Significance of C3 nephritic factor (C3NeF) in non-hypocomplementaemic serum with membranoproliferative glomerulonephritis (MPGN).

C3NeF is an autoantibody of C3 convertase (C3bBb) and is often detected in the serum of hypocomplementaemic MPGN patients. Serum samples from 104 non-hypocomplementaemic MPGN patients (C3NeF) were studied. C3NeF, which cannot activate the alternative pathway, was found in the sera of 6 patients. We examined the C3NeF in purified IgG from five of the non-hypocomplementaemic serum samples (non-hypo C3NeF) and four hypocomplementaemic serum samples (hypocomplementaemic C3NeF) to determine why C3NeF does not induce C3 splitting and hypocomplementaemia. Purified IgG from non-hypo C3NeF stabilized EAC4b3bBb cells in a manner similar to IgG from hypocomplementaemic C3NeF in EDTA gelatin veronal buffer. However, the non-hypo C3NeF IgG did not stabilize C3 convertase (EAC4b3bBb cells) in the presence of control proteins (factors H and I), whereas the hypocomplementaemic C3NeF IgG did. The C3NeF in the hypocomplementaemic serum displayed two characteristics: (i) inhibition of intrinsic decay of Ce convertase (C3bBb); and (ii) inhibition of extrinsic decay by factors H and I. Although the C3NeF in the non-hypocomplementaemic sera did inhibit the intrinsic decay in a manner similar to the hypocomplementaemic C3NeF IgG, it did not inhibit the extrinsic decay. Due to the different characteristics of hypocomplementaemic C3NeF and non-hypo C3NeF in the serum samples, the non-hypo C3NeF did not activate C3. Therefore, we conclude that C3NeF exhibits a heterogeneity which is very important in relation to the pathogenesis of MPGN.     

Monoclonal anti-human C3d antibodies: stabilization of the alternative pathway C3 convertase.

IgG mouse monoclonal antibody (mAb) was prepared by fusion of spleen cells from mice immunized with human C3d (mAb:C3d) using syngeneic thymocytes as feeder cells. mAb:C3d was assessed for its effect on the stabilization of the cell-bound alternative pathway C3 convertase EAC3bBb. It bound to cell-bound C3b and stabilized C3bBb at 30 degrees in the presence of EDTA-GVB. The plasma protein H reduced the stabilization effect of the stabilized C3 convertase. These results suggest that binding of antibody to C3d may stabilize C3bBb. It seems likely that such antibody induces in C3b conformational change, which increases the C3bBb complex stability.     

Modulation of the formation of the human amplification C3 convertase of complement by polycations

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.     

Human anti-idiotypic antibody responses to autoantibody against the alternative pathway C3 convertase

Anti-idiotypic antibodies to autoantibody against the alternative pathway C3 convertase (C3NeF) were isolated and purified from normal human serum as well as from serum from six patients with membrano-proliferative glomerulonephritis (MPGN). All preparations of anti-id antibody blocked C3NeF deposition on EC3bBb as well as C3NeF stabilization of EC3bBb functional activity. The Ka of these ant-id antibodies for C3NeF was 10(9) liters/mol which is comparable to the Ka of C3NeF for its antigen. In addition, 90% of anti-id antibody isolated from patients with MPGN and 20% isolated from normal individuals resembled Bb and bound to C3b as well as to antibody specific for the Bb portion of Factor B. These anti-id antibodies also resembled C3b and bound to antibody specific for the C3c portion of C3b. Immunization of rabbits with this latter form of anti-id antibody led to the production of functionally active C3NeF. These data indicate that C3NeF anti-idiotypic antibodies exist in two distinct forms, with and without internal imagery of C3bBb, and can occur in both normal individuals and patients with MPGN.     

Protection of the classical and alternative complement pathway C3 convertases, stabilized by nephritic factors, from decay by the human C3b receptor

Formation and function of the classical (C4b,2a) and alternative (C3b,Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b,2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b,2a(C4Nef)-bearing cells in a dose-dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell-bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b,Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b,Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.     

