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R B Sim

Publications and source records attributed to R B Sim.

At least 145 records · Page 8Linked to original sources
PubMed1985

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.

Antibodies, Monoclonal↗
PubMed1985

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

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

B-Lymphocytes↗
PubMed1985

Macrophage complement and lectin-like receptors bind Leishmania in the absence of serum.

We have examined the relative roles of the macrophage (M phi) plasma membrane receptor for the cleaved third complement component (iC3b, CR3) and of the mannosyl/fucosyl receptor (MFR) in binding and ingestion of Leishmania donovani. In the absence of exogenous complement, the binding and ingestion of promastigotes, which are good activators of the alternative complement pathway, were inhibited by the anti-CR3 monoclonal antibody M1/70, by the Fab portion of an anti-C3 antibody, or by the nucleophile, sodium salicyl hydroxamate, an inhibitor of C3 fixation. This provides strong evidence that M phi-derived, cleaved C3 (iC3b) present on the promastigote surface mediates binding to CR3. Equivalent inhibition of promastigote binding and ingestion was also observed using the soluble inhibitors of MFR activity, mannan or ribonuclease B. No additive effect for blocking the two M phi receptors simultaneously was observed. For amastigotes, which are poor activators of the alternative pathway, a lesser but nevertheless equivalent effect was observed for the three soluble inhibitors of CR3-mediated binding vs. the two soluble inhibitors of MFR-mediated binding. Modulation experiments in which either CR3 or MFR had been rendered inaccessible demonstrated that both receptors must be present on the segment of M phi membrane to which the parasite binds. The combined function of these two distinct M phi receptors may provide a general mechanism for recognition and ingestion of other pathogenic protozoa known to activate the alternative pathway.

Animals↗
PubMed1985

Expression of complement factor H on the cell surface of the human monocytic cell line U937.

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.

Antibodies, Monoclonal↗
PubMed1985

Interaction of human monocytes, macrophages, and polymorphonuclear leukocytes with zymosan in vitro. Role of type 3 complement receptors and macrophage-derived complement.

Macrophages take up zymosan in the absence of exogenous complement via receptors for iC3b (type 3 complement receptors) acting with or without lectin-like receptors for mannosyl-fucosyl-terminated glycoconjugates. We previously provided evidence that macrophages themselves secrete complement-alternative pathway components able to opsonize zymosan locally (Ezekowitz et al., J. Exp. Med. 1984. 159:244-260). We show here that covalently bound C3 cleavage products C3b and iC3b can be eluted from zymosan particles cultivated with 36-h adherent human monocytes in the absence of serum. The ligand binding site of type 3 complement receptors is involved in macrophage-zymosan interactions as shown by inhibition studies of zymosan binding and uptake with Fab fragments of anti-C3 antibodies and monoclonal antireceptor antibodies M01 and OKM10. In contrast, antibody IB4, which binds to a receptor epitope distinct from the binding site, does not inhibit zymosan uptake. Selective modulation of macrophage receptors onto anticomplement receptor antibody and mannose-rich yeast mannan, respectively, confirms that the complement and lectin-like receptors are distinct. Human polymorphonuclear leukocytes, which express receptors for complement, but are not known to secrete complement proteins, bind and ingest only exogenously opsonized zymosan. Unopsonized zymosan is a poor trigger of respiratory burst activity in neutrophils or 7-d adherent human macrophages, but induces cell aggregation and secretion of large amounts of superoxide anion when these cells are co-cultivated in serum-free medium and challenged with zymosan. Our studies indicate that complement and/or other products synthesized by macrophages at extravascular sites could play an important role in opsonization and lysis of pathogens able to activate the alternative pathway and mediate macrophage-neutrophil collaboration in first-line host defence.

Animals↗
PubMed1985

Preparation of antibodies against the fourth complement component (C4) and development of a direct electroimmunoassay for quantification of C4d.

The specificity of several preparations of antihuman C4 antibodies were examined by crossed immunoelectrophoresis. Two antibody preparations with anti-C4c and anti-"total" C4 reactivity respectively were prepared by immunoadsorption procedures and defined by comparison with reference antibodies of known specificity. These two antibody preparations were used in the development of a rocket immunoelectrophoresis with an intermediate gel for specific and direct quantification of C4d. This method permits the selective quantification of activation of the classical complement pathway as opposed to the alternative pathway activation.

Antibodies↗
PubMed1984

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.

Antibodies↗
PubMed1984

Localisation of a group of antigenic sites in complement component C3, and identification of a new fragmentation pattern.

Immunisation of rabbits or goats with denaturated human C3 reproducibly gives rise to an antiserum of restricted specificity which precipitates native C3, C3b and iC3b, but not C3c or C3d isolated from 'aged' serum. The antigenic sites recognised by the antiserum appear to be localised in a small segment of the polypeptide chain located close to the N-terminus of the C3b alpha' chain. Affinity chromatography using antibodies developed by this procedure reveals an unusual pattern of degradation of C3 which occurs on conventional 'ageing' of serum at 37 degrees C.

