Search

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H-Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H-ficolin in fungal defence. Additionally, we investigated the presence of H-ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H-Ficolin exhibited demonstrable binding to A. fumigatus conidia via l-fucose, d-mannose and N-acetylglucosamine residues in a calcium- and pH-dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H-ficolin opsonization manifested an increase in interleukin-8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen-activated protein kinase MAPK kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. Finally, H-ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H-ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H-ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus-host interactions and modulated immune responses., (© 2015 John Wiley & Sons Ltd.)

The erythrocyte type one complement receptor (E-CR1) mediates erythrocyte binding of complement-opsonized immune complexes (IC), and helps protect against random deposition of circulating IC. Two linked CR1 polymorphisms occur in binding domains, at I643T and Q981H. In Caucasians, the variant alleles (643T, 981H) are associated with low constitutive E-CR1 expression levels. This study was conducted to determine if these polymorphisms affect ligand binding, and if so, represent risk factors for the autoimmune IC disease, systemic lupus erythematosus (SLE). In an ELISA comparing relative ligand binding differences, E-CR1 from individuals homozygous for the variant residues (643TT/981HH) exhibited greater binding to C4b, but not C3b, than homozygous wild-type E-CR1. Analysis of single-binding domain CR1 constructs demonstrated that the 981H residue imparted this enhanced C4b binding. No differences were observed in the 981H allele frequency between Caucasian controls (0.170, n = 100) and SLE patients (0.130, n = 150, P = 0.133), or between African American controls (0.169, n = 71) and SLE patients (0.157, n = 67). In a subset of individuals assessed for CR1 size, excluding from this analysis those expressing at least one B allele revealed a trend for over-representation of the 981H allele in Caucasian controls (0.231 frequency, n = 26) versus SLE patients (0.139, n = 83, P = 0.089), but again no difference between African American controls (0.188, n = 24) and SLE patients (0.191, n = 34). These data suggest that the 981H residue compensates for low constitutive expression of E-CR1 in Caucasians by enhancing C4b binding. This may contribute protection against SLE.

We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2.

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells.

Treatment of patients with septic shock using monoclonal antibodies (mAbs) to endotoxin is still controversial. Clinical trials of E5, one of the mAbs directed against the lipid A moiety of lipopolysaccharide (LPS), are currently in progress. The mechanisms of action of this, and other antibodies under clinical evaluation, are, however, poorly understood. In this study we examined in vitro the ways in which E5 interacted with Gram-negative bacteria, complement, erythrocytes and monocytes. By fluorescence-activated cell sorter (FACS) analysis we showed direct, dose-dependent binding of E5 to Escherichia coli (E. coli) and Salmonella minnesota (S. minnesota). Antibody binding to S. minnesota was enhanced by treatment with the beta-lactam antibiotic amoxycillin, but not by treatment with the aminoglycoside gentamicin. Immune complexes formed between E5 and both species of Gram-negative bacteria activated both classical and alternative complement pathways, but only in the case of S. minnesota did this facilitate binding to erythrocyte CR1 and monocyte CR3. Bacterial C3b and iC3b fixation by E5 was quantified using specific mAbs. These observations suggest that E5 may enhance bacterial clearance in several ways: (1) by facilitating direct complement fixation; (2) by facilitating the binding of opsonized bacteria to cells of the mononuclear phagocyte system; (3) by enabling bacteria to bind to erythrocyte CR1 (CD35), allowing safe carriage in the circulation to the fixed macrophages of the liver and spleen; (4) by acting synergistically with beta-lactam antibiotics.

Ligands such as complement fragments (C3, C4), IgG or alpha 2-macroglobulin, which bind antigen (Ag) before their uptake by antigen-presenting cells (APC), are likely to modulate the different steps of Ag processing and presentation. These ligands contribute to internalization and endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatibility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha 2-macroglobulin family, an intrachain thiolester bond. Conformational alteration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to esterify hydroxyl groups of Ag. Ester-linked complexes including tetanus toxin (TT) and C3b were prepared to analyse the influence of bound C3b on TT processing and presentation by APC. Covalent binding of C3b to TT resulted in increased and prolonged stimulation of specific T-cell proliferation. This effect was observed with non-specific B cells, as well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endosome/lysosome-enriched subcellular fractions prepared from human Epstein-Barr virus (EBV)-transformed B cells, indicated a delay of TT proteolysis when TT was associated to C3b. Treatment of APC with protease inhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using purified cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolysis by cathepsin B was preserved. This finding and the absence of cathepsin B in endosomes may explain a delay in TT processing when it is associated to C3b. Confirming these data, presentation by formaldehyde-fixed cells of C3b-TT proteolysates showed higher stimulation of specific T-cell clones than formaldehyde-fixed TT proteolysates.

