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PubMed1981

Use of cell differentiation effectors to select a human B lymphoblastoid cell line enriched in C3b receptors.

A study was undertaken to establish conditions of growth to increase C3b receptor synthesis on a human B lymphoblastoid cell line (Raji) by use of cell differentiation effectors. It appears that whereas two polar compounds HMBA (2mM) and Me2SO(2%) have no or little effect, 5 BrdU (30microM) and db cAMP (5 x10(-4) M) are able to increase in 48 h and 36 h respectively the synthesis of C3b receptor on Raji cell surface. These two compounds help to select a variant in which 100% of cells have C3b receptors with a high density of receptors per cell. The mechanism of BrdU action on the regulation of C3b receptor synthesis is discussed.

Acetamides↗
PubMed1996

Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae.

The majority of Klebsiella pneumoniae serum-resistant strains activate complement and bind C3b, the opsonic fragment of C3, without C5b-9 formation and bacterial killing. The mechanisms leading to C3b deposition without cell death were studied, and the results indicate that serum-resistant strains activate principally the alternative pathway and that serum-sensitive strains activate both the alternative and classical pathways. Bacterial molecules implicated in C3b deposition are the outer membrane porin proteins and smooth and rough lipopolysaccharides. Porins activate both complement pathways, and the rough lipopolysaccharide activates the classical pathway, causing deposition of C3b in serum-sensitive strains. The smooth lipopolysaccharide of serum-resistant strains activates only the alternative pathway, impeding the binding of C1q to porins (S. Albertí, G. Marqués, S. Camprubí, S. Merino, J. M. Tomás, F. Vivanco, and V. J. Benedí, Infect. Immun. 61:852-860, 1993; S. Albertí, F. Rodríguez-Quinónes, T. Schirmer, G. Rummel, J. M. Tomás, J. P. Rosenbusch, and V. J. Benedí, Infect. Immun. 63:903-910, 1995) and rough lipopolysaccharide molecules and thereby preventing activation of the classical pathway. After its deposition, C3b is quickly degraded to iC3b on both types of strains, but the higher-level deposition of C3b on serum-sensitive strains, resulting from activation of both the alternative and classical complement pathways, supports further complement activation and killing of serum-sensitive strains.

Bacterial Proteins↗
PubMed1979

Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b.

Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase. Analysis by linear regression of the inhibitory effect of each procedure (r = 0.97, r = 0.98, respectively) for adjusting the number of heparin molecules/particle revealed that the dose-response relationships were identical and that complete inhibition occurred with greater than 12 X 10(8) molecules of heparin/zymosan particle. The coupling of heparin to zymosan did not impair the uptake of C3b from the fluid-phase interaction of C3, B, and D, and did not alter the capacity of bound C3b to associate with B so as to permit its inactivation by D. Although the regulatory proteins present in normal serum chelated with EDTA or presented as a combination of purified C3bINA and beta 1H were relatively inefficient in inactivating C3b function on an activating particle of the alternative pathway such as zymosan or zymosan-cyanogen bromide, the control proteins rapidly inactivated C3b on a nonactivating particle wuch as a sheep erythrocyte or zymosan with coupled heparin. The increased numbers of C3b sites susceptible to inactivation by C3bINA in the presence of beta 1H were significantly correlated to the number of molecules of heparin/particle. By linear regression analysis of the correlation (r = 0.99) the number of heparin molecules/particle required to promote total inactivation of bound C3b by purified control proteins was 13.8 X 10(6). This molecular analysis suggests that the action of heparin coupled to an activating particle of the alternative pathway is to promote the interaction between particle-bound C3b and the regulatory proteins, thereby preventing particle-associated amplified C3 cleavage. It is noteworthy that both surface constituents known to maintain a particle as a nonactivator of the alternative pathway, sialic acid and N-sulfated mucopolysaccharide, act by facilitating the inactivation by regulatory proteins of the function of particle-bound C3b.

Complement Activation↗
PubMed1984

Effects of serum on C3b-stimulated release of prostaglandins and thromboxane B2 from human monocytes.

