3dg dissociated with a t1/2 in excess of 120 min. Specific binding of multimeric C3dg was cation independent and was competitively inhibited by molar concentrations of iC3b and C3d that were equivalent to the inhibitory concentrations of unlabeled C3dg; C3b was less able to compete with C3dg for binding to these sites. The capacity of this neutrophil receptor to bind iC3b, C3dg, and C3d suggested its possible identity as CR2 or CR3. However, no specific binding to neutrophils of 125I-labeled HB-5 monoclonal anti-CR2 was detected. Furthermore, uptake of 125I-labeled C3dg was not inhibited by saturating concentrations of rabbit anti-CR1, anti-Mac-1, or OKM10. Thus, a receptor resides on neutrophils that binds the C3d region of iC3b and C3dg and is distinct from CR1, CR2, and CR3.     

Interaction of iC3b with recombinant isotypic and chimeric forms of CR2.

CR2 is a component of a signal transduction complex on B lymphocytes that augments B cell responses to Ag. We have quantitatively assessed binding by the two isotypic forms of CR2 for two of its ligands, the polymerized iC3b (p(iC3b)) fragment of C3, and gp350/220, the EBV membrane protein. The recombinant 15-SCR or 16-SCR forms of CR2 bound p(iC3b) with identical affinities. Full binding activity of CR2 for p(iC3b) was observed with a chimera comprised of SCR-1 and -2 of CR2 fused to SCR-17 through -30 of CR1. Therefore, the alternatively spliced SCR-10a has no role in binding p(iC3b), and the binding activity of wild type receptor for iC3b can be reconstituted with SCR-1 and -2 of CR2. The binding affinities of the two isoforms of CR2 for soluble gp350/220 were also similar. Additional sites in the C3c region of C3 have been postulated also to interact with CR2. However, monomeric iC3b and C3d were equally effective in inhibiting the binding of p(iC3b) to CR2, indicating that the C3c region of iC3b does not contribute to the interaction of iC3b with CR2. Finally, the relative abilities of C3b and iC3b to bind to CR1 and CR2 were compared. The conversion of C3b to iC3b generated a ligand with an approximate 100-fold decrease in affinity for CR1 and a 10-fold increased affinity for CR2, resulting in a 1000-fold greater likelihood for binding to the latter receptor that may then promote B cell activation.     

Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens

The expression of complement receptors by human follicular dendritic cells (FDC) was investigated by immunohistochemical techniques by using polyclonal and monoclonal antibodies to antigenic determinants of CR1, CR2, and CR3. Upon optical immunohistochemical examination of frozen sections from human reactive lymph nodes and tonsils by a three-step immunoperoxidase technique, a strong staining of cell bodies and cytoplasmic extensions of FDC was observed in germinal centers with anti-CR1 and anti-CR2 antibodies. Staining for these antigens was also found on cytoplasmic extensions of FDC in the mantle zone and on the plasma membrane of B cells in the entire follicles. Staining of FDC with anti-CR2 antibody was more intense than that of B lymphocytes. Monoclonal antibodies directed against epitopes of the alpha-chain of CR3 weakly stained FDC in follicles in a similar pattern to that which was observed on adjacent sections with mouse monoclonal antibody KIM4 that only recognizes FDC in human lymph nodes. Immunoelectron-microscopy was performed on frozen sections of a lymph node involved with a centroblastic centrocytic B malignant lymphoma and a reactive tonsil with the use of rabbit F(ab')2 anti-CR1 antibodies and mouse monoclonal anti-CR2 antibody. All the plasma membrane of the cell body and cytoplasmic extensions of FDC in germinal centers and in the mantle zones homogeneously stained for CR1 and CR2 antigens. Fibroblastic reticulum cells were negative. The plasma membrane of tumoral B lymphocytes strongly stained with anti-CR1 and weakly stained with anti-CR2 antibodies. The presence of CR1, CR2, and CR3 on FDC is a unique surface characteristic of these cells that should optimally allow the cells to bind antigen/antibody complexes bearing any type of C3 fragment.     

