Differentiation of C3b receptors on human lymphocytes, phagocytes, erythrocytes and renal glomerulus cells by monoclonal antibodies.

C3b receptor protein was purified form human erythrocytes by 2 M KBr solubilization and affinity chromatography on C3-coated sepharose. This material served as antigen for raising monoclonal antibodies. To investigate the distribution and antigenetic relationship between the receptors for C3b on human erythrocytes, lymphoid and phagocytic cells, as well as kidney cells three monoclonal antibodies were selected which inhibited the binding of EAC14 degrees 23b to complement receptor-bearing cells. This could be shown for human erythrocytes by inhibiting the immune adherence reaction, for tonsil lymphocytes, Raji cells, and guinea-pig spleen cells by inhibition of rosette formation of these cells with EAC14 degrees 23b, and for human renal glomeruli by blocking of the the adherence of EAC14 degrees 23b to kidney sections. In contrast, these monoclonal antibodies were not capable of inhibiting rosette formation of human granulocytes and monocytes with EAC14 degrees 23b. The antibodies only interfered with the rosette formation, of EAC14 degrees 23bi and EAC14 degrees 23d with Raji cells and tonsil lymphocytes-if at all-at high concentrations, whereas the rosette formation of Raji cells and tonsil lymphocytes with EAC14 degrees 23b was influenced by supernatants of the selected clones up to a dilution of 1:10(3) to 1:10(5).     

Human complement (C3b) receptors defined by a mouse monoclonal antibody.

The aim of the present study was to prepare a monoclonal antibody against human C3 receptors. The monoclonal antibody termed C3RTo5, which is characterized in this study, inhibited the ligand binding of C3b receptors of human erythrocytes, neutrophils and lymphocytes, but did not block the ligand binding of C3bi and C3d receptors. C3RTo5 reacted only with cells that express C3b receptors. It did not react with cells that do not express C3 receptors or with cells that express C3 receptors other than C3b receptors. The reactivity of C3RT05 against C3b receptors of human erythrocytes, neutrophils, glomerular cells, tonsil cells, or spleen cells could be removed by absorption with human erythrocytes or tonsil cells, whereas absorption with human peripheral T cells or sheep erythrocytes had no effect. In immunoprecipitation studies, a glycoprotein with a mol. wt of 205,000 could be isolated with C3RTo5 from non-ionic detergent lysate of tritiated tonsil cells. A rabbit antiserum prepared against this glycoprotein was able to stain C3b receptor-positive cells, inhibit C3b receptor ligand-binding activity and, furthermore, to precipitate a 205,000 mol. wt component. The results of this study indicate that C3RTo5 is a monoclonal antibody with selective reactivity to C3b receptors and presumably to the binding sites within the receptor molecule. Using C3RTo5 further strong evidence was obtained that membrane-bound C3b receptors have a mol. wt of 205,000.     

A comparative evaluation of receptor reactivities for C3b, iC3b, and C3d on Raji lymphoblastoid cells