Proteolytic activity of the different fragments of factor B on the third component of complement (C3). Involvement of the N-terminal domain of Bb in magnesium binding

Kinetic experiments measuring the proteolytic activity of Bb and 33Kd fragment (the C-terminal domain of factor B) on C3 were performed in several conditions, in order to assess the role of factor B domains in the catalytic activity and magnesium binding. The experiments were carried out in fluid phase with 125I-C3 or C3(H2O) as substrates and in the presence of nonradioactive C3b as cofactor. The results indicate: (a) The C-terminal domain, 33Kd, possesses proteolytic activity on C3, which is Mg2(+)-independent, whereas proteolysis by Bb is enhanced in 5 mM Mg2+. (b) C3b behaves as cofactor of 33Kd proteolytic activity on C3 and factor H is able to inhibit this activity. (d) Kinetics of C3 proteolysis by 33Kd shows a lag phase which is also displayed by Bb in the absence but not in the presence of Mg2+. Taken together these data are consistent with the involvement of the N-terminal domain of Bb in Mg2+ binding, which results in an enhancement of the proteolytic activity on C3 of the adjacent C-terminal domain. A C3 convertase model accounting for these results is presented.     

Stabilization of homologous and heterologous cell-bound amplification convertases. C3bBb, by C3 nephritic factor

C3 nephritic factor (C3NeF) may be found in the sera of patients with membranoproliferative glomerulonephritis or partial lipodystrophy. It is capable of activating the alternative pathway in normal human serum; purified C3NeF has been shown to bind to the amplification convertase of complement, C3bBb. The binding of C3NeF to C3bBb results in stabilization of the otherwise labile C3 convertase. Decay of the convertase is accompanied by release of Bi and C3NeF from C3b. To determine whether stabilization of C3bBb occurs by the binding of C3NeF to C3bBb itself, to C3b or Bb alone, homologous and heterologous cell-bound convertases were prepared with C3^hu, B^hu, C3^rat and B^rat and exposed to C3NeF or properdin. It was found that properdin induced stabilization of C3b^huBb^hu, C3b^ratBb^hu, C3b^huBb^rat and C3b^ratBb^rat in a dose-dependent manner. On the other hand, nine out of ten C3NeF preparations were only capable of stabilizing C3b^huBb^hu and C3b^ratBb^hu and not C3b^huBb^rat and C3b^ratBb^rat. To determine whether binding of [¹²⁵I]-C3NeF to the various convertases occurred, cell-bound convertase were prepared in the presence of excess B^hu and B^rat, washed and further incubated with [¹²⁵I]-C3NeF; the cells were then washed and the amount of cell-bound [¹²⁵I]-C3NeF was measured. As in the stabilization experiments C3NeF bound only to C3^huBb^hu and C3b^ratBb^hu. The binding of C3NeF was always directly related to the presence of Bb^hu in the convertase. The results obtained with nine out of ten C3NeF preparations suggest that C3NeF is an autoantibody directed against antigenic determinants on Bb^hu, which are exposed after interaction of Bb^hu with C3b^hu or C3b^rat. One out of ten C3NeF preparations showed reactivity with both cell-bound C3b alone and cell-bound C3bBb. These reactivities could be separated by absorption with cell-bound C3b^hu.     

C3 nephritic factor (C3NeF): dissociation of cell-bound and fluid phase stabilization of alternative pathway C3 convertase.

Fluid phase C3 conversion by C3 nephritic factor (C3NeF) is usually easily detectable and forms the basis of the C3NeF screening test. We have described a group of patients with membranoproliferative glomerulonephritis or partial lipodystrophy and hypocomplementaemia who have an unusual C3NeF which stabilizes cell-bound C3 convertase of the alternative pathway (C3bBb) but causes such weak fluid phase C3 conversion that a C3NeF screening test is negative. These patients have low concentrations of C5 in serum.     

