Inhibition of complement alternative pathway in mice with Fab antibody to recombinant adipsin/factor D

Mouse adipsin is a serine protease secreted mainly by adipocytes. Similarly to factor D of human complement, it cleaves factor B. That adipsin is the equivalent of human factor D in the mouse is further suggested by their structural homology. Specific antisera against recombinant mouse adipsin (r-adipsin) were produced in rabbits. Anti-r-adipsin IgG was shown to bind to radiolabeled r-adipsin and to inhibit its hemolytic activity. In vitro, these antibodies Ab and Fab fragments thereof inhibited the adipsin/factor D hemolytic activity of mouse serum. They also blocked C3 activation induced by cobra venom factor (CVF), but did not interfere with classical pathway function. After intravenous injection of antir-adipsin Fab into BALB/c mice, the adipsin/factor D hemolytic activity of serum was abolished during a 4-h period. The C3 depleting effect of CVF injected intravenously was significantly delayed in BALB/c mice which had been pretreated with anti-r-adipsin Fab. These experiments demonstrate that mouse adipsin is the only form of mouse factor D and that anti-r-adipsin antibody can be used to produce a specific inhibition of the alternative pathway in vivo.     

C3 dysregulation due to factor H deficiency is mannan‐binding lectin‐associated serine proteases (MASP)‐1 and MASP‐3 independent in vivo

Uncontrolled activation of the complement alternative pathway is associated with complement‐mediated renal disease. Factor B and factor D are essential components of this pathway, while factor H (FH) is its major regulator. In complete FH deficiency, uncontrolled C3 activation through the alternative pathway results in plasma C3 depletion and complement‐mediated renal disease. These are dependent on factor B. Mannan‐binding lectin‐associated serine proteases 1 and 3 (MASP‐1, MASP‐3) have been shown recently to contribute to alternative pathway activation by cleaving pro‐factor D to its active form, factor D. We studied the contribution of MASP‐1 and MASP‐3 to uncontrolled alternative pathway activation in experimental complete FH deficiency. Co‐deficiency of FH and MASP‐1/MASP‐3 did not ameliorate either the plasma C3 activation or glomerular C3 accumulation in FH‐deficient mice. Our data indicate that MASP‐1 and MASP‐3 are not essential for alternative pathway activation in complete FH deficiency.     

Activation of the alternative complement pathway by extracts of cotton dust

Extracts of cotton dust were tested for their ability to activate the alternative complement pathway in fresh normal human serum (NHS). Alternative pathway activation was determined by a haemolytic assay utilizing glutathione-sensitized human erythrocytes, consumption of alternative pathway components in terms of alternative pathway CH50 units and an immunoelectrophoretic assay to detect split products of activation of factor B. All assays were performed under conditions that have been shown to block the initial steps of classical pathway activation but permit activation of the alternative complement pathway. Results demonstrate that the cotton dust extracts could consume alternative complement pathway proteins in a dose-response manner. The complement activating factor is probably endotoxin since a cotton dust extract obtained by an extraction method for endotoxin yielded the greatest activity.     

Activation of the alternative complement pathway and generation of chemotactic factors by asbestos.

Five types of asbestos plus silica and glass wool fibers were tested for their ability to activate the alternative complement pathway and to generate chemotactic factor activity from fresh normal human serum (NHS). Two assays were used to detect alternative pathway activation by the fibers. The first method was a hemolytic assay that utilized glutathione-sensitized human erythrocytes (GSHE) to detect the C5b-C9 complexes generated by activation of fresh NHS with the various fibers. The second method was an immunoelectrophoretic assay to detect split products of activation of factor B after the fibers were incubated with NHS. Both assays were performed under conditions that have been shown to block the initial steps of classical pathway activation but permit activation of the alternative complement pathway. All five asbestos fibers tested, amosite, anthophyllite, crocidolite, and chrysotiles A and B activated the alternative pathway when tested by both methods. In addition, it was demonstrated that chemotactic factor activity was generated when asbestos fibers were incubated with fresh NHS. In contrast, silica and glass wool fibers did not activate the alternative complement pathway and did not generate chemotactic factor activity. These observations suggest that the complement system may mediate the initial inflammatory response observed upon exposure to certain types of asbestos fibers.     