Amino Acids↗
PubMed1984

Drugs that induce systemic lupus erythematosus inhibit complement component C4.

The syndrome resembling systemic lupus erythematosus (SLE) associated with long-term treatment with hydralazine and isoniazid seems to be due to the drugs themselves rather than their metabolites. This syndrome is associated with deposition of immune complexes; and in the complement system inhibition of C4 is likely to increase deposition of immune complexes. In vitro, hydralazine and isoniazid inhibited 50% of the binding of C4 at 840 mumol/l and 1.05 mmol/l, respectively--ie, within the concentration ranges that have been used in therapy. Acetylated metabolites were not inhibitory at the maximum concentrations tested; and iproniazid, which does not cause SLE, gave 50% inhibition only at a concentration far exceeding that used in therapy.

Acetylation↗
PubMed1984

Substances that can trigger activation of the alternative pathway of complement have anti-melanoma activity in mice.

Intraperitoneal (i.p.) injection of substances that can ignite the alternative pathway of complement, namely isolated human C3b or C3(H2O), guinea-pig C3(H2O) or cobra venom factor, or conventionally prepared zymosan, will reproducibly and very significantly increase the mean survival time of C57BL mice previously inoculated i.p. with melanoma cells. The effect is greater at higher doses and earlier post-inoculation (p.i.) administration, but the substances are active at low doses (30-100 micrograms/mouse) if given early enough. It is likely that C3b or C3(H2O) was the previously unidentified anti-tumour factor activated in serum by S. aureus treatment or serum fractionation and described elsewhere. Activation of the alternative pathway of complement appears to have potential interest for cancer therapy.

Animals↗
PubMed1984

Local opsonization by secreted macrophage complement components. Role of receptors for complement in uptake of zymosan.

We have examined the role of macrophage (M phi plasma membrane receptors for the cleaved third complement component (iC3b; CR3) and mannosyl, fucosyl terminated glycoproteins (MFR) in uptake of unopsonized zymosan. Monoclonal antibodies against CR3, M1/70 (Mac-1) and MO1, each inhibited approximately 50% of uptake of 125I-zymosan by murine and human M phi, respectively. Yeast mannan inhibited 0-50% of zymosan uptake in various M phi, in parallel with their expression of MFR. We demonstrated that M phi were the source of C3 in our assay and that the activity of other components of the complement system, namely a C3 convertase, factor I, and a factor I cofactor were also present in serum-free cultures of human monocytes. Macrophage C3 was deposited rapidly, within 10 min, on the zymosan particles and mediated binding, ingestion, and stimulation of superoxide release in BCG-activated and thioglycollate-elicited peritoneal M phi via CR3. Local secretion of complement proteins by M phi themselves can therefore opsonize pathogens and cells able to activate the alternative pathway and effect their destruction.

Animals↗
PubMed1984

The effect of danazol on the production of C1 inhibitor in the guinea pig.

The production of C1 inhibitor (C1-INH) in guinea pig liver was studied following administration of danazol. Immunochemical assays of the inhibitor were performed on serial serum specimens. An increase in serum C1-INH was observed following danazol treatment. Immunohistochemical studies confirmed that the liver is the sole site of synthesis of C1-INH in guinea pigs. This appears to occur in certain clones of hepatocytes. Ultrastructural studies revealed an increase in coarse endoplasmic reticulum and mitochondria in danazol-treated animals. In in vitro studies with liver slices, however, more C1-INH was detected in culture supernatants from untreated animals than from danazol-treated animals. This observation suggests that secretion of C1-INH from stimulated hepatocytes may require additional factors which are present in vivo but not in vitro.

Animals↗
PubMed1983

Enzymic assay of C3b receptor on intact cells and solubilized cells.

The C3b receptor of human erythrocytes is known to act as a cofactor for the cleavage of the complement protein C3b by the serine proteinase C3b/C4b Ina. The same cofactor activity is shown to be present on human tonsil B-lymphocytes. The cofactor activity of the C3b receptor can be assayed, on intact cells or in solubilized extracts of cells, by determining the rate of C3b cleavage in the presence of fixed concentrations of C3b and of C3b/C4b Ina. This assay method was used to compare the characteristics and relative quantities of C3b receptors on erythrocytes and lymphocytes. The cofactor activities associated with these two cell types resemble each other, but are distinct from the serum cofactor proteins, C4bp and Factor H, in antigenicity and in pH- and ionic-strength-dependence, and are distinct from Factor H in substrate specificity. Assay of cofactor activity in intact cells indicates that there are about 80-fold more receptors per cell on the lymphocyte surface than on erythrocytes. Assays with cells made permeable by detergent show that, whereas essentially all of the receptors on erythrocytes are on the cell surface, B-lymphocytes contain a large internal receptor pool, which makes up more than 80% of the total cofactor activity of the cell.

