Bovine conglutinin binds to an oligosaccharide determinant presented by iC3b, but not by C3, C3b or C3c.

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.     

Prostaglandin E and thromboxane B2 release by PMA-induced U937 cells in response to C3b

The human monocytic cell lines HL-60 and U937 were examined for their ability to release PGE and TxB2 in response to the C3 cleavage fragment C3b. It was found that U937 responds to C3b by releasing both eicosanoids only after a 48 h induction period in the presence of PMA. HL-60 failed to respond. The response of U937 was further studied and characterized. It was found that the continual presence of PMA in culture was not required for the response to C3b, and that by replenishing the culture medium every 48 hr, the response could be maintained for up to 12 days. This contrasts with the previously observed behavior of human monocytes, which fail to respond after 24 hr in culture. Further studies demonstrated that maximal levels of PGE and TxB2 were elicited by 25-50 micrograms/ml of C3b, and that the kinetics of appearance of these eicosanoids in culture supernatants was very similar to that previously observed for monocytes. Maximal levels of PGE and TxB2 occurred following 24-48 hr in culture. In addition, a monoclonal a-CR1 antibody stimulated PG release by these cells. It is expected that this cell line will provide a model for study of the influence of cell differentiation on the response of human mononuclear phagocytes to C3b.     

Deposition of C3b/iC3b leads to the concealment of antigens, immunoglobulins and bound C1q in complement-activating immune complexes

Complement activation by bound IgG in serum at physiological concentrations is reflected in the deposition of C3b/iC3b in the absence of antigenic expression of the IgG or of any bound C1q on the target. The aim of this study was to investigate the functional requirements for this phenomenon and to establish its relationship to a release or concealment of the antigens. Microtiter wells coated with IgG by direct adsorption or by binding of IgG antibodies to pre-adsorbed homologous antigen were incubated with serum or serum reagents at 37 degrees C. The complement reaction was analyzed by ELISA to quantitate bound or released reaction products, and the release of IgG from the coated microtiter wells was gauged radiometrically. In the presence of serum, rapid binding of C1q and C3b occurred and was soon followed by a rapid loss of C1q expression; C3b binding remained high. Loss of IgG paralleled that of C1q.The functional requirement for the reaction was restricted to the activation and deposition of C3b/iC3b but was dependent of the combined function of the classical and alternative complement pathways. The loss of the IgG antigen was solely the result of antigen concealment, whereas the loss of C1q was only partly so. In biological terms, the concealment of bound IgG and C1q may reflect mechanisms by which complement down-regulates leukocyte responses stimulated by ligand-cell membrane receptor interactions.     

Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule.

Incubation of encapsulated and nonencapsulated Cryptococcus neoformans in normal human serum (NHS) leads to activation and binding of potentially opsonic fragments of complement component C3 to the yeast cells. Analysis of the molecular forms of C3 after incubation of encapsulated cryptococci in NHS showed that the percentage of bound C3 occurring as iC3b approached 100% after 8 min. The percentage of bound C3 occurring as iC3b on nonencapsulated cryptococci never exceeded 70%, even after 60 min of incubation in NHS. Conversion of C3b to iC3b was assessed further by incubating C3b-coated cryptococci for various times with a mixture of complement factors H and I at 40% of their respective physiological concentrations. Most, if not all, of the C3b on encapsulated cryptococci was converted to iC3b at a single fast rate. Conversion of C3b to iC3b on nonencapsulated cryptococci did not follow a single rate constant and appeared to have a fast and a slow component. Studies of the requirements for factors H and I in cleavage of C3b to iC3b showed steep dose-response curves for both factors in the case of encapsulated cryptococci and shallow curves with C3b bound to nonencapsulated cryptococci. Taken together, our results indicate that C3b molecules bound to encapsulated cryptococci have a uniformly high susceptibility to conversion to iC3b by factors H and I. In contrast, a significant portion of the C3b bound to nonencapsulated cryptococci is very resistant to conversion to iC3b by factors H and I.     