A monoclonal antibody which blocks the function of factor D of human complement

Factor D is an essential enzyme for activation of complement by the alternative pathway (AP). It has been difficult to obtain mouse monoclonal antibodies (Mabs) which block the function of factor D. We have developed a strategy to obtain such Mabs using a double screening procedure of the initial clones. We selected the clone whose supernatant had the lowest level of anti-factor D Ab by ELISA and abolished factor D haemolytic activity. Addition of this Mab to human serum was shown to abolish conversion of C3 by cobra venom factor, haemolysis of rabbit erythrocytes, and activation of C3 and C5 by cuprophane dialysis membranes.     

Interaction of zymosan and of activated properdin with factor D-depleted guinea pig serum: implications for the mechanism of initial C3 cleavage via the alternative complement pathway

Factor D of the alternative C pathway was specifically removed from guinea pig serum. The resulting serum reagent (RD) supported neither the formation of a C3-cleaving enzyme on zymosan (Z) nor the inactivation of C3 or of factor B in the presence of Z or activated properdin (P). Addition of purified D to RD restored these properties. Studies were limited amounts of purified D were added to RD with Z or P as activating substances, gave the following results: (1) Inactivation of B and of C3 occurs in the presence of minute amounts of D. (2) C3 inactivation is more efficient than B inactivation and proceeds even in the absence of detectable enzymatic B activation. (3) C3 cleavage at any D concentration tested is always accompanied by uptake of C3 fragments onto Z. With respect to initial C3 cleavage via the alternative C pathway these data suggest that the initiating reaction is D-dependent, very efficient in depositing C3 fragments on particulate activating substances such as Z and able to utilize factor B in an apparently uncleaved form.     

Steroids inhibit activation of the alternative-amplification pathway of complement.

Previous studies have demonstrated the ability of methylprednisolone sodium succinate to inhibit complement activation. Two other glucocorticosteroids and three different soluble steroids which lacked glucocorticoid activity were found in the present study to have the capacity to inhibit lysis of sheep erythrocytes by the alternative-amplification pathway. These compounds also inhibited fluid-phase consumption of B in a reaction mixture that contained purified C3b, B, and D, indicating that they exert a direct effect on the generation of alternative amplification pathway convertase. These findings suggest that the capacity to inhibit convertase generation by glucocorticosteroids in high concentration is independent of glucocorticoid activity.     

Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation.

The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.     

Functional deficiency of complement factor D in a monozygous twin

An adult twin with recurrent bacterial infections was found to have a partial functional deficiency of complement factor D. Full restoration of alternative pathway activity and zymosan- or cobra venom factor-induced consumption of C3 and B was found after reconstitution of patient's serum with purified D. Family studies revealed normal D levels in the mother, a brother and another sister. After gel filtration of patient's sera only little D activity could be detected in the fractions, and trypsin activation of the fractions also did not uncover detectable precursor D activity.     

The activation of the alternative pathway C3 convertase by human plasma kallikrein.

Human plasma kallikrein can replace factor D for the activation of the alternative pathway C3 convertase of human complement. The factor B cleavage patterns by factor D and kallikrein are indistinguishable. The ability of kallikrein to cleave factor B is influenced by the magnesium ion concentration and the C3b concentration. Factor D is about ten-fold more effective on a molar basis, for the alternative pathway C3 convertase activation than is kallikrein. The physiological role of the action of kallikrein on the alternative pathway C3 convertase is discussed.     

Activation of factor B of the alternative pathway of complement. Assessment by rocket immunoelectrophoresis

During activation of the alternative pathway of complement, Factor B is cleaved into a smaller fragment Ba and a larger fragment Bb. The Ba in plasma was quantitated by one-dimensional rocket immunoelectrophoresis after precipitating plasma B and Bb with 21% polyethyleneglycol. This method was much more sensitive and faster than other technics with which it was compared. Fragment Ba was detected in 22 of 28 rheumatoid arthritis plasma samples and in 12 of 15 systemic lupus erythematous samples but in only 8 of 68 samples obtained from healthy volunteers. The traditional approach of measuring total Factor B for the assessment of activation of the alternative pathway showed values that were even higher than normal in these diseases. The measurement of plasma Ba promises to be a valuable means of assessment of the activation of Factor B and the alternative pathway of complement in clinical disorders.     