Bovine conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of conglutinin to iC3b, a fragment of the complement component C3. It was reported that conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.

Neutralization of the negative charges of heparin by polycations in the fluid phase suppressed the inhibitory effect of heparin on the generation of the C3b-dependent amplification convertase of complement C3b,Bb. Polymeric polycations alone, whether natural or synthetic, prevented formation of the cell-bound amplification convertase and of the fluid-phase interaction of C3b, B and D, in a dose-related fashion in the concentration range of 1 to 2 X 10(-8)M for poly-L-lysine (PLL) 50,000. The inhibitory effect of PLL on formation of the cell-bound convertase was independent of the presence of P. Percent inhibition of C3b,Bb,P and C3b,B,P formation was constant when the convertases were formed with a fixed concentration of PLL and increasing amounts of B; PLL was more effective in preventing convertase formation on cells bearing low numbers of C3b and developed with high doses of B. The decay of the preformed P stabilized convertase was not altered by PLL whether in the presence or absence of H. Thus, polycations in the fluid phase specifically inhibit formation of the amplification C3 convertase by preventing the association between C3b and B, most likely by acting on C3b. The low-affinity interaction between C3b and B is a privileged site for natural or pharmacological modulation of complement by polyelectrolytes.

Partial degradation of the biologically-active major fragment of the third complement component (C3b) to C3bi is catalysed by the endopeptidase C3b inactivator (I) and its co-factor, beta 1H globulin (H). Complete degradation to the fragments C3c and C3d requires an additional protease, which can be simulated in vitro by trypsin. This study was designed to identify the in vivo correlate of trypsin. Purified and 125I-labelled C3b bound to sheep erythrocytes was used as substrate. Release of label into the supernate served as an index of proteolysis. The chain structure of the peptides in the supernate or remaining bound to the erythrocytes was assessed by SDS polyacrylamide gel electrophoresis. Significant cleavage of cell-bound C3b was obtained by treatment with I, H and extracts from human peripheral blood leucocyte azurophil granules. Purified elastase also removed label in the presence of I and H. The peptide remaining on the cell had the characteristic 33K molecular weight of C3d. The activity of elastase in cleaving was blocked by alpha-1-anti-trypsin, the chloromethyl ketone, MeO-Suc-Ala-Pro-Val-Ch2Cl and by rabbit antibody to elastase. Thus, elastase purified from azurophil granules of human polymorphonuclear neutrophils (PMN) is a potent catalyst of the cleavage of C3bi to C3c- and C3d-like fragments and may contribute in vivo to the control of complement-mediated inflammation.

The generation from arachidonic acid and purification of large quantities of a series of monohydroxy-eicosatetraenoic acids (HETEs) which differed only in the position of the hydroxyl group permitted an in vitro analysis of the relative effects of the HETEs on a variety of human neutrophil functions. All of the HETEs elicited maximal neutrophil chemotactic responses of comparable magnitude, but the chemotactic potencies exhibited a distinct rank order with 5-HETE greater than 8-HETE:9-HETE (85:15, w:w) greater than 11-HETE=12-L-HETE. Peak chemotactic responses were achieved at concentrations of 1 microgram/ml for 5-HETE, 5 microgram/ml for 8-HETE:9-HETE and 10 microgram/ml for 11-HETE and 12-L-HETE. In the absence of a concentration gradient, the HETEs were similar in potency with respect to the stimulation of neutrophil chemokinesis and the enhancement of the expression of neutrophil C3b receptors. At optimally chemotactic and chemokinetic concentrations, none of the HETEs stimulated the generation of superoxide by neutrophils, altered the expression of neutrophil IgG-Fc receptors, or evoked the release of lysosomal enzymes. Methyl esterification of 5-HETE and 12-L-HETE reduced the chemotactic activity to less than 12% of that of the parent compound. The HETE methyl esters competitively inhibited the chemotactic activity of the homologous free acids by approximately 50% at equimolar concentrations, without inhibiting the chemotactic activity of formyl-methionyl peptides or of chemotactic fragments of the fifth component of complement (C5fr). The stimulus specificity of the competitive inhibition of chemotaxis by HETE methyl esters and the functional selectivity of the HETEs as compared to the formyl-methionyl peptides and C5fr, which stimulate neutrophil oxidative metabolism and lysosomal enzyme release, suggest that HETEs activate human neutrophils by a unique mechanism.

