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.     

Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

Phosphorylation of C3 by a casein kinase released from activated human platelets increases opsonization of immune complexes and binding to complement receptor type 1

We have previously demonstrated that complement component C3 is phosphorylated both in vitro and in vivo by a casein kinase released from activated human platelets. In vitro, the studies have shown that cleavage of C3b by factor I is decreased, and binding to various target surfaces is enhanced by affecting the thiol ester. In the present study we have examined the effect of phosphorylation on the binding of C3b to complement receptor 1 (CR1, CD35). Upon phosphorylation by platelet casein kinase, C3b covalently bound to activated thiol Sepharose bound higher amounts of soluble recombinant CR1. Similar effects were demonstrated with two ELISA systems in which microtiter plates were coated with phosphorylated or unphosphorylated purified C3b or with C3 activated by the alternative pathway convertase. Phosphorylated C3b was also four times more efficient than unphosphorylated C3b in inhibiting the binding of complement-opsonized human aggregated gammaglobulin to erythrocytes. A similar increase in binding was found at low serum concentrations when the C3 activation occurred in C3-deficient serum reconstituted with phosphorylated or unphosphorylated C3. In this serum system, using a monoclonal antibody specific for iC3b, we also demonstrated that the phosphorylated C3b was protected against cleavage to iC3b. Corresponding experiments using factor H showed a decrease in binding of both fluid-phase and bound C3b to factor H. We postulate that phosphorylation of C3 by activated platelets amplifies the complement-mediated binding of immune complexes to CR1 by three different mechanisms: decreased cleavage of C3b to iC3b, increased deposition of C3b to immune complexes, and increased binding of C3b to CR1.     

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.     

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD)

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.     

Characterization of a Monoclonal Antibody MoAb bH6 Reacting with a Neoepitope of Human C3 Expressed on C3b, iC3b, and C3c

Activation products of the complement cascade contain neoepitopes that are not present in the individual native components. Monoclonal antibodies detecting neoepitopes have been used for direct quantification of activation at different steps in the cascade. These methods are suggested to be more sensitive and reliable than conventional complement activation tests, which are hampered by precipitation or fractionation procedures. The present study describes production screening and characterization of a monoclonal antibody (MoAb) bH6. MoAb bH6 exhibited a significantly higher binding capacity to ELISA plates coated with zymosan-activated human serum than to plates coated with EDTA plasma. When fixed to the enzyme-linked immunosorbent assay (ELISA) plates, MoAb bH6 retained material from zymosan-activated serum that only reacted with anti-C3 antibodies. Crossed immunoelectrophoresis performed on zymosan-activated serum demonstrated that MoAb bH6 co-precipitated with anti-C3c antibodies. In experiments using highly purified cell-bound fragments MoAb bH6 showed reactivity with C3b and iC3b, but not with C3d. MoAb bH6 reacted in ELISA with purified C3c, but not with C3dg, both as capture antibody and in tests with the fragments absorbed to the solid phase. Thus, MoAb bH6 is highly specific for a neoepitope of human C3 expressed on the cleavage fragments of C3b, iC3b, and C3c.     

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.     

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.     

Studies on the mechanism of C3b inhibition of immune complex induced C1 activation

We had previously demonstrated that in normal human serum (NHS) nascent C3b inhibited C1 activation by immune complexes (IC). We have now investigated the mechanism of this feedback inhibition. For these studies, EA-IgG were added to solutions containing physiological concns of purified C1, C1-In, C2, C3 and C4. Mixtures were then incubated at 37° C for 30 min. Western blot and autoradiographic analyses revealed that almost half of the IgG molecules had become covalently linked to C3b in a 1:1 complex with the C3' chain of C3b being bound to the heavy chain of IgG. IgG-C3b and free IgG were separated by ion exchange chromatography and immune complexes were formed with each. The consumption of complement in NHS by EA-IgG and EA-(IgG-C3b) were then compared. The results indicate that binding of C3b to IgG did not significantly inhibit the C1 activating potential of the IgG. Thus feedback inhibition is not due to the binding of C3b to IgG. An alternative mechanism was next explored. After incubation of EA-IgG with C1 through C3, EA were separated from supernantant fluid by centrifugation. It was determined that one-third to one-half of the IgG had been released from the erythrocytes. Release appears not to have been due to C3b binding to IgG, since the released IgG-C3b readily bind to fresh sheep erythrocyte (E), and since IgG that was free of C3b was also released from EA by complement, it is more likely that C3b binding to the E caused the dissociation of antibody.

These results indicate that under physiological conditions, the C1 activating potential of an immune complex is greatly reduced as the result of the binding of nascent C3b to the antigen moiety of the IC, thereby causing the displacement of complement activating antibody. In addition to IgG, IgG-C3b is also released from the IC.     

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.     

Analysis of C3 deposition and degradation on Neisseria meningitidis and Neisseria gonorrhoeae.

