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.     

Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CR1) and comparison with H, C4b-binding protein (C4bp), and decay accelerating factor (DAF)

Four CR1 variants have been found in the normal population and are designated CR1-A (190,000 daltons), CR1-B (220,000 daltons), CR1-C (160,000 daltons), and CR1-D (250,000 daltons). In the present study, we first developed an improved chromatographic purification scheme for CR1 that does not employ a C3b affinity step. CR1 variants (A, B, and C) were then isolated, and their individual functional activity was assessed. Each possessed similar co-factor activity for I-mediated cleavage of C3(H2O), as well as for the inhibitory activity for fluid phase C3 convertases. These results indicate that, despite relatively large Mr differences, in the purified state these three CR1 variants have similar functional activities. The functional activity of CR1 was also compared with C4bp, H, and decay accelerating factor (DAF) in fluid phase assays designed to assess the inhibition of the C3 convertases and co-factor activity. On a molar basis, CR1 had approximately the same inhibitory activity as C4bp for the classical pathway convertase, and had the same as H for the alternative pathway convertase. These results indicate that CR1 encompasses the functional capabilities of both proteins. They also raise a number of interesting genetic and structural questions in regard to these complement regulatory proteins, because C4bp is thought to have multiple C4b binding domains, whereas H is reported to bind one C3b. DAF was an approximately fourfold better inhibitor of the alternative pathway convertase than CR1 or H, but was a fourfold less efficient inhibitor of the classical pathway convertase than CR1 or C4bp. The effective inhibitory capacity of DAF in these fluid phase assay systems suggests that the DAF substrate specificity is for the convertases. Fluid phase CR1 was twofold less efficient than H in serving as a co-factor for the first cleavage of fluid phase C3b, and hardly mediated the second cleavage. These data are in contrast to the co-factor activity of CR1 on a cell membrane, and provide additional evidence for the local environment being a critical modulator of the function of proteins that regulate the activation of C3.     

CR1, the C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophages

The role of complement receptors on monocyte derived human macrophages in phagocytosis of infective (MP) and noninfective (LP) developmental stages of Leishmania major promastigotes was studied. We compared binding of these specific developmental stages to MO after preincubation in fresh or heat-inactivated serum. Although LP do not require fresh serum for attachment, MP were dependent on serum C opsonization for entry. Inhibition of CR1 substantially abolished binding of the infective MP. In contrast, inhibition of iC3bR (CR3 and p150,95) had no effect on MP binding. Inhibition of both iC3bR, however, did block binding of nonopsonized LP. Attachment of LP to CR3 was blocked by fluid phase addition of mAb OKM1 and M1/70, which inhibit complement-independent binding to CR3, but not by mAb OKM10 which inhibits iC3b binding to this receptor. After fresh serum pretreatment of LP, however, only simultaneous inhibition of CR3 and CR1 effectively blocked their attachment. Addition of mannan did not inhibit attachment of either promastigote stage. Both opsonized and nonopsonized LP trigger a respiratory burst in MO, possibly via the C independent site in CR3, whereas the CR1-mediated uptake of MP does not generate a respiratory burst. The use of this receptor by MP may facilitate their subsequent intracellular survival.     

Translation of the human C3b/C4b receptor mRNA in a cell-free system and by Xenopus oocytes

The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190,000, 220,000, 250,000, and 280,000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220,000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CR1 and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CR1 monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Analysis of C3-receptor activity on human B-lymphocytes and isolation of the complement receptor type 2 (CR2).

The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.     