Capping of complement receptors on human neutrophils induced by group A streptococcal cell walls

Peptidoglycan-polysaccharide polymers derived from group A streptococcal cell walls (PG-PS) were opsonized with either purified C3 or normal human serum and were used as a probe to investigate the mobility of CR1 and CR3, the C3b and iC3b receptors, respectively, on human neutrophils. Incubation of monolayers or cell suspensions of neutrophils with PG-PS opsonized with C3b or serum resulted in capping of PG-PS, as detected by fluorescein-labeled antibody to PS. No binding of PG-PS to neutrophils was observed with heat-inactivated serum. By 30 min the cell walls were internalized and observed in one to three vacuoles. Capping was totally inhibited when PG-PS opsonized with C3b or serum was preincubated with Fab'-anti-C3b. Similar inhibition was observed when C3b-opsonized PG-PS was incubated with neutrophils that were preincubated with anti-CR1 or fluid-phase C3b; only partial inhibition of neutrophil capping was observed by using serum-opsonized PG-PS. Because anti-CR1 blocks only the C3b receptor, the cap formation observed with serum-opsonized PG-PS is probably due to CR3. These results suggest that both CR1 and CR3 on neutrophils cap after stimulation by group A streptococcal cell wall fragments.     

Pre-ligation of CR1 enhances IgG-dependent phagocytosis by cultured human monocytes.

Opsonization of the C3b receptor (CR1) on phagocytic cells with C3b enhances both attachment of targets to the cells and subsequent IgG-dependent ingestion of these targets. To explore mechanisms involved in this increased phagocytosis, we adhered cultured human monocytes to surfaces pre-coated with CR1 ligand or control proteins and quantitated ingestion of sheep E opsonized with IgG alone. Three ligands for CR1 resulted in markedly enhanced phagocytosis of targets when compared individually to a panel of non-ligands, as determined by both the proportion of monocytes ingesting targets (percent phagocytosis) and by the number of targets ingested per 100 monocytes (phagocytic index). The ligands included purified C3b, iC3, and Fab fragments of 1B4, a monoclonal anti-CR1, which resulted in a percent phagocytosis of 56.3 (p less than 0.01), 59.0 (p less than 0.01), and 54.4 (p less than 0.02) and a phagocytic index of 281.2 (p less than 0.01), 281.1 (p less than 0.01), and 247.1 (p less than 0.02), respectively. Control proteins including human serum albumin, hemoglobin, Fab fragments of anti-fibronectin, anti-beta 2 microglobulin, and MOPC 21, and Fc fragments of 1B4 and MOPC 21 produced no significant stimulation of phagocytosis, nor did F(ab')2 fragments of monoclonal anti-CR3, M1/70. CR1-specific augmentation of target ingestion was apparent with monocytes cultured in serum-free medium for 1 to 7 days, but was not seen with freshly elutriated cells. Phagocytosis of unopsonized or IgM-coated targets was minimal. These results suggest that the adherent monocytes are primed by CR1 cross-linking for enhanced FcR-mediated phagocytosis even when the CR1 ligand is not present on the targets. This contrasts with the behavior of CR3, and demonstrated functional divergence between these C3 fragment receptors in the phagocytic process.     

Kinetic analysis of the interactions of complement receptor 2 (CR2, CD21) with its ligands C3d, iC3b, and the EBV glycoprotein gp350/220

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.     

The generation of active fragments of complement receptor type 2 by trypsin digestion

B lymphocytes and Raji cells express the complement receptor type 2 (CR2) of 145 kDa which recognises the C3d fragment of C3. When intact cells are treated with trypsin, CR2 is degraded. There is a parallel loss in C3d-mediated rosetting and in proteins which bind to C3d-Sepharose. Initially 97 and then 83 kDa fragments of CR2 are produced which retain C3d binding activity. These fragments are associated with the cell surface and mediate rosetting. Purified 125I-labelled CR2, solubilised in detergent, produces fragments of apparently identical size on treatment with trypsin. The 83 kDa fragment produced by trypsin treatment closely resembles the major C3d binding protein spontaneously released into Raji cell culture medium.     