Raji cells were described to carry receptors for iC3b, C3d, C3b-beta 1 H and beta 1 H. Controversial opinions, however, exist whether or not these cells carry also receptors for C3b. Using highly purified C3, definitely devoid of beta 1 H and C5, for preparation of C3b intermediates, it could be shown that Raji cells bound to C3b cells. Furthermore, Raji cells reacted with monoclonal antibodies that interfered with binding of C3b to human erythrocytes, lymphocytes and renal cells. The receptor for C3b on Raji cell, however, exhibited some special properties and, therefore, required some distinct experimental conditions for its detection: (1) The origin of the erythrocytes used for preparation of the C3b intermediates seemed to be important; this was not the case when iC3b and C3d receptor reactivity was assessed. (2) Rosettes already formed between Raji cells and EAC1423b showed the tendency to disintegrate within the first 30 min after the rosette formation assay. Again, this effect could not be observed with iC3b- and C3d-dependent rosette formation. (3) Incubation of the Raji cells at 37 degrees C as well as 4 degrees C before rosette formation resulted in a rhythmic loss and reappearance of C3b receptor reactivity. At room temperature (19-22 degrees C) this effect was much less expressed. There was no influence of preincubation at 4 and 37 degrees C, respectively, on the iC3b and C3d receptor reactivity of Raji cells. (4) Diisopropylfluorophosphate (DFP) present during rosette formation enhanced, within a certain range of concentration, the percentage of C3b-dependent rosette formation. iC3b and C3d receptor reactivity was not influenced. A similar reaction pattern was observed with pokeweed mitogen (PWM)-stimulated tonsil lymphocytes. In the concentrations tested, DFP showed no effect on the rosette formation between C3b, iC3b, and C3d cells, respectively, and unstimulated tonsil lymphocytes. The data presented suggest that C3b receptors on Raji cells undergo some special metabolism, possibly controlled by fluid phase or cell-bound proteases. This might be a common property of C3b receptors on blast-like and transformed cells, differing from that of unstimulated small lymphocytes.     

Relationship between the HLA Associated Specificities 4a and 4b and the Lymphocyte C3 Receptor

Contrary to previous reports, we have obtained no evidence that 4a and 4b antisera specifically inhibit C3 rosette formation by human lymphocytes. Pretreatment of lymphocytes with various whole 4a and 4b antisera resulted in partial inhibition which was often nonsepcific. Ultracentrifugation of the sera to remove immune complexes removed C3 rosette inhibitory activity although specific cytotoxic activity remained. It is probable that immune complexes in antisera will have fixed C3 which will have been converted to C3d and so be able to block the C3d receptor. This is the receptor mainly measured in these and in the previous experiments.     

Physicochemical characterization of C3b receptors isolated from human erythrocytes by immunoprecipitation.

A high yield of active C3b receptors was obtained by solubilizing human erythrocyte membranes with 2 M KBr, whereas other solubilization agents yielded no, or significantly less activity. Gel filtration of the KBr lysates revealed that the apparent molecular wieght of biologically active C3b receptor molecules was greater than 1 x 10(6). Immunoprecipitates prepared with radio-iodinated KBr lysates and anti-C3 receptor sera (AC3RS) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or sodium dodecyl gel filtration. Unreduced SDS-PAGE and gel filtration profiles showed three predominant peaks with apparent mol. wts of 1--1.3 x 10(6), 80,000 and 60,000. Whereas the high mol. wt component decreased only slightly after reduction, the 80,000 and 60,000 mol. wt components disappeared and two new peaks with apparent mol. wts of 38,000 and 18,000 appeared in SDS-PAGE profiles. Although the high mol. wt component present in reduced SDS-PAGE profiles was detectable in some of the control experiments, none of the other peaks could be precipitated with control sera, and these components could be demonstrated only when KBr lysates of C3b receptor-positive erythrocytes and AC3RS that were able to inhibit ligand binding of the C3b receptors were used for precipitation. These findings suggest that (a) the C3b receptor of human erythrocytes in its biologically active state is a macromolecule with an apparent mol. wt higher than 1 x 10(6) and (b) the protein moiety consists predominantly of non-covalently linked protein molecules with apparent mol wts of 80,000 and 60,000. These protein molecules are composed of disulphide-bridged polypeptide chains with apparent mol. wts of 38,000 and 18,000.     