B-Lymphocytes↗
PubMed1983

Monoclonal antibodies against the complement control protein factor H (beta 1 H).

Two mouse monoclonal antibodies against the human complement control protein, Factor H (beta 1H), are described. The antibodies are both IgG - gamma 1 - subclass and are directed against different epitopes on the human Factor H molecule. One of the antibodies, MRC OX 24, increases the cofactor activity of Factor H in Factor I-mediated cleavage of soluble C3b. The second antibody, MRC OX 23, which has no effect alone, reduces the increase in cofactor activity observed in the presence of the first antibody. However, MRC OX 24 inhibits the binding of 125I-labelled Factor H to surface-bound C3b (EAC3b). Again MRC OX 23 alone does not have an effect but decreases the inhibition in 125I-labelled Factor H binding to EAC3b observed with MRC OX 24. These studies show clearly that the interaction of Factor H with soluble C3b is different to its interaction with surface-bound C3b. In an indirect immunoprecipitation system using these monoclonal antibodies, single-chain molecules of 150 000 mol.wt. are specifically precipitated from human serum and also from the sera of other primates - rhesus monkey, cynomolgus monkey, and African green monkey. There was no precipitation from sera of cow, pig, sheep, chick, or rabbit. Using a radioimmunoassay with radiolabelled monoclonal MRC OX 23, the concentration of Factor H in human plasma was determined.

Animals↗
PubMed1983

Autolytic fragmentation of complement components C3 and C4 and its relationship to covalent binding activity.

The autolytic cleavage reaction of C3 and C4 and the covalent binding reaction of these proteins, are both aspects of the reactivity of an activated thiolester within these proteins. Autolytic cleavage occurs by internal nucleophilic attack on one face of the planar thiolester, while the covalent binding reaction of the activated proteins follows exposure of the opposite face of the thiolester to attack by external nucleophiles. Although the autolytic cleavage reaction does not occur under physiological conditions, the study of this phenomenon has provided valuable evidence in support of the mechanisms postulated for the physiological covalent binding reactions. The ease with which autolysis can be induced and observed in C3, C4, and alpha 2 M has provided a valuable method for detecting the active forms of these proteins in circumstances where other assays are impracticable, as, for example, in the examination of the uptake of active C3 by lymphocytes. Autolytic cleavage has also been used by Karp and colleagues to produce fragments used in characterizing genetic and biosynthetic variants of mouse C4 and the mouse protein Slp, which is structurally similar to C4. Gross structural comparisons made among C3, C4, and alpha 2 M on the basis of alignment of the autolytic cleavage sites and the protease-activation sites in these proteins were useful in predicting how the alpha-, beta-, and gamma-chains of C4, or the alpha- and beta-chains of C3, were aligned in the single polypeptide chain pro-forms of these proteins. The beta-alpha-gamma alignment deduced for C4 was also found by Goldberger and Colten. Similar alignments of cleavage sites have been used as a basis for evolutionary comparisons of complement proteins and alpha 2 M from species other than man. Although autolytic cleavage has been described only for C3, C4, alpha 2 M, and Slp, it is likely that other proteins will be found that exhibit this phenomenon. A possible candidate is pregnancy-associated plasma protein A (PAPP-A) which resembles alpha 2 M in many respects. The autolytic cleavage reaction will serve as a useful indicator in the detection of other proteins that undergo covalent binding by the mechanism discussed above.

Complement C3↗
PubMed1982

Purification and structural studies on the complement-system control protein beta 1H (Factor H).

An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.

Amino Acid Sequence↗
PubMed1981

Binding of fluid-phase complement components C3 and C3b to human lymphocytes.

It is known that a population of B-lymphocytes has receptors for the third component of complement, C3, and that these lymphocytes may be identified by their ability to form rosettes with sheep erythrocytes coated with covalently bound fragments of complement component C3. Human tonsil lymphocytes, enriched for B-cells, form rosettes with sheep erythrocytes coated with antibody and complement components C1, C4b and C3b (EAC143b cells). Fluid-phase C3 will inhibit rosette formation between EAC143b and human tonsil lymphocytes over the same concentration range as fluid-phase C3b. C3 is not cleaved to C3b during incubation with lymphocytes or with lymphocytes and EAC143b cells. Fluid-phase 125I-labelled C3 and 125I-labelled C3b bind to lymphocytes in a specific manner. The characteristics of binding of both radioiodinated C3 and radioiodinated C3b are very similar, but the binding oc C3 is again not a result of cleavage to C3b. Salicylhydroxamic acid does not inhibit binding of 125I-labelled C3 to tonsil lymphocytes at concentrations that completely inhibit binding of 125I-labelled C3 to EAC142 cells via the nascent binding site of C3b. It is concluded that C3 and C3b share a common feature involved in binding to lymphocytes bearing receptors for the third component of complement.

Animals↗