gp140, a C3b-binding membrane component of lymphocytes, is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4)

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.     

Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine-transfected fibroblasts

CR1 and CR2 are expressed as associated proteins on the B-lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1- and CR1-CR2-expressing cells bound C3b and C3b-dimer whereas CR2- and CR1-CR2-expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1-CR2-positive cells internalized two- to threefold more C3b and 1.5-fold more iC3b than CR1- and CR2-single-positive cells, respectively. Internalization of the anti-CR1 antibody J3D3, or C3de was at the same level, in both double-transfected and single-transfected cells. Furthermore, the internalization of C3b dimer by CR1-CR2 cells was impaired in the presence of OKB7, an anti-CR2-blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.     

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.     

Inhibition by monoclonal anticomplement receptor type 1 on interactions between senescent human red blood cells and monocytic-macrophagic cells

The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (M phi) was investigated. Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated. Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or M phi, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect. Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent rosetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade. As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that monocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.     

Identification of an anti-monocyte monoclonal antibody that is specific for membrane complement receptor type one (CR1)

A monoclonal antibody (E11) was produced by immunization of mice with intact human cells of monocyte lineage. Despite the finding that E11 did not inhibit rosettes with C3b-coated sheep erythrocytes (EC3b), several lines of evidence indicated that E11 was specific for complement receptor type one (CR1). All monocytes, neutrophils, lymphocytes and erythrocytes that reacted with E11 formed EC3b rosettes. The E11 antigen on these cells was shown to be a molecule of 222 +/- 10 kDa. Treatment of lymphocytes, monocytes, and neutrophils with E11 followed by fluorescein-coupled F(ab')2 anti-mouse-IgG at 37 degrees C in buffer lacking sodium azide, led to capping or apparent endocytosis of the E11 antigen and a diminution in CR1 activity of 88%, 59% and 25%, respectively. This same treatment had no detectable effect on monocyte or neutrophil CR3 activity (EC3bi rosettes). Furthermore, with E11-capped lymphocytes, the residual EC3b rosetting was capped directly over the E11-fluorescence cap, whereas EC3d,g rosetting (CR2 specific) was undiminished and distributed evenly around the circumference of cells containing E11-fluorescence caps. Finally, the binding of E11 to cells was inhibited by the prior treatment of these cells with a well characterized rabbit polyclonal anti-CR1. These data indicated that E11 was specific for a site in CR1 that was distal from the C3b-binding site, so that E11 was unable to block CR1 activity. E11 proved to be useful for identifying CR1 on various cells in tissue sections, and for quantitating CR1 on erythrocytes and neutrophils. Erythrocytes and neutrophils from normal individuals were found to bind an average of 610 and 4.6 X 10(4) 125I-labeled E11 molecules per cell. When E11 was visualized in tissues by immunoperoxidase staining, the cells that apparently contained the greatest amounts of CR1 were dendritic reticulum cells and kidney podocytes. The E11 reactive dentritic reticulum cells were characteristic of both follicular and diffuse follicular center cell tumors. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) characteristically expressed little E11, confirming earlier studies that CLL cells lacked CR1 activity detected by EAC1-3b rosette formation. Because normal B cells have been shown to express CR1 at a very early stage of maturation, the absence of CR1 on CLL cells is discordant with the immature nature of CLL cells defined by immunoglobulin expression.     

Modulation of complement receptors of a human monocyte cell line, U-937, during incubation with phorbol myristate acetate: expression of an iC3b-specific receptor (CR3)

The human monocyte line, U-937, derived from an individual with histiocytic lymphoma was studied for the expression of surface C3 receptors, after cultivation in the presence of phorbol myristate acetate (PMA) or T lymphocyte-conditioned medium. Receptors were detected by using EAC4b, EAC3b, EC3b, EAC3bi and EAC3d intermediates. U-937 cells, in exponential growth phase, poorly bound the intermediates; after exposure to PMA or T lymphocyte-conditioned medium, U-937 cells strongly bound both EAC3b and EAC3bi since about 50% of cells rosetted with these intermediates. This binding was totally inhibited by EDTA and by Mac-1 monoclonal antibody, suggesting the presence of only CR3 receptor types on these cells. Although U-937 cells formed rosettes with EAC3b, there was no evidence for the presence of CR1 receptors since no rosette was observed either with EAC4b or with EC3b intermediates (EC3b were prepared by coupling purified C3b to erythrocytes with N-succinimidyl 3-(2-pyridyldithio)propionate. As small amounts of factor H were present on EAC3b intermediates, incubation of EAC3b with U-937 cells induced their transformation into EAC3bi and their binding to CR3. Moreover, U-937 cells did not promote the cleavage of C3b in the presence of factor I alone, suggesting that these cells did not bear a sufficient amount of functionally active CR1. These results demonstrated that U-937 cells predominantly expressed CR3. The study of the kinetics of EAC3bi rosette formation demonstrated that CR3 expression is closely related to PMA activation. We suggest that CR3 activity could result from a phosphorylation of existing receptors.     