Interaction of human lymphocytes with fluid phase human C3b detected by immunofluorescence

Human lymphocytes obtained from tonsils and peripheral blood were found to bind human fluid phase C3b, obtained by trypsin treatment. This binding was detected by indirect immunofluorescence (IIF) using specific anti-C3 antisera. Lymphocytes isolated from thymus tissue scored low percentages in IIF, indicating that the main population of thymus-derived lymphocytes are T cells. The distribution pattern of C3b-binding cells was compared with that of cells forming rosettes with sheep erythrocytes coated with antibody and complement (EAC) and with sheep erythrocytes (E) only, as well as with that of Ig- bearing lymphocytes, as detected by direct immunofluorescence. It appeared that the distribution pattern of lymphocytes which can bind fluid phase C3b is similar to that of EAC rosette-forming and of Ig-bearing lymphocytes. Pre- incubation of the lymphocytes with C3b and pretreatment of the cells with trypsin decreased the capacity to form rosettes and to bind C3b to their sur- face. Human monocytes, granulocytes and erythrocytes did not bind fluid phase C3b, as judged by IIF. Therefore, the selective binding of fluid phase C3b to lymphocytes provides a specific method for the detection of complement-reactive lymphocytes in lymphoid cell preparations.     

Human Membrane-bound C3 Receptors I. Serological and Immunohistological Demonstration of C3 Receptors

The aim of the present study was to present further evidence of the specific reactivity of an anti-C3 receptor serum (AC3RS), to demonstrate membrane-bound C3 receptors by using this AC3RS in different serological and immunohistological methods, and to investigate the relationship between membrane-bound C3 receptor and α₁-antitrypsin. The AC3RS, or F(ab)₂ fragments of the IgG fraction of this antiserum, stained a percentage on various viable cell populations roughly equivalent to the number of cells that bound EAC3b and or EAC3d; C3 receptor-negative T cells and thymocytes were not stained. On frozen sections of tonsils and kidneys it was found that the AC3RS stained the area to which EAC3b adhered. After absorption with neutrophils or E_hu, the AC3RS Inhibited the agglutination of EAC3d with tonsil cells, not the agglutination of tonsil cells or neutrophils with EAC3b; this absorbed AC3RS still stained tonsil cells but not neutrophils, in frozen tonsil sections it Stained only those areas to which AC3d adhered. The absorbed AC3RS did not stain glomeruli. Antisera to α₁-antitrypsin failed to in-hibit EAC agglutination with C3 receptor-bearing cells or to stain C3 receptor-positive cells either in suspension or in frozen sections. Absorption of the AC3RS with purified α₁-antitrypsin did not affect its specific reactivity.     

Complement receptors on Raji cells. The presence of a new type of C3 receptor.

Raji cells in our laboratory did not form rosettes with EAC43hu. When EAC43hu are treated with beta 1H, the treated EAC43hu forms heavy rosettes with Raji cells. Evidence is presented to show that these rosettes resulted from a new type of C3 receptor which is different from either CR1 (C3b receptor), CR2 (C3d receptor) or CR3 (C3bi receptor). Three lines of evidence clearly showed that C3 is implicated in the new rosette formation. C3 receptors isolated from human erythrocytes inhibited the new rosette formation, while they did not inhibit the rosette formation of Daudi cells via CR2, indicating that the new rosette-forming receptor is different from CR2. Anti-Raji cells antiserum inhibited the new rosette formation while it did not inhibit the reaction between human erythrocytes and EAC43 via CR1. This fact indicates that the new rosette-forming receptor is different from CR1 in accordance with the lack of rosette formation of Raji cells with EAC43. The evidence to differentiate the receptor from CR3 comes from no participation of C3b inactivator in the generation of rosette-forming activity of EAC43. Both the mode of action of anti-beta 1H and the effect of modification of bound C3b by N-bromosuccinimide suggest that EAC43 reacts with beta 1H, which in turn results in a conformational change of C3b. Raji cells might have receptors for the beta 1H altered C3b.     

Lymphocyte binding of fluid phase mouse C3b.