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.     

Human eosinophils express CR1 and CR3 complement receptors for cleavage fragments of C3

The functional and antigenic characteristics of C3 receptors expressed on human eosinophils were investigated using rosette assays with sheep erythrocytes coated with C3 fragments and flow cytometric analysis of cells stained with anti-receptor antibodies. Purified peripheral blood eosinophils from 13 patients with hypereosinophilia expressed CR1 antigens. In 8 patients, a mean of 14 + 9.5% eosinophils formed C3b-dependent rosettes that were inhibited by F(ab')2 anti-CR1 antibodies. This number increased to 33% following stimulation with leukotriene B4 (LTB4) (10(-7) M). Similar numbers of C3b rosettes were formed by hypodense and normodense eosinophils. Eosinophils from 2 patients from this group expressed 20,000 125I-labeled monoclonal anti-CR1 antibody binding sites/cell. In another group of patients, 55 +/- 9% eosinophils spontaneously formed C3b-dependent rosettes that could not be enhanced by LTB4. In all patients, a mean of 16 +/- 9% eosinophils formed cation-dependent rosettes with C3bi-bearing intermediates that were inhibited by anti-CR3 antibody OKM1. All eosinophils stained with monoclonal antibodies against the alpha chain of CR3. There was no C3d-dependent rosette formation with eosinophils and no eosinophils stained with monoclonal anti-CR2 antibody. Thus, human eosinophils express CR1 and CR3. Since CR3 is required for the adhesion of granulocytes to surfaces and antibody-dependent cellular cytotoxicity of neutrophils, the interaction of C3 fragments with CR3 and CR1 on eosinophils may be of importance in eosinophil-mediated damage of opsonized targets.     

Characterization of CR1- and membrane cofactor protein-like proteins of two primates

We have identified and characterized C3b binding proteins of two primates, orangutan (Pongo pygmaeus) and gorilla (Gorilla gorilla). Detergent solubilized 125I surface-labeled E and PBMC were subjected to affinity chromatography with homologous or human iC3/C3b. These ligands bound a 225,000 single chain protein from orangutan E and PBMC and a 220,000 protein from gorilla E. Proteins of the same Mr were immunoprecipitated by a rabbit polyclonal and two murine mAb to the human CR1 (CD35). The C3b binding protein of gorilla E aligned with that of the common human CR1 polymorphic size variant. Human or orangutan iC3 was also a ligand for a surface-labeled protein doublet of 59,000 and 65,000 from orangutan E. The doublet pattern and mol wts are similar to membrane cofactor protein (or CD46). Further, this doublet was immunoprecipitated by a mAb to human MCP. The MCP-like protein doublet was not isolated from gorilla or human E. Decay accelerating factor (DAF) of orangutan E was also identified and was structurally and antigenically distinct from the MCP-like protein. Orangutan or gorilla E preparations were a cofactor for the cleavage of human iC3 by human factor I and produced the same cleavage fragments as human CR1. Cofactor activity of orangutan E was partially inhibited by preclearance of CR1 and more completely inhibited by preclearance of MCP. Cofactor activity of gorilla E was inhibited by coincubation with a monoclonal antibody to human CR1. These data indicate that the orangutan and gorilla high m.w. proteins are equivalent to human CR1. The orangutan E membrane protein doublet with m.w. of 59,000 and 65,000 possesses biochemical, antigenic, and functional properties of human membrane cofactor protein.     

Expression of CR2 (the C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes.

The expression of CR2 (the C3dg/EBV receptor, CD21) on normal human T lymphocytes was investigated using purified peripheral blood T cells and indirect immunofluorescence with biotinylated anti-CR2 mAb and streptavidin-phycoerythrin. Thirty to 40% of normal peripheral blood T lymphocytes expressed CR2 Ag. The cells expressed three nonoverlapping epitopes of CR2. The specificity of the staining for CR2 epitopes was demonstrated by the ability of unlabeled anti-CR2 mAb but not of anti-CR1 mAb of the same isotype to compete for the binding of biotinylated anti-CR2 mAb to T cells. The intensity of staining of T lymphocytes with anti-CR2 mAb was approximately 10-fold lower than that of peripheral blood B cells. CR2 was immunoprecipitated from purified T lymphocytes as a single protein of apparent Mr 145,000. The presence of CR2 on normal human T lymphocytes suggests that the receptor may modulate the function of T cells in the immune response and the susceptibility of the cells to infection by lymphocytotropic viruses.     