Previous studies have demonstrated that C3b stimulates the release of prostaglandin E and thromboxane B2 from human monocytes in serum-free cultures. We have examined the influence of serum on this phenomenon, and have found that addition of normal human serum or fetal calf serum to such cultures at concentrations of 0.1-5.0% results in enhanced release of both eicosanoids in C3b-stimulated cultures, while release in control cultures is unaffected or, in many cases, inhibited. Addition of human serum albumin or transferrin to serum-free cultures is not sufficient to mimic the serum effect. Fetal calf and normal human sera increase the efficacy of C3b in stimulating release of both prostaglandin E and thromboxane B2 at all but the lowest doses tested, but fail to significantly alter the kinetics or release or the acquired unresponsiveness of monocytes to C3b that occurs following culture periods of greater than 24 h. By preincubation of purified C3b in normal human serum, it can be shown that such serum degrades C3b stimulatory activity, but at a rate that is slow enough to permit expression of its activity when added at the beginning of the culture period. In contrast, the stimulatory activity of E. coli lipopolysaccharide is unaffected by pretreatment with serum. Thus, in contrast to the inhibitory effect of serum on the activity of C3 fragments in in vitro assays of certain mononuclear cell functions such as lymphocyte transformation and antibody production, serum enhances the ability of C3b to stimulate monocyte prostaglandin release.

Animals↗
PubMed1996

Enhancement of macrophage phagocytosis upon iC3b deposition on apoptotic cells.

Apoptotic cells activate homologous complement and are opsonized with iC3b. We assessed the effect of iC3b opsonization upon phagocytosis of apoptotic Jurkat cells by macrophages, which were differentiated from THP-1 cells by treatment with retinoic acid. Macrophage phagocytosis of apoptotic Jurkat cells was enhanced upon incubation of the apoptotic cells with normal human serum. The enhanced macrophage phagocytosis of normal serum-treated apoptotic cells was decreased by anti-human C3 F(ab')2 and anti-CR3 and anti-CR4 mAbs to the level of phagocytosis of those treated with complement-blocked serum. These results suggest that interaction between iC3b on apoptotic cells and complement receptor type 3 (CR3) and/or complement receptor type 4 (CR4) on macrophages could play an important role for the clearance of apoptotic cells by macrophages in vivo.

Apoptosis↗
PubMed1989

CR3 (CD11b/CD18) expresses one binding site for Arg-Gly-Asp-containing peptides and a second site for bacterial lipopolysaccharide.

Polymorphonuclear leukocytes (PMN) from three patients deficient in the CD18 family of receptors (LFA-1, CR3, and p150,95) exhibited an inability to bind erythrocytes coated with C3bi or bacterial LPS. These observations confirm that the CD18 family, and CR3 in particular, can bind the structurally dissimilar molecules C3bi and LPS. Further studies showed that LPS and C3bi bind to CR3 at distinct sites. mAb OKM10 against CR3 blocked binding of C3bi to PMN but did not block the binding of LPS. In contrast, mAb 904, directed against a different epitope on CR3, blocked binding of LPS to PMN but not binding of C3bi, thus suggesting that different regions of CR3 were involved in binding these two ligands. In addition, synthetic peptides based on the sequence in C3bi recognized by CR3 competitively blocked the binding of C3bi to CR3 but did not block the binding of LPS. Rather, occupation of the peptide binding site on CR3 by the synthetic peptides enhanced binding of LPS. These results indicate that CR3 has two distinct binding sites, one that recognizes ligands composed of protein and a second that recognizes LPS.

Antibodies, Monoclonal↗
PubMed1991

Inhibition of Epstein-Barr virus infection in vitro and in vivo by soluble CR2 (CD21) containing two short consensus repeats.

The extracellular domain of CR2, the Epstein-Barr virus (EBV)/C3d receptor of B lymphocytes, contains 15 or 16 tandemly arranged short consensus repeat elements (SCR). Recombinant CR2 proteins containing SCR 1 and 2 fused to Staphylococcus aureus protein A (PA-CR2) and to murine complement factor H SCR 20 (CR2FH) were expressed in Escherichia coli and in insect cells, respectively. These recombinant CR2 molecules retained functional activity as indicated by their ability to bind to C3dg in an enzyme-linked immunosorbent assay and to inhibit EBV gp350/220 binding to B cells. PA-CR2 and CR2FH were as efficient in blocking EBV gp350/220 binding as the full-length CR2 extracellular domain, indicating that the first two SCR of CR2 contain the majority of the ligand binding activity of the receptor. PA-CR2 and CR2FH inhibited EBV-induced B-cell proliferation in vitro and blocked the development of EBV-induced lymphoproliferative disease in severe combined immunodeficient mice reconstituted with human lymphocytes. These studies indicate that soluble forms of truncated CR2 proteins may have potential therapeutic value in the treatment of EBV-induced lymphoproliferative disorders in humans that involve viral replication.