Isolated fluid phase mouse C3b adhered to human and to mouse lymphocytes. On human, but not on mouse cells it could be stained by fluoresceinated F(ab')2 anti-mouse C3. Binding to mouse cells was, however, shown by inhibition of C3-dependent rosette formation and by direct staining with fluorescein-conjugated C3b (FITC-mouse C3b). Optimal staining of lymphocytes by FITC-mouse C3b depended on a sufficient intensity of fluorochrome conjugation and on a degree of aggregation of the C3b. FITC-mouse C3b preparations which initially stained weakly, stained strongly after being aggregated with glutaraldehyde. The failure of immunofluorescent techniques to demonstrate binding by mouse lymphocytes of mouse C3b which had not been so aggregated was apparently due to inaccessibility to anti-C3 of the receptor-bound C3b. Aggregated mouse C3b-FITC induced sequential patching and capping of lymphocyte complement receptors followed by endocytosis, all of which was inhibited completely by cold (4 degrees), sodium azide (2 x 10(-3) m), cytochalasin B (28 microgram/ml) and chlorpromazine (10(-4) m), and partially by lignocaine (2 x 10(-3) m), and colchicine (10(-4) m). That mouse C3 which has interacted with mouse complement receptors may not be demonstrable by anti-C3 antibody may have important implications for immunohistochemical localization of C3 bound in vivo to lymphoid cells.     

Role of β1H for the binding of C3b-coated particles to human lymphoid and phagocytic cells

Coating of EAC14^oxy23b with highly purified human serum β1H globulin (β1H) led to acceleration of rosette formation with human peripheral blood lymphocytes (PBL), tonsil lymphocytes, B lymphoblastoid (Raji) cells, granulocytes and monocytes. This reaction was discernible from C3bi-dependent rosette formation. Enhancement of rosette formation of C3b cells by β1H was most effective at limiting amounts of C3 per EAC14^oxy23b. The β1H effect was not due to trace contamination with C3b inactivator. β1H-dependent rosette formation with the various lymphoid and phagocytic cells could be suppressed by the F(ab′)₂ fragment of anti-β1H suggesting β1H-mediated binding of β1H-coated particles to complement receptor-positive (CR⁺) cells. In turn, binding of fluid-phase β1H to lymphoid and phagocytic cells could be demonstrated by fluorescence and by ¹⁴C-labeled β1H. In addition, the functional status of these cells with respect to their receptor reactivity was altered. Treatment of normal lymphocytes (PBL, tonsil lymphocytes) and of granulocytes with β1H improved their rosette formation with both EAC14^oxy23b and EAC14^oxy23b-β1H. The reaction of monocytes was hardly affected. The β1H effect on Raji cells resulted in reduced rosette formation with EAC14^oxy23b-β1H, while binding of EAC14^oxy23b remained unchanged. These results suggest the presence of sites on CR⁺ cells, to which soluble and particlebound β1H can bind, leading to alteration of the functional status of the cells. In all likelihood, EAC14^oxy23bi can attach to the β1H-binding sites on CR⁺ cells.     

Methods for producing T- and B-lymphocyte receptor-specific antisera.

Spent tissue culture medium from two continuous lymphoblastoid cell lines, FL-74 and CT45-S, expressing the T-lymphocyte receptor for guinea pig E and the B-lymphocyte receptor for EAC respectively were used to produce receptor-specific antisera. Anti-E receptor sera blocked E rosette formation on FL-74 cells, canine and feline lymphocytes and canine and feline thymocytes but not EAC rosette formation by CT45-S cells or canine and feline lymphocytes. Anti-EAC receptor sera blocked EAC rosette formation on CT45-S cells and canine or feline lymphocytes. Absorption of antisera will the appropriate lymphoblastoid cell line removed E or EAC-blocking activity. The results of this study suggest that similar methods may be used to produce lymphocyte subpopulation-specific antisera in other species including man.     

C3b receptors in glomerular disease.

Using two indicator systems--sheep erythrocytes or fluoresceinated S. typhi coated with C3b the presence of a receptor for C3b (but not C3d) in the normal human glomerulus is confirmed. No receptor could be detected in other species tested (mouse, rat, guinea-pig, rabbit and rhesus monkey). Binding of indicator particles was reduced or lost in diseases associated with glomerular capillary deposition of C3, but not in those with mesangial deposition alone. However in some cases the receptor was lost in the absence of detectable C3 deposition. No receptors were detected in proliferating cells in glomerular crescents.     