Identification and characterization of the Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2).

In pursuing studies on the early events in the infection of human B cells by Epstein-Barr virus (EBV), we examined the host cell attachment phase with a panel of B-cell-specific monoclonal antibodies. One of the monoclonal antibodies, OKB7, directly blocked the attachment of purified EBV to B lymphocytes in the absence of a second anti-immunoglobulin antibody and thereby prevented EBV infection of tonsil and peripheral blood B cells. Although earlier studies have shown a close association of the EBV and complement receptor (CR2), an anti-CR2 monoclonal antibody, anti-B2, did not directly block the binding of EBV to B cells. A comparison of the structures recognized by these monoclonal antibodies on various cell types and their functional and physiochemical properties was undertaken. Flow cytometric analysis revealed that the molecules detected by OKB7 and anti-B2 were coexpressed to the same extent on B cells but were not expressed on T-cell lines. OKB7 and anti-B2 both immunoprecipitated a 145,000-molecular-weight membrane protein with an isoelectric point of 8.2 from membrane extracts of Raji lymphoblastoid cells. OKB7 and, to a lesser extent, anti-B2 directly blocked the attachment of C3d,g-coated fluorescent microspheres and sheep erythrocytes bearing C3d to B cells, indicating that these antibodies also react with CR2. These studies indicate that the EBV-CR2 receptor is a single membrane glycoprotein which possesses multiple antigenic and functional epitopes.     

Functional properties of membrane-associated complement receptor CR1

It was previously shown that membrane receptors for C3b (CR1) purified from human erythrocytes were powerful inhibitors of the complement cascade and that they encompass the regulatory functions of the serum proteins beta 1H (H) and C4-binding protein (C4bp). In the present report we study the functional properties of membrane-associated CR1. When tonsil lymphocytes, which contain between 30 and 60% of CR1-bearing B cells, are incubated with the red cell complement intermediate EAC14oxy2lim or EAC14oxy23lim, they inhibit both C42 and C423 in a dose-dependent manner. These effects are mediated by membrane-associated molecules. Indeed, mild trypsinization of the lymphocytes abolishes their activity, and formaldehyde-fixed cells are as effective as viable cells. The inhibitory effects are in part mediated by CR1. The lymphocyte activities are reversed about 60% if monoclonal antibodies to CR1 or fluid phase C3b are present in the incubation medium. Moreover, upon addition of C3b-inactivator (l), lymphocytes release C3c fragments from EAC14oxy23b. The release of C3c was also abolished by antibodies to CR1. These results support the idea that CR1, as well as other molecules from the lymphocyte membrane, can function as inhibitor(s) of complement activation in their vicinity.     

Isolation of complement-fragment-iC3b-binding proteins by affinity chromatography. The identification of p150,95 as an iC3b-binding protein.

The proteins from labelled human spleen membranes and polymorphonuclear leucocytes which bind to the iC3b fragment of complement component C3 were prepared by iC3b-Sepharose chromatography in the presence of bivalent cations. Complement receptor type 3(CR3) was eluted from iC3b-Sepharose by removal of bivalent cations. Complement receptors type 1 and 2 (present in spleen but not in polymorphonuclear leucocytes) were sequentially eluted by an NaCl gradient. An additional protein of Mr 135 000 was eluted from iC3b-Sepharose under the same conditions as those used to elute CR3. Preabsorption of the starting material on an anti-(CR3 beta-subunit) antibody column before iC3b-Sepharose chromatography removed the alpha- and beta-chains of CR3 and the 135 000-Mr protein. Preabsorption with iC3b-Sepharose before the anti-(CR3 beta-subunit) antibody column showed that iC3b binds CR3 and p150,95, the smallest member of the group of three homologous proteins that share the same beta-subunit.     