Amino Acid Sequence↗
PubMed1980

Significance of glomerular C3b receptors in human renal diseases.

In order to elucidate their role in mediating renal immune injury in man, the glomerular C3b receptors have been analyzed in frozen sections of 205 kidneys biopsied from 199 patients affected by various well-defined renal diseases. Using fluoresceinated C3b-coated bacteria as the indicator system, the receptor activity has been compared with that found in 10 normal human kidneys. In 76 out of 98 cases (77%) of glomerulonephritis (GN) with capillary wall abnormalities, a significant loss of receptor activity has been observed. No difference was present between the 89 patients affected by GN presumably due to immune complexes (ICX) parietal deposition (membranoproliferative, membranous, acute poststreptococcal GN and proliferative GN of SLE and cryoglobulinemia) and the 9 patients affected by GN presumably due to anti-GBM antibodies (Goodpasture's syndrome and extracapillary GN). In the group of ICX-GN, no correlation could be demonstrated between inhibition of C3b receptors and the presence of parietal C3 deposits. A significant loss of receptors activity has been also demonstrated in 12 out of 13 patients with amyloidosis or diabetic glomerulosclerosis. On the contrary, inhibition of C3b receptors has been observed in only 3 out of 20 patients with nonglomerular renal diseases (interstitial and vascular nephropathies) and in only 11 out of 57 patients (19%) affected by mesangial GN with no capillary wall abnormalities (Berger's disease, Henoch-Schonlein purpura and mesangial GN of SLE). C3b receptors were normal in 13 out of 17 patients affected by lipoid nephrosis (minimal change GN or focal glomerulosclerosis). In conclusion, the analysis of C3b receptors in renal diseases do not support the hypothesis of their involvement in parietal localization of C3b-bearing ICX: these results rather suggest that the receptors become undetectable in many glomerular diseases (immune-mediated or not) with lesions of capillary walls, because of the loss of integrity of C3b receptor-bearing visceral epithelial cells.

Antigen-Antibody Complex↗
PubMed1989

The relationship between CR3 deficiency and neutrophil actin assembly.

Polymorphonuclear leukocytes (PMN) with a deficiency of the complement receptor type 3 (CR3) membrane glycoprotein family have impairments in the ability to adhere to surfaces as well as chemotactic and phagocytic defects, processes that require a functional contractile apparatus. PMN from the patient with neutrophil actin dysfunction (NAD) displayed similar functional characteristics to those with CR3 deficiency suggesting the two disorders may be the same disease. In order to evaluate the relationship between CR3 deficiency and actin assembly, actin filament assembly was measured in PMN from six previously reported homozygotes (two severe and four moderate CR3-deficient patients) as well as five heterozygotes for CR3 deficiency. PMN from all patients had normal unstimulated concentrations of F-actin and after exposure to the chemotactic peptide FMLP (5 x 10(-7) mol/L for 5 to 40 seconds at 25 degrees C) assembled actin normally. Pretreatment of normal PMN with concentrations of monoclonal anti-alpha CR3 antibody, capable of blocking PMN adherence, also failed to impair FMLP-induced actin filament assembly. CR3 glycoprotein expression was measured in PMNs from the mother, father, and older sister of the NAD patient (N Engl J Med 291:1093, 1974). Actin filament assembly was recently shown to be defective in PMNs from all three family members. The total concentrations of the alpha and beta CR3 subunits were below normal in PMN detergent extracts from the mother (25% of simultaneous controls) and older sister (56% of control). PMN surface expression of these two subunits was also found to be depressed (mother, 50%; older sister, 63% of control). These findings suggest these two NAD family members are heterozygote carriers for CR3 deficiency as well as NAD. Simultaneous studies of the father, however, demonstrated normal total concentrations of both the alpha and beta CR3 subunits (126% of controls) as well as normal surface expression of both subunits after phorbol myristate acetate stimulation and incubation at 37 degrees C (mean, 112% of controls) but slightly lower than normal levels after FMLP stimulation (mean, 83%). These findings indicate that CR3 deficiency generally is not associated with defective actin filament assembly and support the conclusion that NAD represents a unique kindred in which PMN actin function differs from previously reported genotypes of CR3 deficiency.