Influence of glycosylation on allelic and cell-specific Mr variation, receptor processing, and ligand binding of the human complement C3b/C4b receptor

The human complement C3b/C4b receptor (CR1), a single chain membrane glycoprotein of Mr approximately 200,000, has several cell-specific Mr variations as well as an allelic variation involving four phenotypes whose Mr values span a range of 90,000. We investigated the role of glycosylation in these structural variations and in receptor metabolism. In the human HL-60 promyelocytic leukemic cell line differentiated toward granulocytes or monocytes and in Epstein-Barr virus-transformed B lymphoblastoid cell lines of the common phenotype, CR1 is synthesized as a precursor of Mr = 222,000 that is converted into mature CR1s with differing Mr values. Endoglycosidase F treatment of mature CR1 from all these cell types produced the same lower Mr band. Additionally, the previously noted 5,000 higher Mr of CR1 from human peripheral granulocytes versus erythrocytes was abrogated by endoglycosidase F. Hence these cell-specific variations are due to differences in N-glycosylation. Lectin affinity chromatography shows that these N-linked oligosaccharides are mostly tri- and tetraantennary complex-type species with specific differences in fractionation patterns that correlate with their differing Mrs. In lymphoblastoid cell lines, the four allelic variants each have a precursor 6,000 lower in Mr than the respective mature CR1. In the presence of tunicamycin, each of the CR1 allelic products is 25,000 lower in Mr than the glycosylated receptor. The failure to radiolabel CR1 with [3H]glucosamine in the presence of tunicamycin indicates the lack of O-linked oligosaccharide on CR1. These data, taken together, strongly suggest that the CR1 polymorphism resides at the polypeptide level. Nonglycosylated CR1 synthesized in the presence of tunicamycin had twice the turnover rate of glycosylated CR1. The efficiency of surface membrane insertion and of ligand binding to hemolytically inactive C3 were markedly reduced for nonglycosylated CR1, suggesting that glycosylation is important for the proper expression of CR1 function.     

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.     

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.     

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.     

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.     

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.     

Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d,g (alpha 2D), and C3g

The fragments that result from the inactivation of C3b have not been completely characterized. Initial inactivation is catalyzed by the protease factor I, which, in the presence of its cofactor (factor H), cleaves two peptide bonds in the alpha'-chain of C3b. This results in the release of a small peptide (C3f, Mr 3000) from iC3b, which consists of the C3 beta chain covalently bonded to two alpha'-chain-derived peptides (Mr 68,000 and Mr 43,000). Surface-bound iC3b is cleaved at a third site by factor I to produce C3c and C3d,g (or alpha 2D). The factor I cofactor for this cleavage is the C3b receptor that is present on erythrocyte and leukocyte membranes. This report describes the isolation and initial structural characterization of C3c and C3d,g generated in whole blood after complement activation with cobra venom factor. These fragments were compared with the C3 fragments isolated from the serum and plasma of a patient with complement activation in vivo. The fragments were isolated with two solid phase monoclonal antibodies, one of which recognizes a determinant on C3g (clone 9) and one of which recognizes a determinant on C3c (clone 4). C3c isolated from normal blood showed three polypeptides that had apparent m.w. of 75,000, 43,000, and 27,000. The C3d,g consisted of a single polypeptide chain with a m.w. of 40,000. Amino terminal sequence analysis showed that the Mr 27,000 peptide from C3c is derived from the amino terminal portion of the alpha'-chain of C3b, whereas the Mr 43,000 peptide is derived from the carboxy terminus of the same chain. Amino terminal sequence analysis showed also that C3g is derived from the amino terminus of C3d,g. The C3 fragments isolated from a patient with partial lipodystrophy, nephritic factor activity, low serum C3 levels, and circulating C3 cleavage products showed a more complicated pattern on SDS-PAGE. The fragment isolated with clone 9 had an apparent m.w. of 40,000, identical to C3d,g generated in vitro, and it had the same amino terminal sequence as C3d,g generated in vitro. The eluate from insolubilized clone 4, however, showed prominent bands with Mr of 75,000, 56,000, 43,000, and 27,000, together with a triple-banded pattern at 68,000 and a minor band at 80,000. This eluate thus appears to contain C3c, and iC3b or an iC3b-like product. The origin of the Mr 56,000 and Mr 80,000 peptides have not yet been determined. These studies, with previous data, definitively order the C3c and C3d,g peptides in the alpha-chain of C3.(ABSTRACT TRUNCATED AT 400 WORDS)     

The superfamily of C3b/C4b-binding proteins

The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively, which have more limited homology with the repetitive regions in this family. All available data indicate that multiple gene duplications and exon shuffling have been important features in the divergence of this family of proteins with the 60-amino-acid repeat.     