The deposition and degradation of human complement component C3 on the cell surfaces of Neisseria meningitidis and Neisseria gonorrhoeae were studied. Bacteria were incubated in human serum, and ester-linked C3 fragments were analyzed by hydroxylamine release and immunoblot detection. Similar patterns of C3 degradation were found for both serum-resistant and serum-sensitive meningococcal strains of serogroups A, B, C, Y, and W135, as well as for serum-sensitive gonococcal strains and their sialylated serum-resistant variants. The predominant fragments in all cases were the 40-kDa alpha' 2 chain of iC3b and the 75-kDa beta chain common to both C3b and iC3b. The 67-kDa alpha' 1 chain of iC3b was also detected. The 105-kDa alpha' chain of intact C3b represented a minor proportion of deposited C3. Capsule-specific immunoglobulin G or immunoglobulin A1 did not alter the observed degradation patterns, nor did incubation of meningococci in properdin-deficient serum. The degradation of C3 in C5-, C6-, or C8-deficient serum was the same as that in normal serum, although the deposition of C3 was severely limited, based as indicated by the intensity of the fragments. With the use of an enzyme-linked immunosorbent assay that measured total iC3b and C3, I found that both iC3b deposition and C3 deposition varied among meningococcal and gonococcal strains and that the amounts of iC3b and C3 were independent of the relative quantities of cell surface sialic acid and of serum sensitivity for meningococci but not for gonococci. I conclude that complement activation on neisserial cell surface results in the formation of an identical repertoire of predominantly iC3b fragments of ester-linked C3b molecules regardless of the presence of sialic acid in either the capsule or the lipooligosaccharide or of the sensitivity of the organism to complement-mediated lysis but that the quantities of both ester- and amide-linked iC3b molecules deposited exhibit strain variability.     

Formation of covalently linked C3-C3 dimers on IgG immune aggregates

Upon activation of the complement system by IgG immune aggregates several components become tightly bound to the aggregates. The covalent interaction of C3 with immune complexes is essential for the solubilization and inhibition of immune precipitation of the complexes. It has recently been reported that on erythrocytes that have a fixed complement, activated C3 can become involved in the formation of C3b-C3b covalent dimers, which acts as high-affinity binding sites for C5 (Kinoshita, T., Takata, Y., Kozono, H., Takeda, J., Hong, K. and Inoue, K., J. Immunol. 1988 141: 3895). To characterize the molecular composition of immune aggregates that have fixed complement by the alternative pathway, we have investigated whether such C3b-C3b dimers are formed in IgG immune complexes. For this purpose immune aggregates bearing covalently bound C3 were analyzed by two-dimensional gel electrophoresis and the resolved bands transferred to polyvinylidene difluoride membranes and sequenced. When immune aggregates were incubated with serum for 15 min at 37 degrees C, the major high-molecular mass bands detected by gel electrophoresis corresponded to heavy chain-C3 alpha 65 and C3 alpha 65-C3 alpha 43 (derived from iC3b-iC3b-IgG) covalent complexes. If K76COONa, an inhibitor of factor I, was added to the serum, before incubation with the immune complexes, then the major C3 alpha fragment detected on the complexes corresponded to the C3 alpha' chain (105 kDa) and not C3 alpha 65. Hence C3b-C3b covalent dimers are readily formed on the immune aggregates incubated with normal human serum, and are degraded to iC3b-iC3b by factor I. The second C3b molecule was shown to be bound to the C3 alpha 43 region (C-terminal portion of the C3 alpha' chain) of the first C3b molecule, which was itself covalently bound to the heavy chain of IgG. Covalent complexes of heavy chain-(C3 alpha 65)2 molecular composition were also detected, but their precise bonding pattern has not been established.     

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.     

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.     

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.     

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.     

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigenpresenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,χ and IgG2a,χ murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and puritied in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of χ light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigenpresenting cells.     

Rat polymeric IgA binds C1q, but does not activate C1

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.     

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.     

Anti-C1q column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between C1q and immune complexes

An efficient and reproducible procedure has been developed for the specific isolation of immune complexes. PEG precipitation of EDTA serum or plasma was an essential preliminary step to separate complex-bound from free C1q. PEG had no discernible effect on the molecular weight size of the extracted complexes. Redissolved complexes were incubated with a Sepharose-4B column coated with anti-human C1q antibodies and following removal of unbound material the bound complexes were sequentially eluted with 0.02 M EDTA, 0.5 M NaCl and 1 M propionic acid. Characteristics of the affinity column were established by the purification of 125I-labelled BSA-anti-BSA complexes and heat-aggregated IgG (HAGG) incubated in normal human serum (NHS). EDTA and NaCl eluted complexes were of similar molecular size and contained antigen, specific antibody, as well as human IgM, IgG, albumin, C3, C3c, C3d and C1q. Acid eluted complexes contained the highest yield of specific antigen and antibody and comprised in addition human C1q and C3d. Activation of complement components after C1q made the bond between C1q and immune complexes resistant to 0.5 M NaCl and interfered with the binding between solid phase anti-C1q and complex bound C1q. Using BSA-anti-BSA complexes and HAGG activated in NHS it was apparent that only a minority of the complexed material was isolated via the C1q ligand and this probably applies to the C1q binding assay. Most complexed material could be isolated using an anti-C3 affinity column.