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.

Immunoglobulin G (IgG) provides an efficient acceptor site for nascent C3b, and complement activation on the surface of IgG-coated bacteria has been shown to generate significant numbers of C3b-IgG complexes. We have studied the relative efficiency of IgG alone, C3b-IgG complexes, and similar densities of IgG and C3b residues deposited independently, in mediating ingestion of sheep erythrocyte (E) targets by human phagocytes. Human 125I-C3b covalently bound to rabbit anti-Forssman IgG was generated as described elsewhere (Fries et al., 1985). E,EIgMC4b, or EIgMC4b3b (prepared with IgM antibody and purified complement components) were sensitized with radiolabelled anti-Forssman IgG or C3b-IgG heterodimers to generate targets bearing IgG alone, C3b-IgG covalent complexes, or C3b and IgG in equivalent numbers but not bound to each other. Phagocytosis by monocytes and polymorphonuclear leucocytes (PMN) of targets bearing C3b-IgG was markedly enhanced relative to those bearing IgG alone, especially at levels of less than 2000 opsonin residues/target cell. Uptake of C3b-IgG-bearing targets was also significantly more resistant to competitive inhibition by ambient monomeric IgG. Phagocytosis of EIgMC4b + C3b-IgG by monocytes was superior to the uptake of either EAC4b + IgG or EAC4b3b + IgG bearing equivalent amounts of C3b and IgG not in covalent complex (P less than 0.05, n = 10). Similar results were obtained with PMN. Thus, generation of C3b-IgG complexes in vivo may not only promote complement activation and enhance C3b deposition, but also produce a compound opsonic residue which is a more potent promoter of phagocytosis than an equal number of C3b and IgG residues randomly distributed relative to each other.

The effects of propamidine and their dose dependency, on utilization of the third and fifth complement components in immune haemolysis have been compared. While C3 utilization is not disturbed that of C5 is markedly inhibited by propamidine in concentrations as low as 0.5 mM. Both, binding of C5 to surface-fixed C3b and cleavage of C5 by convertases C42 and C3bBb, are also inhibited in the presence of propamidine. Since neither C3 cleavage by these enzymes nor even C5 cleavage by the cobra venom factor-supported convertase CFVBb is significantly reduced a general convertase-inhibiting effect of propamidine is ruled out. Rather the effect on utilization of C5 is the result of interference with binding of C5 is the result of interference with binding of C5 to C3b and hence impairment of its accessibility to the convertases. These findings thus further support the role of surface fixed C3b in C5 activation proposed earlier.

By use of a batch microcalorimeter of the thermopile type, heat production was measured in isolated populations of human peripheral blood cells exposed to defined immune complexes formed in vitro. It was found that most of the heat production recorded in whole blood after admixture of immune complexes occurs in the granulocytes. Under these conditions small but constantly higher activation values were found in the absence of active complement. It was shown that complexes consisting of antigen and F(ab)2 fragments prepared from the specific antibodies were able to initiate heat production in the cells only in the presence of active complement. These experiments indicate that immune complexes are able to induce increased heat production in the cells either by binding to Fc receptors or by activation of complement through the alternative pathway and subsequent binding of the generated C3b to C3b receptors on the heat-producing cells.