C3bi/CR3 is a Main Ligand-Receptor Interaction in Attachment and Phagocytosis of C3-Coated Particles by Mouse Peritoneal Macrophages

We investigated the relative role of C3bi-CR3 interaction in the binding and phagocytosis of EAC43 by mouse peritoneal macrophages. Anti-Mac-1 F(ab')2 markedly inhibited the binding and lymphokine-induced phagocytosis of both EAC43b and EAC43bi. Fifty per cent inhibition of attachment and phagocytosis occurred at 1 microgram/ml of anti-Mac-1 F(ab')2 in the incubation media. On the other hand, EIgG binding and phagocytosis were not inhibited at all even at a concentration of 10 micrograms/ml. Depletion of divalent cations from the incubation media abolished EAC43b and EAC43bi rosettes but not EIgG rosettes or phagocytosis. These data suggested that both EAC43b and EAC43bi binding to macrophages were mediated via CR3. Because a drastic decrease of EAC43bi rosettes was observed in the case of EAC43bi cells prepared with smaller amounts of C3, a small contamination of C3bi molecules on EAC43b, itself, cannot explain the efficient attachment of EAC43b. We propose that EAC43b on the macrophage surface can be quickly converted to EAC43bi, forming EAC43bi rosettes, and that those erythrocytes are vigorously ingested by lymphokine-activated macrophages. In accordance with this hypothesis, we demonstrated that EAC43b was converted to EAC43bi in the medium in which macrophages had been incubated.     

Human liver Kupffer cells express CR1, CR3, and CR4 complement receptor antigens. An immunohistochemical study

The expression of complement receptor antigens by human Kupffer cells (KC) was investigated by immunohistochemical techniques in seven normal human liver biopsies. Polyclonal and monoclonal antibodies were revealed by double labeling of cells using indirect immunofluorescence and immunoenzymatic techniques or by using double immunoenzymatic techniques. In most experiments, one antigen was revealed by streptavidin-biotin-peroxidase complexes whose reaction product was examined by light microscopy and the second antigen stained using the alkaline phosphatase antialkaline phosphatase method visualized by fluorescence microscopy using fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate filters. KC were identified using monoclonal antibody EBM11 that recognizes 100% of KC in hepatic lobules and was paired with each antibody directed against complement receptors. CR1 and CR3 (alpha- and beta-chains) were found to be the predominant receptor antigens expressed by human KC. CR4 (p150,95) was expressed on all KC, but staining with anti-CR4 monoclonal antibodies was consistently weaker than that observed with anti-CR3 antibodies. No staining of KC was observed with anti-CR2 (CD 21) antibodies. Expression of CR1, CR3, and CR4 complement receptors on KC provides the cells with an optimal capacity to bind and phagocytize particles or immune complexes coated with any type of ligands for C3 receptors.     

Enhanced binding of immune complexes processed by erythrocyte CR1 (CD 35) receptors to purified CR2 (CD 21) receptors from tonsillar mononuclear cells

The binding of immune complexes (IC) opsonized by serum complement (C) and IC processed by CR1 (CD 35) receptors on human erythrocytes (E) to purified CR2 (CD 21) receptors was compared. Soluble CR2 was prepared from tonsillar mononuclear cells and purified by antibody affinity chromatography. Solid phase CR2 as well as CR2 subjected to PAGE and blotted onto nitro-cellulose membranes bound 125I-labelled BSA anti-BSA IC which had been opsonized by C and processed by CR1 up to ten times more efficiently than IC reacted with serum only. Radiolabelled monomeric C3d also bound to solid phase CR2. The binding of IC to purified and solid phase bound CR2 could be inhibited by anti-CR2 antibodies or by preincubation of the IC with polyclonal antibodies reacting with C3d or C3b/iC3b. Thus, both C3dg and iC3b appeared to mediate binding of IC to CR2. Preincubation of solid phase CR2 with purified monomeric C3d did not inhibit the subsequent binding of E-CR1 processed IC. The data indicate that E-CR1 have an important role in generating IC which bind effectively to CR2 receptors on B lymphocytes.     

Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.     

Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

Contribution of CR3, CD11b/CD 18 to cytolysis by human NK cells

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.     

Expression of CRl and CR2 Complement Receptors following Epstein-Barr Virus Infection of Burkitt''s Lymphoma Cell Lines

Epstein-Barr virus (EBV) infection of human B lymphocytes involves a specific receptor closely associated with, or identical to, the C3d complement receptor, CR2. Thus, 25 out of 29 EBV-positive Burkitt's lymphoma (BL) cell lines but none of 15 EBV-negative BL lines were found to express C3 receptors. Furthermore, in vitro infection with EBV of six EBV-negative cell lines resulted in the expression of C3 receptors in association with that of EBV-determined nuclear antigen (EBNA). Rosette assays using erythrocytes coated with human C3b, C3bi, and C3d, inhibition of rosette formation with anti-receptor antibodies, and flow cytometry analysis of stained cells demonstrated that EBV-converted lines expressed C3b and C3d receptors, CR1 and CR2. Anti-receptor antibodies recognized an average of 40,700 anti-CR1 and 140,000 anti-CR2 binding sites on an EBV-converted line (BL41/B95), whereas no specific binding occurred on the corresponding EBV-negative (BL41) cells. Because CR1 and CR2 are involved in B-cell proliferation and/or differentiation, enhanced expression of C3 receptors following the interaction between EBV and B cells and/or subsequent infection of the cells by EBV may provide a basis for positive control of B lymphocyte proliferation by EBV.     

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble β-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein–Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble β-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I⁺ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcγRIIIA (CD16).     

Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes.

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. Two macrophage receptors, the mannose-fucose receptor (MFR) and the receptor for complement component C3bi, CR3, were examined for their roles in the attachment and ingestion of L. donovani by human monocyte-derived macrophages. Two monoclonal antibodies which bind to the human CR3, anti-Mo1 and anti-Mac-1, inhibited both attachment and ingestion of L. donovani promastigotes after preincubation with human monocyte-derived macrophages; attachment was inhibited by 40 and 62% by anti-Mo1 and anti-Mac-1, respectively, and ingestion was inhibited by 34 and 51% by anti-Mo1 and anti-Mac-1, respectively. The interaction between promastigotes and CR3 may not have involved the C3bi-binding site on CR3, however, because a monoclonal antibody which exhibits specificity for this site, OKM10, inhibited promastigote attachment by only 18%. In contrast, OKM1, which is believed to react with the alternate lectinlike binding site on CR3, inhibited ingestion by 65%. MFR activity was inhibited using the soluble MFR ligands, mannan and mannosylated bovine serum albumin, which also inhibited promastigote attachment by 40 and 37%, respectively. The simultaneous inhibition of both CR3 (by anti-Mac-1) and the MFR (by either mannan or mannosylated bovine serum albumin) resulted in a greater decrease in promastigote attachment than inhibition of either receptor alone. Additionally, the reduction of MFR activity by allowing macrophages to adhere to a mannan-coated surface followed by the addition of anti-CR3 antibodies resulted in an 81% inhibition of promastigote ingestion, a greater decrease than was obtained by manipulation of either receptor alone. The results suggest that the MFR and CR3 independently participate in the attachment and ingestion of L. donovani promastigotes by human macrophages.     