Activation of the alternative pathway of human complement by autologous cells expressing transmembrane recombinant properdin

Properdin (P) is a serum glycoprotein that stabilizes the labile C3 convertase (C3bBb) of the alternative pathway of the complement system (AP). Thanks to its oligomeric nature, P specifically upregulates AP on surfaces without activating AP in the fluid-phase. We investigated whether human cells, displaying P at their membrane, could activate autologous AP. The cDNAs encoding human P and the transmembrane domain of human platelet derived growth factor receptor were fused together and expressed in human embryo kidney cells (HEK-293). Selected cells displayed P at their surface as shown by FACS. In contact with human serum at 37 degrees C, they triggered AP-mediated C3 deposition. SDS-PAGE analysis showed C3 covalently bound to various membrane proteins, but not to P itself. However, displayed P affinity could bind to serum or purified C3i at 4 degrees C. C3 binding was restricted to the cells displaying P, was inhibited by an anti-P mAb, and did not require serum P. Bound C3 allowed further C5, C7 and C9 deposition as well as cell lysis after blocking CD59 function. In contrast, wild-type cells, cells displaying factor D or truncated P (deleted from its 6th thrombospondin-like repeat) did not activate AP. We hypothesize that displayed P activates AP by stabilizing bystander C3b and/or by capturing serum C3iBb convertase. Finally, we suggest that P could be used for retargeting autologous complement to AP-resistant pathogens and tumor cells.     

Regulation of the C3 nephritic factor stabilized C3/C5 convertase of complement by purified human erythrocyte C3b receptor

Activation of complement may result in the generation of the amplification convertase C3bBb. This convertase can be stabilized by properdin (P) or C3NeF. C3bBbP is susceptible to inactivation by beta 1H, while C3bBbNeF is relatively resistant. Since it has been shown that the human erythrocyte C3b receptor (CR1) is able to inactivate C3bBbP, the inactivating action of CR1 on C3bBbNeF was investigated CR1 is at least five times more efficient than beta 1H in inactivating C3bBbNeF. Kinetic studies revealed that CR1 induces an enhanced biphasic kinetics of decay of C3bBbNeF; further purification of this C3NeF preparation by cation exchange chromatography showed that this phenomenon is dependent on the population of C3NeF. Finally CR1 is also able to inactivate fluid phase C3bBbNeF.     

The cofactors required by C3 nephritic factor to generate a C3 convertase in vitro.

The mechanisms by which a C3 convertase is generated by C3 nephritic factor (NeF) were investigated using purified NeF, C3, C3b, factor B and factor D of the alternative pathway of complement activation. NeF could generate a C3 convertase with C3 and B in the absence of D, and without cleavage of B. At lower concentrations of NeF the addition of D was required to generate a C3 convertase, and B cleavage now occurred. The generation of both the D-independent and D-dependent C3 convertases with NeF was inhibited by preincubation of the C3 source with C3b inactivator (KAF); isolated C3b was more efficient than the C3 preparations used in generating the D-independent C3 convertase with NeF. These experiments indicate that C3b is required for the formation of both convertases, and that the reaction occurring with apparently native C3 is due to trace amounts of C3b. It is concluded that the C3 convertase generated by NeF in the absence of D is C3bB (NeF), and that generated in the presence of D is the feedback convertase C3bBb. The relevance of these experiments to reactions which may occur in vivo is discussed.     