Granulocyte-Dependent Extracellular Cytotoxicity is Enhanced by Complement C3bi and Independent of Phagocytosis

We have evaluated phagocytosis, C3-binding and cytotoxicity of human leukocytes simultaneously, using IgM-sensitized C3-fragment-bearing sheep erythrocytes (EAC3b, EAC3bi) as targets. In this method, 51Cr-labelled EAC3b or EAC3bi were added to human peripheral granulocytes anchored by fibronectin onto microtitre plate wells. The degrees of haemolysis, binding and ingestion of the target cells were estimated from the radioactivity released. We found that the granulocytes predominantly lysed EAC3bi but not EAC3b or EAC4b. EAC3bi lysis elicited from granulocytes was as effective as that from lymphocytes under the same assay conditions. However, one difference in the cytolysis of the two effector cells was that EAC3bi bound efficiently to all of the granulocytes similarly to EAC3b, whereas it bound to only 4% of the lymphocytes. The bound cells did not appear to be efficiently phagocytosed by the granulocytes. Blocking studies using antibodies suggested that C3bi receptors, CR3 and CR4, but not other C3-binding proteins, C3b/C4b receptor (CR1), membrane cofactor protein (MCP) or decay-accelerating factor (DAF), are involved in granulocyte-mediated haemolysis. We speculate that simultaneous stimulation of the C3bi receptors and the fibronectin receptor results in elicitation of cytotoxicity by granulocytes.     

Lymphoblastoid cell supernatants increase expression of C3b receptors on human polymorphonuclear leucocytes: direct binding studies with 125I-C3b

Human PMN incubated in culture supernatants of the Raji long-term human lymphoblastoid cell line showed increased rosette formation with sheep erythrocytes coated with C3b (EIgM C4b3b) but no change in rosette formation with IgG-coated erythrocytes. This suggested a specific increase in cell surface C3b receptors, which was further investigated using 125I-C3b for direct binding studies. The results confirmed that specific binding of 125I-C3b to PMN incubated in culture supernatants increased up to three- to four-fold over binding to PMN incubated in control media alone. Scatchard analysis revealed that the apparent Ka for supernatant-treated cells, 3.36 +/- 0.89 X 10(7) L/M did not differ from the Ka for cells incubated in control media, 3.76 +/- 0.75 X 10(7) L/M, suggesting an increase in a single class of C3b receptors. Kinetic studies revealed that the active factor was present within 24 hr of culture of the Raji cells, and that neutrophils incubated in culture supernatants increased their C3b receptors continuously for up to 4 hr, the longest interval tested. The effect of the culture supernatant was lost with dilution beyond eight- to 10-fold. The results suggest that culture supernatants of this long-term lymphoblastoid cell line contain soluble factors that induce increased expression of C3b receptors on PMN and may thus serve as a model for study of important physiologic effects of lymphocyte products on PMN in vivo.     

Complement (C3) receptors on dendritic reticulum cells of normal and malignant lymphoid tissue.

The aim of this study was to investigate whether dendritic reticulum cells (DRC) in normal lymphoid tissue and in malignant non-Hodgkin's lymphomas possess receptors for the third complement component (C3 receptors). For this purpose we studied the in situ expression of C3 receptors in lymphoid tissue by staining frozen tissue sections with an antiserum specific for human C3 receptors (AC3RS), using a modified immunoperoxidase method. The results indicate that DRC of normal and malignant lymphoid tissue express large amounts of C3 receptors. Absorption experiments revealed that the C3 receptors of DRC are identical with, or at least share common antigenic sites with, the C3 receptors expressed by tonsil B cells.     