Activation of the alternative pathway of complement by monosodium urate crystals

Monosodium urate crystals (MSU) have been shown to activate the alternative pathway of complement in a dose- and time-dependent fashion at 37 degrees C. Activation was maximal upon addition of 10-20 mg/ml monosodium urate crystals to C2-deficient human serum (C2D) or normal human serum containing 5 mM MgEGTA. Immunoelectrophoretic analysis of such treated sera demonstrated cleavage of C3 and factor B. Incubation of highly purified C3 and factor B with 10 mg/ml MSU did not, however, affect their immunoelectrophoretic pattern, suggesting that cleavage of either factor B or C3 in serum requires an intact alternative complement pathway. The fluid-phase control proteins, Factor H and Factor I, were not found to be diminished upon incubation of C2D serum or NHS containing MgEGTA with MSU. Thus activation appeared to be surface dependent and not a consequence of control protein depletion. It was also found, in agreement with earlier observations, that the classical complement pathway is activated, with concomitant depletion of C1 and C4. We conclude that MSU crystals activate both the classical and alternative pathways, and that such activation may participate in the pathogenesis of gouty arthritis.     

Role of fibrinogen in complement inhibition by streptococcal M protein.

M protein, the major virulence factor of group A streptococci, has antiopsonic activity in that it inhibits activation of the alternative complement pathway on the streptococcal surface. Two properties of M protein have been claimed to account for the inhibitory activity, namely, (i) its binding affinity for complement factor H, which is an inhibitor of alternative pathway activation, and (ii) its high binding affinity for fibrinogen. We have recently shown that fibrinogen, like M protein, inhibits alternative pathway activation by possessing binding affinity for factor H. Here we report that fibrinogen effectively competes with factor H for binding to M protein but retains its own binding affinity for factor H. The presence of fibrinogen did not significantly affect alternative pathway inhibition on the streptococcal surface.     

In vitro synthesis of factor B of the alternative pathway of complement activation by mouse peritoneal macrophages.

Factor B of the alternative pathway of complement activation was shown to be synthesized and secreted by unstimulated mouse peritoneal macrophages. The activity of B in the culture supernatants from macrophage monolayers was detected by consumption of C3 in reaction mixtures containing supernatant and guinea pig factors C3, D and insoluble C3b. Using a monospecific antiserum, factor B in concentrated culture supernatants was shown by immunodiffusion and immunoelectrophoresis to be identical to factor B in mouse plasma and to form a characteristic complex with cobra venom factor in the presence of D. A steady rate of factor B secretion was observed for 4 days providing the medium was changed every 24 h. Cycloheximide (0.5 mug/ml), an inhibitor of protein synthesis, caused inhibition (90%) of factor B production. Incubation of culture medium containing 14C-labeled amino acids with the macrophage monolayer resulted in incorporation of radioactivity into factor B as detected by autoradiography of precipitation lines formed with anti-B antiserum; This indicated that synthesis of factor B had occurred. In the same culture supernatants the presence of newly synthesized C3 was also demonstrated.     

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.     

Inhibition of the alternative pathway of complement by glomerular chondroitin sulphate proteoglycan.

Cultured rat glomerular epithelial cells (GEC) synthesize and secrete a complement inhibitory chondroitin sulphate B proteoglycan (termed GCRF). As proteoglycans and their component glycosaminoglycans may affect several different steps of complement activation, the functional properties of GCRF were investigated in this study. GCRF inhibited preformed alternative pathway convertases (C3bBbP), but did not substantially accelerate their decay. In contrast to other polyanions, GCRF did not affect the activity of human or rat factor H, nor did it inhibit the terminal complement proteins. GCRF inhibited the effect of decay-accelerating factor (DAF) on C3bBbP when the two were incubated together and DAF was in excess, but it did not affect the decay of DAF of classical pathway convertases. However, when DAF was first incorporated into the erythrocyte membrane, the effect of GCRF on C3bBbP was additive to that of DAF. Thus, GCRF inhibits the activity of C3bBbP and blocks the action of DAF, but not that of factor H, perhaps by binding to factor B in the alternative pathway convertase.     

Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP).

Human C3b bound to the ghost of sheep erythrocytes (E*) via activation of the alternative complement pathway (E*AC3b) consists of four major constituents on SDS-PAGE of 350, 260, 210 and 180 kDa. 350 kDa C3b is a dimeric form of C3b in which the alpha' chain of one C3b binds covalently to that of the other C3b. This complex is presumed to serve as a core for the alternative pathway C5 convertase. The other C3b populations are monomers complexed with membrane proteins or sugars. Using E*AC3b (C3b labeled) as a substrate, we have investigated functional properties of membrane cofactor protein (MCP), which is an integral membrane protein with C3b-binding and factor I-dependent cofactor activities. In conjunction with factor I, MCP was found to degrade the protein-bound C3b preferentially including the 350 kDa dimer. There was a similar but lesser tendency of this selective cleavage of C3b-dimer by CR1 but not by factor H or C4bp. In contrast to CR1 and factor H, detergent solubilization of EAC3b was required for MCP to fully express its cofactor activity for this selective degradation of C3b. We next separated the C3b dimer from the monomers and assessed their ability to assemble the alternative C5 convertase. The C3b dimer but not the monomers expressed C5 convertase activity following the addition of factors B and D, C5 and Ni2+. Kinetic analysis of the degradation of the C3b dimer by MCP and factor I suggested that only one C3b was efficiently converted to C3bi and this occurred concomitant with a decrease in C5 convertase activity. These results suggest that MCP has the ability to more efficiently interact with protein-bound C3b and that this may relate as well to its preferential ability to irreversibly inactivate the C5 convertase.     

Evaluation of alternative pathway and factor B haemolytic activities in patients with systemic lupus erythematosus: correlations with the alternative pathway regulatory proteins.

The haemolytic activities of the complement alternative pathway and factor B were studied using a 51Cr-release assay in 77 sera from patients with systemic lupus erythematosus (SLE). These values were compared to those of several complement components and of the regulatory proteins beta H and C3b INA. There was a significant decrease in the alternative pathway activity, which correlated with a decrease in the haemolytic activity of factor B and with the activity of the classical pathway. There was a significant decrease in the level of one regulatory protein beta 1H, while the level of the other regulatory protein, C3b INA, was increased; however, there was a positive correlation between these two parameters. The results obtained suggest that the decreased activity of the alternative pathway observed in SLE reflects a consumption due to a triggering of the C3b amplification loop through the activation of the classical pathway. In one patient, an acquired deficiency of the capacity to activate the alternative pathway in the presence of inulin was observed.     

Low-molecular weight inhibitors of the alternative complement pathway

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases.     

Interaction of poly(2-acrylamido 2-methylpropane sulfonate)-grafted polystyrene beads with cationic complement proteins.

Influence of various biomaterials on the complement system in serum has been intensively studied by many research groups, since activation of the complement pathway in vivo has been known to give rise to some pathological conditions, such as inflammation and anaphylaxis. Much effort has been devoted to develop new materials that do not activate or deteriorate the complement system. The present work is aimed at revealing the mode of reactions of anionic poly(2-acrylamido 2-methylpropane sulfonate) grafted on polystyrene bead (PAMPS-g-bead) with serum complement. Complement activity assay, determination of complement proteins levels, and immunoblot analysis were carried out for sera pretreated with PAMPS-g-beads. The results clearly showed that, when PAMPS-g-beads were incubated with serum, those beads adsorbed several complement proteins, i.e. C1q, factor D, factor P, C6, and C8, but the generation of activation fragments of complement components was not observed. Especially, factor D was most effectively removed from serum, resulting in potential inhibition of the alternative pathway. A larger amount of PAMPS-g-beads was needed to decrease the serum CH50 level. That may be caused by removal of C6. Although some polyanions, such as dextran sulfate, were reported to activate the complement system, the obtained results indicate that the PAMPS-g-bead is not an activator of the complement pathway, but acts as an adsorbent of complement components. One possible clinical application of the PAMPS-g-beads is adsorption of serum factor D by extracorporeal treatment of patients with renal failure with a high level of factor D, because the increased quantity of factor D in serum may cause consistent activation of the alternative pathway.