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.     

Streptococcus pneumoniae Capsular Serotype Invasiveness Correlates with the Degree of Factor H Binding and Opsonization with C3b/iC3b

Different capsular serotypes of Streptococcus pneumoniae vary markedly in their ability to cause invasive infection, but the reasons why are not known. As immunity to S. pneumoniae infection is highly complement dependent, variations in sensitivity to complement between S. pneumoniae capsular serotypes could affect invasiveness. We have used 20 capsule-switched variants of strain TIGR4 to investigate whether differences in the binding of the alternative pathway inhibitor factor H (FH) could be one mechanism causing variations in complement resistance and invasive potential between capsular serotypes. Flow cytometry assays were used to assess complement factor binding and complement-dependent neutrophil association for the TIGR4 capsule-switched strains. FH binding varied with the serotype and inversely correlated with the results of factor B binding, C3b/iC3b deposition, and neutrophil association. Differences between strains in FH binding were lost when assays were repeated with pspC mutant strains, and loss of PspC also reduced differences in C3b/iC3b deposition between strains. Median FH binding was high in capsule-switched mutant strains expressing more invasive serotypes, and a principal component analysis demonstrated a strong correlation between serotype invasiveness, high FH binding, and resistance to complement and neutrophil association. Further data obtained with 33 clinical strains also demonstrated that FH binding negatively correlated with C3b/iC3b deposition and that median FH binding was high in strains expressing more invasive serotypes. These data suggest that variations in complement resistance between S. pneumoniae strains and the association of a serotype with invasiveness could be related to capsular serotype effects on FH binding.     

Pattern of C3, iC3b, and C3d in patients hospitalized for acute asthma.

Twenty-three patients hospitalized for acute asthma were studied for a peripheral blood complement profile consisting of C3, C4, C3d, iC3b, C4d, and Bb concentrations. Compared with normals (n = 22) and patients (n = 10) with acute bacterial infections (ABI), asthmatic patients had significantly higher serum C3 concentrations (P less than .001). Plasma C3d levels and iC3b in asthmatic patients were both comparable to those observed in normal controls, whereas patients with ABI had significantly higher iC3b levels than both other groups. The ratio of iC3b to C3 concentrations were similar in asthmatic patients and controls, and iC3b levels were correlated with total serum C3 levels in asthmatic patients (r = .55, p less than .001) as well as in normal (r = .69, p less than .001). Both of these groups had significantly lower iC3b to C3 ratios compared with the ABI group (P less than .0001). Also observed in asthmatic patients were a significant correlation between serum C4 and C3 levels (r = .83, P less than .001) and a lower mean ratio of plasma C4d to C4 compared with normals (P less than .005). This profile of complement alterations is distinct from that observed in acute bacterial infection. These changes in asthmatic patients may relate to an acute phase reaction phenomenon affecting complement and/or complement regulatory proteins.     

Appearance of acceptor-bound C3b on HLA-DR positive macrophages and on stimulated U937 cells; inhibition of Fc gamma-receptors by the covalently fixed C3 fragments

The appearance and the functional role of acceptor-bound C3b during differentiation of human monocytes into macrophages were studied. Acceptor-bound C3b could be detected by the immune adherence (IA) test parallel to the expression of antigenic determinants specific to mature cells--i.e. on days 4-5 of culture. Consequently, the capacity of these phagocytes to fix C3b covalently via C3b-acceptors (C3bAs) can be considered as one of the signs of their activation/differentiation. All the mature macrophages positive in the IA test were also found to express HLA-DR antigens on their membrane. Using solubilized extracts of stimulated, 35S-cysteine-labelled cells of the human monocytic cell line, U937, we demonstrate that C3 synthesized by these cells can bind to C3bAs of the same cells. Covalently fixed C3 fragments were found to inhibit Fc gamma-receptor-mediated ingestion of immune complexes and also antibody-dependent cellular cytotoxicity of monocyte-derived macrophages.     