Actins↗
PubMed1988

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line.

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.

B-Lymphocytes↗
PubMed1982

Impaired antibody-dependent cell-mediated cytotoxicity in cryptogenic fibrosing alveolitis (synonym: idiopathic pulmonary fibrosis).

Mononuclear cells from the blood of 26 patients with the 'autoimmune' connective tissue disorder cryptogenic fibrosing alveolitis (CFA) were examined in Chang cell cytotoxicity assays for their capacity to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The results showed an impairment for the group by comparison with a group of 45 normal healthy controls (P less than 0.01). The impairment was greater in patients with associated connective tissue disorders of other systems (CFA+CT) than in those having the lung disorder alone (lone CFA); (P less than 0.001). The reduction in ADCC showed a correlation with reducing counts of cells bearing Fc gamma G surface receptors (P less than 0.05), and with increasing levels of soluble immune complexes in the blood of these patients by C1q binding (P=0.05). Non-specific esterase staining indicated that the Fc gamma G rosetting cells were subpopulations of lymphocytes not monocytes. We therefore suggest that the observed ADCC impairment may be due to impairment of lymphocyte Fc receptor function, and we speculate that this may influence immune regulation.

Adult↗
PubMed1978

The role of C1s, C1r and properdin in the initiation of the C3b-dependent feedback mechanism of the complement system.

The influence of activated C1s, C1r and properdin in the fluid phase initiation of the C3b-dependent feedback mechanism of the human complement was studied. It was found that C1s caused conversion of C3 and factor B in a normal serum, but not in a serum genetically deficient in C4 or in a serum to which Na2EDTA had been added. When a normal serum was incubated with C1r before incubation with C1s, only C3 was converted, whereas factor B remained in the unaltered native state. Properdin did not influence the C1s mediated conversion of C3 and factor B. When activated properdin was added to a properdin-depleted serum, both C3 and factor B were converted. Activated properdin was also incubated with purified C3 and purified C3b. It was shown that C3 was converted to C3b, but C3b was not degraded despite prolonged incubation.

Chromatography, Affinity↗
PubMed1988

Enhancement of the function of neutrophil type-1 and type-3 complement receptors by human leukocyte inhibitory factor (LIF).

The lymphokine leukocyte inhibitory factor (LIF) has previously been documented to enhance several neutrophil (PMN) functions, including stimulated chemotaxis and superoxide generation, phagocytosis and adherence of opsonized targets, and antibody-dependent cellular cytotoxicity. The present studies were designed to investigate the effects of LIF on PMN function mediated by the complement components C3b and C3bi. LIF induced a dose-dependent increase in superoxide production generated by opsonized zymosan (up to 97.1 +/- 31.4% at 16 U LIF/ml; P less than 0.01). While neither control nor LIF-treated PMN were capable of inducing phagocytosis of either C3b- or C3bi-opsonized sheep erythrocytes (E) directly, exposure to LIF caused a significant (P less than 0.05) increase in their adherence to E (137.4 and 59.4%, respectively). Specificity for complement receptor function was confirmed by the ability of anti-CR1 antibody to block adherence of LIF-treated PMN to EAC3b (77.0% inhibition) and anti-CR3 antibody to block adherence to EAC3bi (70.2% inhibition). Increased C3b and C3bi function may have been due, at least in part, to increased expression of their respective surface membrane receptors. Thus, using indirect immunofluorescence, LIF induced a 38.2% increase in fluorescence of the anti-CR1 antibody and a 96.1% increase in anti-CR3 binding. These studies describe an additional mechanism through which LIF may have an important pro-inflammatory role in vivo.

Antibodies, Monoclonal↗
PubMed1984

Reticulo-endothelial function in glomerulonephritis.