Human Membrane-bound C3 Receptors

The aim of the present study was to define the physicochemical structure of C3b and C3d receptors of lymphoid cells, C3b and C3b receptors were isolated from KBr lysates of the 20,000 g fraction of human tonsil homogenates by immunoprecipitation with an anti-C3 receptor serum (AC3RS). Sodium dodecyl sulphate (SDS) gel filtration and polyacrylamide gel electrophoresis (PAGE) of unreduced immunoprecipitates revealed a highly predominant component with an apparent molecular weight (mol.wt.) greater than 1 × 10⁴ and a small component with a mol.wt. of 80,000. After reduction, the SDS-PAGE profile was made up of a constant major 38,000 mol.wt. component and an inconstant smaller 18,000 mol.wt. component. The 38,000 (and also the 18,000) component could he isolated only from C3 receptor-active lysates, and not from C3 receptor-negative lysates. Taken together, the results of this study suggest that the active C3 receptor molecule of tonsil cells is a lipoprotein complex with a mol.wt. greater than 1 × 10⁴; its protein moiety consists predominantly of disulphide-bridged polypeptide chains with a mol.wt, of 38,000; C3b and C3d receptors are composed of equal-sized polypeptide chains, but the specific binding sites for C3b and C3d are located on different molecules.     

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.     

Maturation-linked expression of C3b and C3b' receptors on developing human bone marrow and peripheral blood granulocytes.

The sequential expression of granulocyte membrane receptors for C3b, C3b' and C3d have been investigated in normal human bone marrow and peripheral blood. In particular the relationship between receptor expression and morphological characteristics of granulocyte maturation has been assessed. The result indicate that C3b receptor development precedes that of the Fc-IgG receptor and is present on all neutrophil precursors with the exception of the agranular myeloblast. Furthermore, there is, in contrast to Fc-IgG receptors, no difference (P greater than 0.35) in C3b receptor expression between marrow and peripheral blood segmented neutrophils. An apparent subpopulation of granulocytes also appear to have receptors for C3b' whereas receptors for C3d were not detected at all during granulocyte maturation. The application of these findings to the normal in vivo functions of granulocytes and to the study of abnormal granulocyte populations in myeloproliferative disorders is discussed.     

Inhibition of neutrophil function by fluid phase C3b of complement.

A high-molecular-weight fragment of C3 was isolated from normal human serum by column chromatography, was generated by incubation of serum at 37 degrees C with inulin, and was produced from highly purified C3 by limited digestion with trypsin. This product was shown to inhibit the antibacterial function of neutrophils by using Escherichia coli O75 as the main test organism. The inhibitor reacted with anti-C3b and anti-C3c, but not with anti-C3B (anti-native C3) or anti-C3a. The manner of preparation of the inhibitor, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, and the amino acid composition of the inhibitor indicated that it was fluid phase C3b. The inhibitor of neutrophil function (fluid phase C3b) was shown to bind to C3b receptors or acceptors on sheep erythrocytes in a model system.     

Studies of the presence of membrane receptors for complement,IgG and the sheep erythrocyte rosetting capacity on the same human lymphocytes

Normal peripheral blood lymphocytes were tested by a mixed rosette method, employing different sized erythrocytes as indicators to identify lymphocytes simultaneously possessing membrane markers found commonly on B and T cells. Only small populations of these lymphocytes were detected regulary in normal lymphocyte preparations. One type of lymphocyte (ranged from 0.5%-8%) was shown to possess the following markers: receptors for human and rabbit IgG, receptors for the third complement component C3b and C3b inactivator-cleaved C3b (C3d), and the capacity to rosette spontaneously with uncoated sheep erythrocytes (SRBC). Another lymphocyte cell type was shown to possess both SRBC and IgG receptors but lack membrane immunoglobulins and complement receptors. This population was detected in lymphocyte preparations depleted of complement receptor cells, and an increased number of these cells was found in rosette preparations incubated with human serum. The possible presence of some lymphocytes possessing both complement and SRBC receptors and lacking other markers was considered. The possibility that these small populations of human lymphocytes are sub-populations of T cells with certain cytotoxic function is postulated.