C3 nephritic factor protects bound C3bBb from cleavage by factor I and human erythrocytes

C3 nephritic factor is an autoantibody to the alternative-pathway C3 convertase (C3bBb) which increases the half-life of the convertase both in the presence and absence of serum regulatory proteins. Human erythrocytes contain membrane proteins which also can regulate C3bBb. One of these proteins, the C3b/C4b receptor (CR1), plays an important role in the processing of soluble immune complexes. C3b which is fixed to immune complexes binds to CR1 and is cleaved by factor I to C3c and C3dg. We have tested the effectiveness of the nephritic factor in protecting bound C3b from cleavage by factor I and human erythrocytes. Sheep erythrocyte intermediates EAC1423b were prepared using 125I-labeled C3 and incubated with factors B and D in the presence and absence of nephritic factor. Breakdown of C3b was measured by release of 125I-C3c following incubation with human erythrocytes and factor I. Purified IgG from two patients with nephritic factor prevented C3c release in a dose-dependent manner. Normal human IgG was ineffective as was nephritic factor in the absence of factor B. Factor P also inhibited the release of C3c in the presence of factor B with equivalent activity at approx. 20-fold higher concns than nephritic factor. These results indicate that nephritic factor can impair human erythrocyte dependent degradation of C3b in alternative-pathway-activating immune complexes.     

A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.     

A reexamination of the role of magnesium in the human alternative pathway of complement

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.     

C2 by-pass: Cross-talk between the complement classical and alternative pathways

Several disorders associated with the total or partial absence of components of the human complement system are known. Deficiencies of classical pathway (CP) components are generally linked to systemic lupus erythematosus (SLE) or SLE-like syndromes. However, only approximately one-third of patients who lack C2 show mild symptoms of SLE. The relatively high frequency of homozygous C2 deficiency without or with minor disease manifestation suggests that there might be a compensatory mechanism which allows the activation of the CP of complement without the absolute requirement of C2. In this study we show that factor B (FB), the C2 homologue of the alternative pathway (AP) of complement, can substitute for C2. This was confirmed by using C4b as immobilised ligand and FB as analyte in Surface Plasmon Resonance (BIACORE). C2 binding to the immobilised C3b-like molecule C3(CH₃NH₂) was not seen. The estimated binding constant for C4bB complex formation was 2.00 * 10^?5 [M]. We were further able to demonstrate that C4b supports the cleavage of Factor B by Factor D. Finally, cleavage of ¹²⁵I-C3 by C4bBb was evaluated and gave strong evidence that the “hybrid” convertase C4bBb can cleave and activate C3 in vitro. Cleavage activity is very low, but consistent with some of the “C2-bypass” observations of others.     

FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a

We examined the inhibitory effect of FUT-175 on the C3/C5 convertase activity of the cobra venom factor-derived enzyme CVF,Bb by measuring C5b6-mediated reactive lysis of unsensitized guinea pig erythrocytes and by measuring directly the released fragments C3-des-Arg and C5a-des-Arg. In this study, we showed that the concentration of 4.5 X 10(-6) M of FUT-175 caused 50% inhibition of C5 convertase activity of CVF,Bb in reactive hemolysis assays, and that 4.0 X 10(-6) M FUT-175 caused 50% inhibition of the production of C3a and C5a generated by the C3/C5 convertase activity of CVF,Bb.     

The ability of H to modulate C5 utilization by the cell-bound alternative pathway C5 convertase differs on sheep and rabbit erythrocytes

The ability of H to inhibit formation of the alternative pathway C3 convertase differs on the non-activating cells sheep erythrocytes (E^s) and on the activating cells rabbit erythrocytes (E^r) or desialated sheep erythrocytes (E^des). C3 convertase sites are converted to C5 convertase sites by deposition of additional C3b molecules and C5 binds to cell-bound C3b in a reaction that is inhibited by H. C5 convertase sites P(C3b)_nBb were formed on E^s, E^r, E^des and revealed by incubation of the cells with purified C5 and with rat serum treated with KSCN and hydrazine. When incremental amounts of H were added to the reaction, a dose-dependent inhibition of C5 utilization was observed. The amount of H that inhibited 50% of C5 utilization was the same for a given cell regardless of the number of C5 convertase sites that were expressed on the cell. However the 50% inhibitory dose of H was 92 ng/10⁷ E^sP(C3b)_nBb, 87 ng/10⁷ E^desP(C3b)_nBb while it was at least 380 ng/10⁷ E^rP(C3b)_nBb. These results suggest that H competes with C5 for uptake within the C5 convertase site and that the regulatory effect of H on C5 cleavage depends on the surface to which the convertase is bound.     