IgG- and C3b-coated erythrocytes were used to investigate splenic Fc-receptor function and hepatic C3b-receptor function by measurements of their blood clearance. Most patients with glomerular disease including those who were HLA, A1, B8, DR3 had normal Fc-receptor function. Occasional individuals with persistent glomerulonephritis had delayed clearance unrelated to disease activity. In rapidly progressive glomerulonephritis, the phenomenon of 'reticulo-endothelial blockade' was confirmed. C3b-receptor function was normal in most patients with persistent glomerulonephritis. A defect in splenic Fc-receptor function hepatic C3b-receptor function is unlikely to underlie persistent glomerulonephritis.

Adult↗
PubMed1990

Immobilization of C3b with retention of functional activity.

We compared eight commercially available, pre-activated affinity chromatography supports for ability to immobilize C3b that would retain functional activity. Pre-activated supports that we studied were: cyanogen bromide activated agarose, N-hydroxysuccinimide activated agarose, Reacti-Gel HW-65, Actigel A aldehyde activated agarose, thiopropyl activated agarose, 1,4-bis(2,3-epoxypropoxy) butane activated agarose, Reacti-Gel GF-2000 and tresyl activated agarose. The amount of C3b immobilized by each support varied from 81% for Actigel A aldehyde activated agarose to only 19% for Reacti-Gel GF-2000. We examined the functional capacity of the C3b immobilized on these various supports to participate in the alternative pathway. Immobilized C3b was mixed with factors D and B of the alternative pathway and examined over time for ability to consume factor B hemolytic activity. C3b immobilized on thiopropyl activated agarose consumed factor B at a rate comparable to unbound fluid phase C3b. C3b immobilized on other supports was less active in participating in factor B consumption. Thus, we have demonstrated the ability to immobilize C3b onto a solid matrix with the immobilized C3b retaining the ability to participate in the alternative pathway. This immobilized C3b can be used to fractionate substances with high C3b binding affinity.

Animals↗
PubMed1981

Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes.

C3b receptors on human polymorphonuclear leukocytes (PMN) were nonrandomly distributed in small clusters on the plasma membranes of these cells when assessed by indirect immunofluorescence at 0 degree C using monospecific rabbit Fab' or F(ab')2 anti-C3b receptor and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat IgG anti-F(ab')2. When PMN were incubated with the bivalent anti-C3b receptor at 37 rather than at 0 degree C, almost no immunofluorescence was observed, which indicates that the C3b receptor-F(ab')2 complexes had been rendered inaccessible to TRITC-IgG anti-F(ab')2. Endocytosis of the anti-C3b receptor ligand was quantitated by measuring the binding 131I-IgG anti-F(ab')2 by PMN that had previously taken up 125I-F(ab')2 anti-C3b receptor at 0 and at 37 degree C, respectively. There was a constant 2: 1 molar ratio of anti-F(ab')2 to anti-C3b receptor with PMN that had been incubated with the first antibody at 0 degree C. In contrast, when increments of F(ab')2 anti-C3b receptor were taken up by the cells at 37 degree C, there was a dose-related decline in this molar ratio to a minimum of 0.2 molecules of anti-F(ab')2 anti-F(ab')2 bound per molecule of PMN-associated anti-C3b receptor. 125I-F(ab')2 anti-C3b receptor taken up by PMN at 37 degree C was also inaccessible to release by proteolytic treatment of the cells with pronase. The rate of endocytosis of 125I-F(ab')2 anti-C3b receptor was rapid as the PMN-bound antibody fragment became inaccessible to 131I-IgG anti-F(ab')2 within 10 min during incubation of the cells at 37 degree C. In contrast to these findings, 125I-Fab' anti-C3b receptor that was taken up by PMN at 37 degree C remained accessible to both 131I-IgG anti-F(ab')2 and to proteolytic release by pronase, which suggests that monovalent interaction of ligand with C3b receptors was not sufficient for induction of endocytosis. The requirement for multivalency was also demonstrated using the C3b-OR, the normal ligand for the C3b receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell receptor. 125I-C3b-OR was specifically bound by PMN but remained on the cell surface, as determined by its accessibility to pronase, unless it was cross-linked with F(ab')2 anti-C3. Although C3b receptors on PMN do not mediate internalization of adsorptive pinocytosis of soluble ligand indicates their potential for the clearance of C3b-bearing immune complexes without recruitment of other cell surface receptors.

Antigen-Antibody Complex↗