Ligand binding by the p150,95 antigen of U937 monocytic cells: properties in common with complement receptor type 3 (CR3)

U937 cells (a monocytic cell line) grown in the presence of phorbol myristate acetate were surface labeled with 125I and the iC3b-binding proteins isolated by affinity chromatography on iC3b-Sepharose in the presence of divalent cations. Three polypeptides of 170, 150 and 95 kDa were found to bind specifically to iC3b-Sepharose. The polypeptides of 170 and 95 kDa were identified as the alpha and the beta subunits of CR3 by immunoprecipitation with OKM1 monoclonal antibody. The 150-kDa polypeptide was not immunoprecipitated by antibodies to the alpha subunit of CR3 or LFA-1. However, the 150-kDa polypeptide, together with the 95-kDa polypeptide, was immunoprecipitated with an anti-beta subunit-specific antibody IB4, which immunoprecipitates LFA-1, CR3 and p150,95. These results indicated that the 150-kDa polypeptide is the alpha subunit of the p150,95 antigen. The binding of p150,95 and CR3 to iC3b-Sepharose is specific as neither binds to C3u-Sepharose. A monoclonal antibody, 3.9, which immunoprecipitated the 150 and a 95-kDa polypeptide from U937 cells was characterized as being directed against the alpha-subunit of the p150,95 antigen. Phorbol myristate acetate-stimulated U937 cells from rosettes with EAC3b and EAiC3b but not with EAC3d cells. Monoclonal antibody 3.9 does not inhibit either type of rosetting, but we were unable to exclude a role for p150,95 in adherence of iC3b-coated particles. Since there is no rosetting with C3d-bearing particles it is unlikely that p150,95 is a receptor for C3d, a role for p150,95 which has been suggested by others (Wright, S.D., Licht, M.R. and Silverstein, S.C., Fed. Proc. 1984. 43: 413).     

CR3-dependent resistance to acute Toxoplasma gondii infection in mice.

Studies were performed to determine whether resistance to acute Toxoplasma gondii infection in mice depends on a mechanism involving CR3, the type 3 complement receptor. Nineteen of 22 mice (86%) given multiple injections of the anti-CR3 monoclonal antibody, 5C6, prior to and after intraperitoneal inoculation of cysts of the ordinarily mildly virulent ME49 strain of T. gondii died within 8 to 12 days, whereas control antibody-treated mice survived. All (five of five) anti-CR3-treated BALB/c mice infected via the natural peroral route died within 8 days of infection. Flow cytometric analysis of cells recovered from peritoneal lavages of anti-CR3-treated T. gondii-infected mice revealed that the percentage of Thy-1+ CD4- CD8- cells was reduced to about 50% of that of control antibody-treated mice and to about 20% of the number of such cells in controls. The numbers of macrophages, polymorphonuclear leukocytes, and lymphocytes recovered from the peritoneal cavities of T. gondii-infected mice were all reduced in anti-CR3-treated mice to about 40% of those of controls. In addition, anti-CR3-treated mice had less than 25% of the induced NK cell activity of the controls, and gamma interferon was reduced to undetectable levels. Thus, the rapid death of anti-CR3-treated mice was probably caused by impaired preimmune defenses. Histological examination of anti-CR3-treated T. gondii-infected mice revealed extensive liver pathology compared with that of infected mice given a control antibody or uninfected mice given anti-CR3. The inflammation, degeneration, and necrosis in most of the anti-CR3-treated mice were severe enough to account for the observed mortalities.     

Modulation of neutrophil expression of C3b receptors (CR1) by soluble monomeric human C3b

Upon incubation at 37 degrees C with purified human C3b (500 micrograms/ml), polymorphonuclear neutrophils (PMN) were found to express up to 50% more C3b receptors (CR1) than PMN incubated with buffer alone. This up-regulation of CR1, assessed by the binding of radiolabeled CR1-specific monoclonal antibody, was dependent on the dose of C3b, occurred within 10-20 min and was stable for at least 90 min. PMN incubated with C3b also demonstrated enhanced CR1-dependent binding functions, such as EC3b rosette formation and phagocytosis of EIgGC3b particles. C3b at a concentration of 500 micrograms/ml induced up to 90% increase in the attachment or the phagocytic index. However, CR1 remained unable to promote phagocytosis of EC3b intermediates. Fc receptor-mediated functions were unaffected by the treatment with C3b. The active factor was characterized as monomeric C3b and, in particular, shown to be distinct from C5a. C3b purified by anion-exchange fast protein liquid chromatography on a Mono Q column or eluted from a monoclonal anti-C3b-Sepharose retained its modulating activity, while native C3 or C3 fragments such as iC3b, C3c or C3d,g were ineffective.