Expression, molecular association, and functions of C3 complement receptors CR1 (CD35) and CR2 (CD21) on the human T cell line HPB-ALL.

We have investigated the expression, molecular association, ligand binding properties, and ability to transduce intracellular signals of CR1 and CR2 C3 receptors on cells of the human HPB-ALL T cell line. CR1 and CR2 on HPB-ALL cells bound polymeric C3b and C3dg and several anti-CR1 and anti-CR2 mAb recognizing different epitopes of the receptors on normal peripheral blood cells. Immunoprecipitated CR1 and CR2 exhibited similar m.w. to those of the receptors on normal peripheral blood T and B lymphocytes. CR1 and CR2 were partially associated in the form of CR1/CR2 complexes in the cell membrane as assessed by the ability of the receptors to cocap and cointernalize and to form a detergent-sensitive complex upon immunoprecipitation analysis. Triggering of CR2 with mAb OKB7 that recognizes an epitope associated with the ligand binding site of the receptor induced an increase in intracellular free calcium concentration in HPB-ALL cells. The signal provided by mAb OKB7 did not synergize with that triggered by anti-CD3 mAb UCHT1. Triggering of CR1 did not result in changes in intracellular free calcium concentration. Our observations have significance for the biology of normal human T cells because the majority of peripheral blood T cells that express CR1 also expressed CR2 and because a change in (Ca2+)i was induced by mAb OKB7 in purified normal T cells. These functions may be relevant for the regulatory role of C3 fragments on the immune response to T-dependent Ag and for the penetration into T cells of lymphocytotropic viruses.     

Characterization of a soluble form of the C3b/C4b receptor (CR1) in human plasma

A radioimmunoassay with the use of soluble 125I-Fab monoclonal anti-CR1 and rabbit IgG anti-CR1 bound to Staphylococcus aureus particles was employed to detect and quantitate CR1 antigen in human plasma. Among 16 normal individuals the concentration of soluble CR1 in plasma ranged from 13 to 81 ng/ml, and a similar range of concentration was found in plasma from 15 patients having systemic lupus erythematosus (SLE). The amount of plasma CR1 in normal donors, but not in SLE patients, significantly correlated with the number of CR1 sites on erythrocytes (r = 0.90, p less than 0.001), and was 7.1% of the amount of receptor that was present on erythrocytes in blood. The concentration of soluble CR1 was not diminished by ultracentrifugation or ultrafiltration of plasma, was not affected by various modes of anti-coagulation or even by clotting of blood, and did not change during incubation of blood at 4 degrees C for up to 4 hr. On sucrose density gradient ultracentrifugation of plasma the CR1 was distributed as a broad peak that overlapped the plasma protein profile. The Mr of plasma CR1 was identical to that of erythrocyte CR1 when assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunoblotting. In addition, the plasma form of CR1 exhibited the same structural phenotype as did receptor from erythrocytes of the same individual. CR1 antigen purified from plasma was as active as CR1 from erythrocytes in promoting the cleavage by factor I of C3b to iC3b, C3c, and C3dg. Therefore, a functionally and structurally intact form of soluble CR1 resides in plasma.     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR1)

We identified on the membrane of mouse spleen cells a polypeptide of Mr 190,000 (S190), with binding affinity for the mouse third component of the complement system (C3). S190, purified by affinity chromatography on C3-Sepharose, has properties resembling those of the human C3 receptor type 1 (CR1). Thus, S190, like CR1, served as a cofactor for the C3b inactivator (I)-mediated cleavage of fluid-phase C3b into iC3b, and had cofactor activity comparable to that of serum factor H (H). S190 also acted as a cofactor for the cleavages of membrane-bound C3b or membrane-bound iC3b into C3c (Mr 140,000) and C3dg (Mr 40,000) by serum factor I. As is the case with CR1, the specific activity of S190 for the cleavages leading to C3c-C3dg formation was approximately 100-fold greater than that of H. We therefore conclude that S190 and CR1 are analogous proteins.