Expression of functional cell surface C1-inactivator by U937 cells

We have previously shown that the human monocyte-like cell line U937 synthesizes C1-INA and expresses cell surface C1-INA. In this report we provide evidence that this surface-expressed C1-INA is functionally active. Intact U937 cells demonstrated functional C1-INA activity in a hemolytic assay. This activity was blocked when the cells were incubated with monospecific antibody to C1-INA, and was not detectable in cell-free supernatants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purified by anti-C1-INA affinity chromatography revealed two distinct bands. One protein had a Mr of 105 kDa identical to plasma C1-INA, and the second had a Mr of 200 kDa. We were unable to determine the identity of the 200 kDa protein by Western blotting with anti-C1-INA. However, the possibility exists that this 200 kDa molecule may represent a C1-INA receptor, a dimeric form of C1-INA, or an unrelated cell surface protein with affinity for C1-INA. Furthermore, we show that treatment of U937 cells with phorbol ester resulted in an increase in the percentage of cells expressing surface C1-INA. These results suggest that U937 cells express functional cell surface C1-INA, which could function in vivo to protect these human tumor cells from lysis by host complement.     

Binding of multivalent CD147 phage induces apoptosis of U937 cells

CD147 is a broadly expressed cell-surface molecule and serves as a signaling receptor for extracellular cyclophilins. CD147 also appears to interact with immune cells, but its counter-receptor on these cells has not been clearly described. In the present report, we displayed multiple copies of the CD147 extracellular domain (CD147Ex) on VCSM13 phage to study the interaction of CD147 with its ligand. Recognition of phage containing fusion protein of CD147Ex and gpVIII (CD147Ex phage) by four different anti-CD147 mAbs indicated that at least parts of the CD147 are properly folded. Specific binding of CD147Ex phage to various cell types was demonstrated by flow cytometry. Morphological changes, however, were observed only in U937, a monocytic cell line, after 24 h incubation with multivalent CD147Ex phage. After 48 h, U937 cell propagation ceased. Staining with annexin V and the presence of cleaved caspase-3 indicated that many of the CD147Ex phage-treated cells had lost viability through apoptotic cell death. The above results suggest that CD147 induces apoptosis in U973 cells and that at least a portion of this cell death program involves a caspase-dependent pathway.     

Inefficient Binding of IgM Immune Complexes to Erythrocyte C3b–C4b Receptors (CR1) and Weak Incorporation of C3b–iC3b into the Complexes

The binding of soluble complement-reacted IgM immune complexes (IC) to erythrocyte (E) C3b–C4b receptors (CRI) and the incorporation of C3b–iC3b into solid phase IgM-IC was investigated. The optimal binding of liquid phase IgM-IC to E-CRI was obtained with IC formed at moderate antibody excess, but the binding was low (2–3%) when compared to the binding of the corresponding IgG-IC (50–60%). Solid phase IC were prepared by coming microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37°C. The binding of C3b–iC3b was determined by use of biotinylated F(ab')₂ antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b–iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching a maximum after about 2 h at 37°C. The binding of C3b–iC3b to the IgM-IC was remarkably inefficient when compared to the incorporation into IgG-IC reacted with the same amounts of BSA-precipitating antibody.     

Macrophage triggering by aggregated immunoglobulins. II. Comparison of IgE and IgG aggregates or immune complexes.