Culture supernatants (c.s.) collected from thioglycollate-elicited macrophages were concentrated and incubated with purified C3. In this reaction mixture loss of haemolytic C3 according to classical enzyme kinetics was observed. As revealed by SDS-PAGE, c.s.-catalysed fragmentation of the C3 alpha-chain occurred. The cleavage products were identified by size and function as C3a and C3b. The apparent molecular weight of this C3-activating enzyme in c.s. was approximately 220,000 according to ultracentrifugation studies. This large enzyme showed the following characteristics: it had no activity against C5; it was inhibited by EDTA; Mg2+ was required for its optimal function; its half-life at 37 degrees was approximately 35 min; it was completely inhibited by anti-B IgG. Thus, we were able to detect a C3-activating enzyme in c.s. containing B but differing otherwise from a preformed C3 convertase of the alternative pathway. The exact component composition of this new enzyme is under further investigation.

Factor B was isolated from fresh rat plasma by sequential chromatography on QAE-A50, Biorex-70, gel filtration on Sephadex G-200 superfine and rechromatography on QAE-A50. That Brat was isolated in its native form was indicated by its migration during immunoelectrophoresis and by its capacity to react with cobra venom factor (CoVF) in the presence of human D to form a C3 convertase capable of cleaving purified rat C3 and human C3. The recovery of Brat was between 8 and 15%; the final material was homogeneous according to SDS-PAGE analysis. Reduction of Brat with DTT in the presence of urea and SDS did not produce detectable peptides of lower molecular weight. Both reduced and unreduced Brat had an apparent molecular weight of 100,000. An antiserum against Brat induced in rabbits recognized only one protein in fresh rat plasma as indicated by immunodiffusion and immunoelectrophoretic analysis. Zymosan treatment of rat serum resulted in the cleavage of Brat into two fragments with alpha and gamma mobility. Native Brat has a beta electrophoretic mobility. The plasma concentration of Brat in Wistar rats was 215 +/- 38 microgram/ml (mean +/- SD).

We determined in binding assays using monomeric fluid-phase iC3b and Scatchard analysis that iC3b binds to human polymorphonuclear leucocyte type 3 complement receptor (CR3), a low-density/high-affinity receptor (28,200 binding sites, affinity constant (Ka) = 2.1 +/- 0.47 X 10(6) L/M), and to the C3b receptor (CR1), a high-density/low-affinity receptor (54,700 binding sites, Ka = 1.7 +/- 2.04 X 10(5) L/M. Binding of iC3b to CR1 was confirmed by blocking experiments with polyclonal F(ab')2 antibody against CR1, and competitive binding experiments with C3b. Binding of iC3b to CR3 was demonstrated by blocking experiments with the monoclonal antibody OKM10 against the ligand binding site of CR3. Inhibition of both CR1 and CR3 did not completely reduce iC3b binding, indicating the existence of additional iC3b-binding sites on PMN. Using flow cytometric analysis of receptor expression, no positive or negative co-operativity was observed between CR1 and CR3. Expression of both receptors increased in a dose-dependent manner after incubation with f-met-leu-phe or phorbol myristate acetate; however, only CR3 expression was enhanced at very low concentrations of these stimuli. iC3b/CR3 interactions probably play a central role in host defence against microorganisms.

Previous studies have shown that the degradation of soluble immune complexes or aggregates (AIgG) by normal peritoneal macrophages can be enhanced by complement. The enhancement of degradation was shown to be at least in part dependent on the number of C3b molecules bound per complex. The present investigations indicate that the enhanced degradation is not found with thioglycollate-stimulated macrophages, and that at high concentrations of complement, inhibition may even occur. The Fc receptor-mediated degradation of soluble immune complexes and AIgG by stimulated macrophages was at least twice as high as that by normal macrophages. This increase was compatible with the increased number of Fc receptors on the stimulated macrophages. The inhibitory effect of high concentrations of serum, as a complement source, on the degradation of AIgG was dependent on the number of C3b molecules bound per AIgG. Although there was also a two-fold increase in the number of C3b receptor sites on the stimulated macrophages, more than 11 C3b molecules per AIgG40 caused significant inhibition of degradation. This phenomenon may be dependent on shielding of Fc-Fc receptor interaction by varying numbers of C3b molecules per complex.