Investigation of the mechanisms of anti-complement activity in Ixodes ricinus ticks

The feeding success of a tick upon a host depends on its ability to suppress host anti-tick responses which include activation of the complement system. We investigated the mechanism of inhibition of the alternative pathway of complement by salivary gland extract (SGE) of the ixodid tick species, Ixodes ricinus. SGE treatment strongly inhibited C3a generation and factor B cleavage in serum when rabbit erythrocytes were used as complement activator, but not when cobra venom factor (CVF) was used as an activator. SGE treatment strongly inhibited C3b deposition on rabbit erythrocytes, and the turnover of C3 (to C3b/iC3b) in serum. However, there was no significant effect upon the formation, stability or activity of C3 convertase (C3bBb) when formed from purified C3b, factor B and factor D. SGE treatment of isolated C3 resulted in a shift in mobility of the alpha-chain (by about 5 kDa). N-terminal sequencing of this species suggests that cleavage occurs at the C-terminus of the alpha-chain of C3. Consistent with this hypothesis, the modified alpha-chain was still a substrate for pre-formed convertase. The activity was specific for the alpha-chain of C3 but not of C3(H2O) nor the alpha'-chain of C3b. It is proposed that SGE-modified C3 does not participate in convertase formation, probably having a reduced affinity for factor B.     

In vivo modulation of rat complement activities by infusion of anti-H antibodies

Regulation of the activity of the alternative pathway of complement occurs by the plasma proteins I and H. H is able not only to prevent formation of the amplification convertase C3bBb but also to cause the decay-dissociation of Bb from C3bBb. The present studies have investigated the role of H in vivo. Affinity-purified goat (Fab')2-anti H was infused intravenously in rats, and its effect on C4, C3 and CH50 activities was assessed. In addition, H, C3, C4 and B were quantitated by immunochemical methods. H depletion was dependent on the dose of anti-H, and maximal consumption in vivo occurred between 5 and 15 min. A maximum of 51% and 77% hemolytic C3-consumption was seen after 15 min with 3.8 and 8.4 mg anti-H, respectively. The injection of 6.3 mg affinity-purified goat IgG anti-rat H caused 96.8 +/- 0.7% and 85.0 +/- 1.4% consumption of CH50 and C3 hemolytic activity, respectively. Using this dose of IgG anti-rat H, it was found that the clearance in rats of rat erythrocytes sensitized with 8000 molecules of guinea pig IgG2 per E was impaired as compared to the clearance of these intermediates in control rats injected with a comparable dose of normal goat IgG. The results indicate that H functions as a potent regulator of C in vivo and that infusion of anti-H can be used as a method to achieve depletion of circulating complement components.     

C3 convertase of the human alternative pathway of complement: modulation of enzyme activity and stability by mouse monoclonal antibodies to Bb

To further characterize functional sites on C3b,Bb, and C3 convertase of the alternative pathway of complement, we examined the effect of four monoclonal antibodies on its activity and stability. These antibodies recognized antigenic epitopes on human Bb which were not fully exposed in intact factor B. Three of the monoclonal antibodies inhibited lysis of rabbit erythrocytes by normal human serum in the presence of Mg2+ and EGTA. Two of these antibodies markedly inhibited the activity of purified C3b,Bb deposited on rabbit erythrocytes. However, all four monoclonal antibodies increased the half-life of the C3 convertase. Thus, these results demonstrate that binding of antibodies to Bb may concomitantly stabilize C3b,Bb and abate its activity. It is likely that such antibodies induce in Bb conformational changes which increase the C3b,Bb complex stability but may also hinder its catalytic site.