Macrophages incubated with complexed or aggregated IgE released beta-glucuronidase (beta-G) within 30 min. In contrast in the presence of aggregated or complexed IgG, macrophages liberated equivalent amount of beta-G only after 6 h incubation. In addition the rapid macrophage stimulation induced by aggregated IgE was also followed by a faster 3H-glucosamine incorporation when compared to the delayed activation caused by aggregated IgG. However, macrophages stimulated either by IgG or by IgE oligomers produced the same percentage of plasminogen activator at 24 h. In contrast, while the interaction between macrophages and aggregated IgE was only followed by a peak of cyclic GMP and a beta-G release during the first 30 min of incubation, the interaction between macrophages and IgG oligomers was accompanied by a simultaneous increase of cyclic GMP and AMP nucleotides and by an absence of beta-G exocytosis. Moreover, the beta-G release induced by aggregated IgE was increased when macrophages were preincubated with aggregated IgG. This additive effect was not observed in the reverse situation. Finally macrophages activated by IgG oligomers were demonstrated to exert a cytotoxic effect on tumour cells and to kill schistosomula in the presence of a low level of complement. Taken together these results underline the peculiar ability of aggregated or complexed IgE to trigger rapidly the macrophage activation compared to aggregated IgG and can explain the important role of complexed IgE in some macrophage dependent cytotoxicity mechanisms (i.e. in parasitic diseases).     

Selective and efficient inhibition of the alternative pathway of complement by a mAb that recognizes C3b/iC3b

The alternative pathway (AP) of the complement system plays an important role in tissue damage and inflammation associated with certain autoimmune diseases and with ischemia-reperfusion injury. Selective inhibition of the AP could prevent such pathologies while allowing the classical and lectin pathways of complement activation to continue to provide protection. Here we present data describing selective inhibition of the AP of complement by anti-C3b/iC3b monoclonal antibody (mAb) 3E7, and by a chimeric, "deimmunized" form of this mAb, H17, which contains the human IgG1 Fc region and was further modified by substitution of amino acids in order to remove T cell epitopes. Both mAbs block AP-mediated deposition of C3b onto zymosan or Sepharose 4B, and they also inhibit AP-promoted lysis of rabbit erythrocytes. MAbs 3E7 and H17 also successfully compete with both factors B and H for binding to C3b-opsonized substrates, and the ability of both mAbs to inhibit the AP is blocked by pre-incubation with two different sources of C3(H2O). Kinetic measurements demonstrate that mAb 3E7 effectively stops progression of C3b deposition after AP activation is initiated. Our results therefore suggest that these mAbs block activation of the AP by binding to both C3(H2O) and to C3b, and thus prevent binding and activation of factor B. Based on these and other observations, mAb H17 may find future use in therapeutic applications focused on selective inhibition of the AP.     

Candida albicans and Candida stellatoidea, in contrast to other Candida species, bind iC3b and C3d but not C3b.

It was demonstrated that complement-coated sheep erythrocytes bind to Candida albicans cells grown in serum-free RPMI 1640 medium. Testing of purified complement components proved that iC3b and C3d were responsible for the reaction, whereas C3b and C3b-H reacted only slightly if at all. Binding occurred only to C. albicans and C. stellatoidea, not to other species pathogenic to humans. There was evidence of a lectinlike nature of the effect.     

Antibody-Mediated Complement C3b/iC3b Binding to Group B Streptococcus in Paired Mother and Baby Serum Samples in a Refugee Population on the Thailand-Myanmar Border

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations.     

Characteristics of iC3b binding to human polymorphonuclear leucocytes.

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.     

Cleavage of complement C3b to iC3b on the surface of Staphylococcus aureus is mediated by serum complement factor I

Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 and 8 S. aureus strains after incubation in human serum. Using purified classical activation pathway complement proteins and the Western blot assay, we showed that when C3b was generated on the S. aureus surface no iC3b fragments were found, suggesting that other serum proteins may be required for cleaving C3b to iC3b. When C3b-coated S. aureus was incubated with serum factor I, a complement regulatory protein, iC3b was generated. Purified factor H, a serum protein cofactor for factor I, did not enhance factor I-mediated cleavage of C3b. These findings suggest that C3b cleavage to iC3b on S. aureus is mediated by serum factor I and does not require factor H.