A steady state algebraic Mass Action analysis of the factors controlling the concentration of the alternative pathway C3 convertase is presented. The positive feedback loop does not make this portion of the complement system insensitive to variation in control protein concentrations; on the contrary, a hyperbolic relationship is revealed between C3 convertase and a complex product of the control protein concentrations which contains some mathematical non-linearities. The consequences of this relationship are explored. It is found that variations in the concentration of any one control protein will have an effect which can only be predicted if its initial concentration and those of the other control proteins are known. The system is capable of responding with a considerable range of amplitudes to a given signal which raises the question as to whether the feedback loop confers biological advantage, not only by augmenting the activation of C3 to C3b, but through its potential for flexibility in responding to an activating stimulus. The analysis may also prove useful in the design and evaluation of experiments to determine the reaction rate constants governing the interactions between the control proteins in the fluid phase in vitro.

C3b acceptors (C3bAs) of human peripheral blood mononuclear cells (PBMC) reacting with the labile binding site of nascent C3b(C3bx) have been investigated by the immune adherence (IA) test. In non-cellular systems some conventional chemical groups (OH-, NH-2) have been reported to be the target of the covalent binding of C3bx. Thus it should be assumed that every cell can fix C3bx via its labile binding site and C3bAs are barely saturable. Contrary to this expectation, however, normal human PBMC were found to be heterogeneous from this point of view, as 57 +/- 4% of B cells and 21 +/- 2% of Null cells possess C3bAs while T cells do not. C3bAs of human PBMC are saturable and trypsin-sensitive structures. The covalent nature of the C3bx-C3bA interaction has also been proved. Studying the effect of acceptor-bound C3b on the function of other cell-surface structures, the inhibition of the Fc gamma receptor function and the abolishment of the enhancement of pokeweed mitogen-stimulated blastogenesis by immune complexes were found.

The interactions of monomeric IgG, monomeric C3b, dimeric C3b, and C3b covalently bound to IgG (C3b-IgG) with neutrophils were examined. C3b-IgG heterodimers exhibited an increased affinity of binding to neutrophils, relative to C3b, both at half normal ionic strength (five-fold enhancement) and at normal ionic strength (six-fold enhancement). Binding of monomeric IgG to neutrophils was barely detectable and did not permit direct measurements of affinity in our assay conditions. The increased affinity of C3b-IgG for neutrophils was the result of divalent interactions with CR1, the C3b receptor, and the Fc gamma receptor of the cells, as could be demonstrated by competition studies using unlabelled IgG in the medium or monoclonal antibodies against the neutrophil Fc gamma receptor. This double receptor-ligand interaction allowed C3b-IgG to bind to the cells not only at normal ionic strength, but also in the presence of a 315-fold excess of IgG (near plasma concentration)--conditions that would preclude the binding of C3b or IgG alone. Furthermore, this interaction leads to the internalization of the C3b-IgG complex by the cells. C3b-IgG is internalized with kinetics similar to those found using dimeric C3b, with resting cells demonstrating a slow initial phase, which is accelerated by phorbol ester activation in both cases. Uptake of the homodimeric C3b was greater at 15 min than that of heterodimeric C3b-IgG, but both were significantly greater than that of monomer C3b, which showed no net internalization. The physiological implications of these findings are discussed.

Quantitative studies of the interaction between a fragment of the third component of complement (C3b) and its receptor on human cells have been undertaken with a recently developed radioligand binding assay. Specific binding of fluid phase monomeric C3b was direct and saturable under low (mu = 0.0513) but not physiological (mu = 0.15) ionic strength. Dimeric C3b had a greater affinity for the C3b receptor at either ionic strength. Complexing of monomeric C3b with another protein (human IgG) did not result in increase in binding to human erythrocytes when compared with monomeric C3b alone. These data suggest that ionic forces are involved in C3b receptor-ligand interactions and that the stable C3b-receptor binding site is present in monomeric C3b. Furthermore they suggest that the greater affinity of dimeric or oligomeric C3b is a result of multimeric binding to preclustered C3b receptors. These findings provide an explanation for the relatively stable interaction of particle bound C3b and C3b receptor-bearing cells in the immune adherence reaction.

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).