Beta-glucan, a "specific" biologic response modifier that uses antibodies to target tumors for cytotoxic recognition by leukocyte complement receptor type 3 (CD11b/CD18).

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.     

Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11b/CD18).

The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.     

C5a-mediated leukotriene B4-amplified neutrophil chemotaxis is essential in tumor immunotherapy facilitated by anti-tumor monoclonal antibody and beta-glucan

Intravenous and orally administered beta-glucans promote tumor regression and survival by priming granulocyte and macrophage C receptor 3 (CR3, iC3bR and CD11b/CD18) to trigger the cytotoxicity of tumor cells opsonized with iC3b via anti-tumor Abs. Despite evidence for priming of macrophage CR3 by oral beta-glucan in vivo, the current study in C57BL/6 and BALB/c mice showed that granulocytes were the essential killer cells in mAb- and oral beta-glucan-mediated tumor regression, because responses were absent in granulocyte-depleted mice. Among granulocytes, neutrophils were the major effector cells, because tumor regression did not occur when C5a-dependent chemotaxis was blocked with a C5aR antagonist, whereas tumor regression was normal in C3aR(-/-) mice. Neutrophil recruitment by C5a in vivo required amplification via leukotriene B(4), because both C5a-mediated leukocyte recruitment into the peritoneal cavity and tumor regression were suppressed in leukotriene B(4)R-deficient (BLT-1(-/-)) mice.     

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.     

Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation

Although surface membrane density of complement receptor type one (CR1) on erythrocytes (E) is probably an inherited trait among normal individuals, recent evidence from our laboratories suggests that the reduced number of CR1 per E observed in patients with systemic lupus erythematosus (SLE) results from acquired as well as genetic factors. In the present investigation, the number of CR1 per E was quantitated with 125I-monoclonal anti-CR1 and was found to vary inversely with disease activity in patients with SLE who were followed serially for as long as 14 mo. Although evidence for E surface-bound immune complexes or fixed C3b/iC3b was not obtained, periods of disease activity and low amounts of CR1 per E correlated with the presence of 100 to 800 molecules per E of fixed C3dg fragments (less than 100 C3dg per E in normal subjects). Reduced CR1 and excess fixed C3dg on E also were observed in patients with other disorders associated with complement activation, including chronic cold agglutinin disease, autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH), Sj?gren's syndrome, and mycoplasma pneumonia. A significant negative correlation (r = -0.498) between CR1/E and fixed C3dg/E was demonstrable in 255 individual assays evaluated by regression analysis. CR1 decreased and fixed C3dg increased during active disease; the converse was obtained during remission. In patients with active SLE, both serum complement activity and E CR1 decreased, whereas fixed C3dg fragments increased. By piecewise linear regression analysis, the appearance of 100 to 400 C3dg molecules on patients' E corresponded to a 27 to 60%, reduction in the number of CR1 per E (p less than 0.0002), confirming that fixation of C3 to E was correlated with a loss of CR1. In patients with PNH, low values for CR1 were observed on moderately complement-sensitive PNH type II E in association with increased fixed C3 fragments; however, the markedly complement-sensitive PNH type III E had essentially normal amounts of CR1 and bore little fixed C3. The addition of soluble DNA/anti-DNA immune complexes to normal blood generated levels of fixed C3dg fragments on E comparable to those observed on E from patients with SLE. Kinetic experiments indicated that C3b was fixed to E during the process of immune complex binding and release from E CR1, and that this fixed C3b was subsequently degraded rapidly to fixed iC3b and more slowly to fixed C3dg without the loss of CR1 that occurs in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)